101st Annual Meeting | Abstracts | Hot Topics | Keynote Address | Schedule of Events | Sponsors | Short Courses
BIO 1: Sterols I
Chair(s): R. Moreau, USDA, ARS, ERRC, USA; and P.J.H. Jones, University of Manitoba, Canada
The Role of a Disordered Steroid Metabolome in the Elucidation of Sterol and Steroid Biosynthesis and Metabolism. C. Shackleton1,2, 1Centre for Endocrinology, Diabetes and Metabolism (CEDAM), University of Birmingham, Birmingham, UK, 2Children’s Hospital Oakland Research Institute (CHORI), Oakland, CA, USA
In 1937 Butler and Marrian found large amounts of the steroid pregnanetriol in urine from a patient with the adrenogenital syndrome, a cortisol deficiency condition. This introduced the concept of altered production of metabolites as a tool for understanding sterol and steroid biosynthesis. From the cyclized lanosterol to the most downstream product estradiol, there are around 50 steps. Based on a distinctive metabolome clinical disorders have now been attributed to about 6 post-squalene cholesterol (C) biosynthetic steps and 10 en-route to steroid hormones. 20 Years ago it was thought that almost all steroid biosynthetic defects were known but interest rekindled as novel metabolomes were documented. We have been involved in two “new” steroid disorders, P450 oxidoreductase deficiency (ORD) and apparent cortisone reductase deficiency (ACRD). Interestingly, these result not from synthetic enzyme mutations, but in ancillary ones necessary for co-factor oxido-reduction. Our third interest is Smith-Lemli-Opitz syndrome (SLOS), a C synthesis disorder caused by 7-dehydrocholesterol reductase mutations. The late George Schroepfer, in whose honor this talk is given, contributed greatly to defining the sterol metabolome of this condition. Finding the cause of clinically severe disorders can lead to improved treatments. We are now involved in murine gene therapy studies which, if successful could in the future offer an alternative therapy for this severe condition.
Biological Activity of Phytosterol Glycosides. Richard E. Ostlund, Jr.1, Xiaobo Lin1, Susan B. Racette2, Lina Ma1, Robin Fitzgerald1, Catherine L. Anderson Spearie3, Robert Moreau4, 1Division of Endocrinology, Metabolism and Lipid Research, Washington University, St. Louis, MO, USA, 2Department of Physical Therapy, Washington University, St. Louis, MO, USA, 3Institute of Clinical and Translational Sciences, Washington University, St. Louis, MO, USA, 4Agricultural Research Service, USDA, USDA
Of phytosterols contained in common diets, 9-22% are glycosylated. In order to determine whether glycoside forms are bioactive and should be included in phytosterol measurements we conducted both human and animal experiments. Acylated phytosterol glycoside (ASG) was prepared by selective extraction from soy lecithin. In a randomized, crossover design 11 healthy subjects were given 3 single-meal cholesterol absorption tests in random order that contained 30 mg cholesterol-d7 and either no phytosterols, 300 mg phytosterols as esters (PE) or 300 mg phytosterols as ASG. Compared to no addition, ASG reduced cholesterol absorption 37.6 ± 4.8% and PE 30.6 ± 3.9% (both p<0.001). Similar results were seen in mice where, in contrast to PE, ASG phytosterols demonstrated little incorporation into plasma or liver sterols. ASG was quantitatively recovered in the stool as deacylated phytosterol glycoside. Cleavage of glycosidic linkages is not required for biological activity of ASG.
Long Term Cholesterol-lowering Efficacy of Phytosterols. I. Demonty, E.A. Trautwein, Unilever Research & Development, Vlaardingen, The Netherlands
The LDL-cholesterol (LDL-C) lowering efficacy of phytosterol (PS) (encompassing plant sterols and stanols)-enriched foods is well established. Meta-analyses of intervention studies confirmed that PS lower LDL-C by about 9% for a 2 g/d dose. Most studies included in meta-analyses were short-term (typically 4-12 wks). Nevertheless, longer-term studies suggest that the LDL-C lowering effect of PS is maintained over time. Two 1-yr studies with spreads enriched in plant stanols (Miettinen et al 1995) or sterols (Hendriks et al 2003) showed sustained LDL-C reductions over the treatment phase. Plant sterol- and stanol-enriched spreads were recently shown to similarly lower LDL-C in statin users over a 1.5 yr period (De Jong et al, 2008). Additional support for the sustained efficacy of PS is provided by the Portfolio diet studies, where 2 g/d plant sterols in spread combined with soy protein, dietary fibres and almonds were effective over a 1-yr period, with the main LDL-C lowering effect (9%) being due to plant sterols (Jenkins et al 2006, 2008). Observational data (Wolfs et al 2006) and community intervention studies (deJong et al 2007) provide further support to the long-term effectiveness of PS-enriched spreads. Overall, there is sufficient evidence from longer-term human efficacy studies lasting for up to 1.5 yr that the LDL-C lowering effect of PS intake via enriched foods is sustained.
Plant Stanol Ester Enriched Foods Lower LDL-C and Serum Triacylglycerol Concentrations. J. Plat, R.P. Mensink, Maastricht University, Maastricht, The Netherlands
Plant stanol enriched foods are known for lowering serum LDL-C. In an earlier meta-analysis, we found that in subjects with high baseline serum triacylglycerol (TAG) concentrations, plant stanol esters may lower serum TAG. We therefore initiated two placebo-controlled studies in subjects with elevated serum TAG concentrations. In study 1, a 9-weeks placebo-controlled study with a yogurt drink (2.0 g stanols/d) in 18 patients with the metabolic syndrome we found a 28% reduction in serum TAG (P=0.044) in the stanol group vs placebo. Moreover, the number of large TAG-rich VLDL-1 particles was significantly lower, suggesting a reduced hepatic VLDL-1 production underlying the effect on TAG concentrations. In study 2, a 3-weeks placebo-controlled study with margarine (2.5 g stanols/d) in 28 familial combined hyperlipidemia patients we again found a reduction in serum TAG vs placebo (-11%) but only in those with TAG levels > 2.3 mmol/L (P=0.009). This illustrates that effects on TAG can only be shown in subjects with elevated TAG levels. Although the number of VLDL-1 particles was not significantly lowered in study 2, a significant correlation was found between the plant stanol-induced changes in TAG and VLDL-1 particles.We conclude that foods enriched with plant stanols not only lower serum LDL-C, but also TAG levels in subjects with overt hypertriglyceridemia.
The Occurrence and Biological Roles of Steryl Glucosides in Foods, Vegetable Oils, and Biodiesel. R. Moreau, M.J. Haas, Sustainable Biofuels and Co-Products Research Unit, Eastern Regional Research Center, ARS, USDA, Wyndmoor, PA, USA
In animals, cholesterol, C27H46O, is the major sterol. Plants usually contain a mixture of several C28 and C29 sterols, which can occur in several forms, including as steryl glucosides (SGs) and acylated steryl glucosides (ASGs). In plants, ASG and SG are components of biological membranes and may be involved in signal transduction. We recently found that in corn ASG is a major lipid in the membrane surrounding the triacylglycerol-storing organelles. In vitro studies with pancreatic enzymes suggested that the fatty acid portion of ASG is hydrolyzed during digestion, but the glycosidic bond appears to remain intact, as recently confirmed by in vivo studies. Although SGs and ASGs are effectively extracted with polar solvents, they are also partially extracted with hexane and are thus detectable in unrefined plant oils. Conventional refining/bleaching/deodorization (RBD) removes most of the ASG and SG, but both are still detected in RBD oils. Biodiesel is produced by converting the triacylglycerols in oils to methyl (or ethyl) esters. Some of the ASG or SG present in plant oils used to make biodiesel will be carried into the biodiesel product. During this process most of the ASG will be converted to SG. If SG remains dissolved in the biodiesel it has no affect, but under certain conditions, especially low temperatures, the SG can precipitate and cause fouling during storage and in diesel engines.
Oxidation of Plant Sterols in the Industrial By-products of Edible Fats and Oils. Paresh Dutta, S.J. Kumari A. Ubhayasekera, Department of Food Science, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden
In addition to fatty acids, oxidation also affect other molecules with or without double bond in their chemical structures, such as plant sterols (phytosterols) in food lipids or bulk fats and oils. A wide range of phytosterol oxidation products (POPs) can be formed in vegetable fats and oils during the refining steps of degumming, neutralization, bleaching, deodorization, and during food preparation, heating, storage and handling. Heat treatments often at temperature of 250°C and higher are used in the refining steps. A number of valuable by-products are also formed while refining crude fats and oils for human consumption. Some of the by-products are used as ingredient of animal feed formulation. The quality of the fatty sources in feed formulation is very important in the production of high-quality food of animal origin. We have studied the levels of POPs in various types of by-products e.g. acid oils from chemical refining, acid oils from physical refining, lecithins, oils extracted from exhausted bleaching earth, hydrogenated fats from by-products, and recycled cooking oils from the food chain. A varieties of POPs such as 5,6-epoxyphytostanols, epimers of 7-hydroxy-, and 7-ketophytosterols, were identified and quantified by GC and GC-MS. Large variations in the levels of POPs were observed which was possibly due to the origins and different processing conditions.
Public Perception of Values of Plant Sterols in Foods. J. Cranfield, University of Guelph, Guelph, Ontario, Canada
Increasingly consumers are using food products containing bioactive compounds as a means to enhance their health and well-being. Plant sterol containing foods and natural health products offer an interested example in this respect. Such products are currently available in a number of regions of the worlds, but are still unavailable in a number of countries, despite their proven efficacy in alleviating elevated levels of serum blood cholesterol. This presentation will review the economic and consumer science literature related to consumer understanding, acceptance of use of plant sterol containing food and natural health products. Focus is placed on the motives and barriers to use of these products, the role of health information in shaping acceptance and use, and characterizing the profile of consumers who use these products.
Global Regulations and Health Claims on Phytosterol-containing Functional Foods. Jerzy Zawistowski, Food, Nutrition and Health, University of British Columbia, Vancouver, BC, Canada
Phytosterols represent one of the most intensely and actively researched groups of bioactives in the area of cardiovascular diseases. This group of compounds has been clinically proven to reduce total and LDL blood cholesterol. Based on their efficacy and safety profile, sterols are approved for use in foods in many countries. One of the challenges food formulators face is following current regulations and laws specific to the target market. The regulations have a significant impact on choice of food matrix, number of serving sizes that are used for delivering phytosterols, ingredient and nutritional composition of food products, as well as allowable maximum daily doses. Manufacturer and marketers are responsible for ensuring that a food label and associated advertising material is truthful, non-misleading and substantiated. To market a new sterol-containing food product, the majority of international jurisdictions require a cumbersome dossier containing information regarding food composition, formulation and processing, as well as clinical and safety substantiation. This talk will focus on the regulatory aspect of sterol/stanol containing foods and health claims, their substantiation, and the authorization systems in various countries.
Economic and Societal Benefits of Plant Sterols in Foods. P.J. Jones, Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, MB, Canada
Plant sterols are naturally occurring chemical compounds that have the potential to lower serum LDL-cholesterol levels in humans when fortified into foods. Currently plant sterol fortified foods are not legal in Canada, despite being permitted for sale in over 35 other countries. The health-related and economic savings of introduction of plant sterols into the diets of Canadians has not yet been assessed. The objective of this study was to quantify the potential economic savings which can be attributed to the consumption of plant sterol enriched foods to allow for informed policy decisions surrounding legalization of plant sterols in Canada. A four step economic simulation model involved i) assessment of a sterol intake “success” rate, ii) estimation of blood cholesterol reduction due to consumption of foods containing plant sterols, iii) determination of the reduction in the incidence of coronary heart disease (CHD) due to decrease in blood cholesterol levels, and iv) calculation of the reduction in costs associated with CHD due to a reduction in the overall incidence of CHD. The model used varying assumptions to provide both conservative and less conservative estimates. The savings in direct health care expenditures and lost human capital was assessed as ranging from $38 million to $2.4 billion. This reduction represents a significant economic saving for Canada. Supported by CAPSIC.
Phytosterols and Phytosteryl Conjugates in Grains. Anna-Maija Lampi1, Tanja Nurmi1, Laura Nyström1,2, Vieno Piironen1, 1University of Helsinki, Helsinki, Finland, 2ETH Zurich, Zurich, Switzerland
The aim is to give an overview of phytosterol composition of cereal grains and their fractions, and present data on their steryl conjugates. Phytosterols were included in a diversity screen study (www.healthgrain.org) where 150 wheat lines and 50 other cereals were analyzed for bioactive compounds. There were differences in phytosterol composition among species, and the highest contents were found in rye (1098-1420 μg/g dm). The contents in various wheat types were 670-1187 μg/g dm. The most abundant phytosterol was sitosterol followed by campesterol and stanols. A study on environmental factors on 26 wheat genotypes showed that growing location had a significant effect on phytosterols.Localization of phytosterols in grains was also studied. Dry milling experiments showed that white wheat flour contained only ca 50% of the phytosterols of whole grains whereas the contents in the bran fractions were up to 300%. During sequential debranning highest phytosterol levels were found in the intermediate and aleurone layers. Milling fractions were also characterized for steryl conjugate profiles. Whole grain wheat flour contained 53%, 35% and 12% of free, esterified and glycosylated sterols, while the proportions in rye flour were 42%, 48%, and 12%, respectively. Profiles varied greatly within a grain. In wheat germ oil, the proportion of steryl glycosides was <1% while it increased to 8% in the bran and up to 15% in the refined flour.
BIO 1.2 / PCP 1: Process and Co-products of Biofuel and Industrial Production
Chair(s): K. Liu, USDA, ARS, PWA, USA; D. Solaiman, USDA, ARS, ERRC, USA; J. Wanasundara, Agriculture & Agri-Food Canada, Canada; and H. Wang, Iowa State University, USA
Product Opportunities from Algae. Joel Butler, Joanna Money, Solix Biofuels, Ft. Collins, CO, USA
While much of the recent interest in algae has been driven by its value as a potential fuel, algae has the potential to deliver a large number of additional products including nutraceuticals, food supplements, feed for animals and fish and bioplastics. Some of these products are more valuable than biocrude but their extraction from the algae is complex and therefore their viability as a product is still unclear. This presentation will describe these potential products in more detail and discuss some of the challenges facing algae producers in extracting their value.
In situ Transesterification of Algae for the Production of Fatty Acid Methyl Esters for Use as Biodiesel. M.J. Haas, K.M. Scott, Eastern Regional Research Center, USDA, Wyndmoor, PA USA
There has recently been considerable interest in the use of lipids synthesized by algae as feedstocks for the production of renewable fuels. One possibility is the production of biodiesel, which consists of the simple alkyl esters of fatty acids, from algal acylglycerols. This would typically involve the dewatering of the algal biomass, recovery of its lipid by pressing or solvent extraction, and transesterification of the resulting lipid preparation to form biodiesel. An alternative to this approach, termed 'in situ' transesterification, is to conduct the transesterification reaction directly on the dried algal biomass. This eliminates the need for pressing or solvent extraction steps to recover the oil from the algal mass before transesterification, and could therefore simplify and reduce the cost of biodiesel production. We have investigated this approach for the synthesis of biodiesel from algal oil, and will present the results of this investigation.
Glycerine - A Valuable Biodiesel Coproduct for Fermentation Processes. R.D. Ashby, D.K.Y. Solaiman, T.A. Foglia, USDA, ARS, ERRC, Wyndmoor, PA, USA
One of the impediments in the widespread use of biodiesel is the prohibitive cost involved in removing and/or refining the glycerine coproduct. Attempts to impart value to crude glycerine have resulted in the development of a number of technologies designed to utilize crude glycerine as a precursor for value-added product synthesis. The focus of our research group is the bioproduction of green polymers and surfactants with potential as substitutes for petroleum-based materials. We have focused our attention on the utilization of low-value feedstocks (e.g. glycerine, soy molasses) to improve process economics of fermentation-based syntheses. In this presentation, we will discuss our continued efforts to improve product yields of polyhydroxyalkanoates (PHA; bacterial polyesters) and sophorolipids (SL; glycolipid surfactants) from both refined and crude glycerine through manipulation of fermentation parameters and the potential for property modification of the bioproducts based on the absolute content of the crude glycerine. Lastly, we have demonstrated the antibacterial properties of SL towards Propionibacterium acnes by using SL:Poly-3-hydroxybutyrate (PHB)glycerine composite films and showed, through scanning electron microscopy, the physical effects of SL addition on the surface topography and porosity of PHB polymer films.
Advances in Corn Ethanol Enzyme Technology, Effect on DDGS and Opportunities for Animal Feed Industry. M. Hruby, Danisco Animal Nutrition, Woodbury, MN, USA
Enzymes are crucial in production of ethanol from corn and other starch sources. Enzymes facilitate production of sugars used by yeast to yield ethanol through a fermentation process. Enzymes can also function as processing aids to improve throughput, reduce slurry viscosity and salt deposits in the processing equipment, water and the use of other compounds such as acids. Ethanol production in United States comes mainly from dry mill grind plants. Besides ethanol and CO2 , dry mill grind plants also produce distillers dried grains with solubles (DDGS). DDGS are increasingly counted as economically important products of ethanol production process. A deliberate focus on higher nutrient digestibility, low antinutrient levels and high uniformity of DDGS could result in a significant increase of ethanol plant′s revenues. Targeted focus on DDGS quality can be crucial at times when ethanol production gives lower return on investment. Nutritionists have been using greater quantities of DDGS in animal diets. Understanding the effect of various enzymes used in ethanol production process on DDGS quality and uniformity can provide final users, whether domestic or in export markets, with a valuable information to achieve a greatest economic benefit in production of animal protein when offered specific DDGS to include in animal feeds.
Techno-economic Analysis of Brassica Protein and Co-product Extraction Technologies for Food and Biofuel Applications. Edmund K Mupondwa, Janitha Wanasundara, Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada
This study presents a techno-economic analysis of an aqueous extraction process to recover proteins of Brassica oilseeds, focusing on napin (2S) and cruciferin (11S) proteins, both of which have distinct biological, physicochemical, and structural properties as well as industrial applications. This is distinct from other processes whose derived protein extracts are mixtures of 11S and 2S proteins. The aqueous extraction process provides various fractions of different physicochemical and functional properties that are expected to significantly enhance high-value application of Brassica proteins, beyond current traditional uses. A mass transfer study of a pilot plant setup was conducted together with an economic analysis of the extraction process. Simulations were conducted for capital equipment sizing and costs to predict various commercial-scale plant capacities. A full economic and financial analysis was completed including capital investment, annual manufacturing costs, primary and co-product revenues, product sales, risk-adjusted return on investment, and payback period, using an appropriate rate of return. The study shows that the economics of the aqueous extraction process are positive notwithstanding the fact that the aqueous extraction operating conditions had not yet been optimized.
Manufacturing New Food Co-Product(s) Using a Novel Ethanol Corn Fractionation System. T. T. Lohrmann, D.J. Hammes, Quality Technology International, Inc, Elgin, IL USA
A majority of the ethanol plants today are designed such that the entire corn kernel is ground prior to fermentation. The remaining non-fermentable fraction after fermentation is commonly dried and sold into the livestock markets as a lower value feed product termed distiller dried grains with solubles. The current focus of our technology is to convert ethanol plants from a traditional dry grind system into a next generation fractionation bio-refinery capable of making bulk food products such as high TDF corn bran and high purity corn germ for further processing to corn oil. Since our wet fractionation systems for ethanol do not utilize sulfur dioxide, production of high purity food products are attainable. Recent work by our group on second generation products has shown that corn germ proteins can be isolated and purified in to novel commercial food products. Other second generation, further-processed food products are in development which will bring even greater consumer benefits and value from this evolving industry.
Identification of Novel Co-product Opportunities From the Low Temperature Fermentation of Grains to Ethanol. D. Bressler, R. Zijlstra, A. Gibreel, University of Alberta, Edmonton, AB, Canada
With the support of the fermentation industry, research focused on determining the biofuel potential of several non-traditional grains including barley, triticale, pulses and other crops with wheat and corn serving as benchmark feedstocks. Research into the fermentation of these non-traditional grains via both traditional jet-cooking and low temperature enzymatic approaches was conducted. An emphasis was placed on freeze-drying the resultant distillers grains and the final product were chemically profiled and assessed in terms of feed value as well as opportunities for the extraction of high value components. Chemical profiling clearly demonstrated that with newer low temperature enzyme approaches, several high-value components survived fermentation and were in fact concentrated three to five fold in the resulting solids fractions. The high-value components identified included tocopherol, tocotrienols, polyphenols, and phytosterols. Additionally, the fatty acid composition of the grains was preserved through fermentation. The identification of these potential co-products as an extraction opportunity prior to high temperature drying of distillers grains provides an opportunity for diversification of value-addition in the ethanol industry.
The Composition of Crude Corn Oil Recovered after Fermentation via Centrifugation from a Commercial Dry Grind Ethanol Process. R.A. Moreau1, K.B. Hicks1, D.B. Johnston1, N.P. Laun2, 1ERRC, USDA, Wyndmoor, PA, USA, 2Western New York Energy, Medina, NY, USA
A study was conducted to examine the chemical composition of corn oil obtained via centrifugation after fermentation of corn to make fuel ethanol, and compare its composition to that of corn germ oil (commercial corn oil) and experimental corn oils. The levels of free fatty acids in the post fermentation corn oil were high (13-16%), as previously reported. The levels of free phytosterols and hydroxycinnamate steryl esters (similar to oryzanol in rice bran oil) were higher than those of corn germ oil and were comparable to those of ethanol extracted corn kernel oil. The levels of tocopherols and tocotrienols were lower in post-fermentation oil than in either corn germ oil or ethanol extracted corn kernel oil. The levels of lutein and zeaxanthin in post-fermentation were much higher than those in corn germ oil and were comparable to those in ethanol extracted corn kernel oil. Overall, exposure to all upstream processes of a fuel ethanol plant, including high-temperature liquefaction, saccharification and fermentation appeared to have the greatest effect on tocopherols and tocotrienols, but it had little effect on the levels of free phytosterols, hydroxycinnamate steryl esters, lutein and zeaxanthin.
Changes in the Oil Concentration, Fatty Acid Composition, and Functional Lipid Profiles During Dry Grind Ethanol Production from Corn. Keshun Liu1, Robert Moreau2, Jill Moser3, 1USDA, ARS, Aberdeen, ID, USA, 2USDA, ARS, ERRC, Wyndmoor, PA, USA, 3USDA, ARS, NCAUR, Peoria, IL, USA
Demand for alternatives to fossil fuels has resulted in a dramatic increase in ethanol production from corn. The dry grind method has been the major process, resulting in a large volume of dried distiller grains with solubles (DDGS) as a co-product. This presentation reports our study to monitor concentration changes of various types of lipids during the entire dry grind process of corn. Samples of ground corn, intermediate products and DDGS were provided by three commercial plants in Iowa. After lyophilization, the moisture and crude oil levels as well as the fatty acid, phytosterol, tocopherol, and tocotrienol composition were measured. Results from Plant 1 samples show that the three steps that caused significant increases in oil content as compared with ground corn were slurrying, fermentation, and centrifugation (only into thin stillage). However, for all other lipids measured, including fatty acid composition (relative to total fatty acids), and composition of phytosterols, tocopherols and tocotrienols (relative to total lipid mass), the process did not cause significant changes, even at the three critical steps that caused significant increases in oil content. The conclusion is that the dry grind process caused a significant change in lipid quantities, but not lipid quality. Data from Plant 2 and 3 confirmed those found with Plant 1 samples.
Changes in Physical Properties During Dry Grind Processing of Corn. K.A. Rosentrater1, K. Liu2, 1USDA, ARS, Brookings, SD, USA, 2USDA, ARS, Aberdeen, ID, USA
Corn-based ethanol has dramatically increased in the U.S. in recent years. So too has the quantity of coproducts. These are composed of nonfermentable components (i.e., protein, lipid, fiber, and ash) from the corn kernel. These materials are separated from the ethanol and then subjected to various separations and drying processes. The most common coproduct is distillers dried grains with solubles (DDGS). DDGS has become widely used as a livestock feed ingredient. One of the key issues associated with DDGS is poor flowability. The objective of this study was to identify where in the manufacturing process stickiness is imparted to the coproduct material. Eight samples were collected from two corn-based fuel ethanol plants, and included raw corn, cooked slurry, liquefied mash, whole stillage, thin stillage, wet cake, distillers dried grains (i.e., no added solubles), and DDGS. Each of these samples was dried at 50ºC for 24 h, then milled to 0.5 mm. Properties tested included Carr Testing (angle of repose, aerated bulk density, packed bulk density, compressibility, angle of spatula, total flowability index, angle of fall, angle of difference, dispersibility, floodability index), thermal conductivity, thermal diffusivity, and color (Hunter L, a, b). Results of these tests will be presented and compared, and implications for the industry will be discussed.
Integrated Production of Ethanol and Succinic Acid in a Biorefinery. Nhuan P. Nghiem, Kevin Hicks, David Johnston, Eastern Regional Research Center USDA ARS, Wyndmoor, PA, USA
Production of succinic acid from glucose by Escherichia coli strain AFP184 was studied in a batch fermentor. The bases used for pH control included NaOH, KOH, NH4OH, and Na2CO3. The yield of succinic acid without and with carbon dioxide supplied by an adjacent ethanol fermentor using either corn or barley as feedstock was examined. The carbon dioxide gas from the ethanol fermentor was sparged directly into the liquid media in the succinic acid fermentor without any pre-treatment. Without the CO2 supplement, the highest succinic acid yield was observed with Na2CO3, followed by NH4OH, and lastly by the other two bases. When the CO2 produced in the ethanol fermentation was sparged into the media in the succinic acid fermentor, no improvement of succinic acid yield was observed with Na2CO3. However, several-fold increases in succinic acid yield were observed with the other bases, with NH4OH giving the highest yield increase. The yield of succinic acid with CO2 supplement from the ethanol fermentor when NH4OH was used for pH control was equal to that obtained when Na2CO3 was used, with or without CO2 supplementation. The benefit of sparging CO2 from ethanol fermentation on the yield of succinic acid demonstrated the feasibility of integration of succinic acid fermentation with ethanol fermentation in a biorefinery.
Assessment of a High Purity Zein Product from Commercial Zein. David J. Sessa, Kristen Kruger Woods, Plant Polymer Research, NCAUR, USDA, ARS, Peoria, IL, USA
Successful utilization of commercial zein products for food, pharmaceutical, cosmetic and medical applications requires a decolorized/deodorized zein that is substantially undenatured protein. A zein protein with those qualifications has already been developed by a patent pending process. The objective of this presentation is to devise methodologies to assess the required attributes. Zein purity was assessed by FTIR and circular dichroism (CD) of commercial zein before and after further purification. Spectral differences were observed in the amide I (1650 cm^1^) peak, amide II region (1530 and 1515 cm^1^) and the amide III peak at 1240 cm^1^, where ratios of dominant peaks were strongly dependent on purity of sample. CD analyses validated FTIR results by showing increased α-helical content for purified zein. We defined off-odor removal by UV spectroscopic ratio of 280nm:325nm of ≥8, where diferoylputrescine is the major contributor. Color removal, attributed to xanthophylls in zein was followed by a visible spectroscopic assay. These results are a major contribution for achieving a consistent zein product.
BIO 1.1: Biocatalysis I
Chair(s): C. Hou, Renewable Product Technology Research Unit, NCAUR, ARS, USDA, USA; and J. Ogawa, Kyoto University, Japan
A Novel Aliphatic Amino Acid Metabolism in Bacteria Generating a Potential Insulinotropic and Anti-obesity Amino Acid. J. Ogawa1,2, T. Kodera3, S. V. Smirnov4, N. N. Samsonova4, M. Hibi5, K. Yokozeki5,6, S. Shimizu1, 1Division Applied Life Science, Graduate School of Agriculture, Kyoto University, Kyoto, Japan, 2Res. Div. Microb. Sci., Kyoto University, Kyoto, Japan, 3Inst. Life Science., Ajinomoto Co., Inc., Kawasaki, Japan, 4Ajinomoto-Genetika Res. Inst., Moscow, Russia, 5Lab. Ind. Microbiol., Grad. Sch. Agric., Kyoto Univ., Kyoto Japan, 6Aminosci. Lab., Ajinomoto Co., Inc., Kawasaki Japan
4-Hydroxyisoleucine (HIL) is a polar non-charged amino acid and was found to be an active component of fenugreek seeds, which are used in spices and in traditional medicines as a remedy for diabetes and hypercholesterolemia. Based on its insulinotropic and anti-obesity effects, HIL might be considered as a novel orally-active drug with potential for the treatment of insulin-independent diabetes mellitus and obesity. Here we show that newly isolated Bacillus thuringiensis strain 2e2 operates a novel metabolic pathway for L-isoleucine via 4-hydroxyisoleucine (HIL) and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL synthesis was found to be catalyzed stereoselectively by a novel alpha-ketoglutaric acid-dependent (succinic acid-producing) dioxygenase and to be useful for efficient production of a naturally occurring HIL isomer, (2S,3R,4S)-HIL. The metabolic pathway functions as an effective by-pass pathway that compensates for the incomplete TCA cycle in B. thuringiensis, which lack the alpha-ketoglutaric acid dehydrogenating activity being necessary to generate succinic acid, and also explains how AMKP, a vitamin B12 antimetabolite with antibiotic activity, is synthesized. These novel findings pave a new way for commercial production of HIL and also for AMKP.
Chiral Technologies for Single Enantiomer Drug Intermediates Through Biocatalysis. Ramesh N. Patel1,2, Sandip J. Parekh2, 1SLRP Associates, Bridgewater, NJ USA, 2Unimark Remedies, Ltd, Mumbai, India
Biocatalytic processes for producing chirally pure pharmaceutical intermediates are of growing importance. Biocatalysis is rapidly evolving into a key technology for the production of fine chemicals and chiral intermediates where high yielding chemo-, regio-, and enantioselective reactions are critical. Advances fermentation technology and development in molecular biology methods such as over expression of enzymes and directed evolution technologies have now lead to improvement in activity, selectivity and stability of biocatalyst under process conditions. Biocatalysis provides advantage offering higher yields, fewer side reactions, elimination of protection and de-protection steps and reduced environmental waste. Operational advantages, including the ability to carry out reactions under mild operational conditions, avoiding extremes of pH, temperature, and pressure that often require the use of expensive equipment or energy intensive processing. Furthermore this minimizes problems of undesired side-reactions such as decomposition, isomerization, racemization and rearrangement which often plague traditional methodology. In this presentation various biocatalytic processes for the preparation of chiral intermediates for drugs will be described.
Characterization of Milkweed Oil and Cis-vaccenate Biosynthesis. Thomas McKeon, Frank Rittig, Charlotta Turner, USDA, ARS, WRRC, Albany, CA 94710, USA
We evaluated the fatty acid composition in seeds of 22 Asclepias (milkweed) species, using supercritical fluid extraction to obtain the seed oil. We then compared transesterification methods, using methanolic HCl and lipase catalyzed transesterification in supercritical CO2. We found that under the conditions we used, the lower pressure of 470 bar was effective for quantitative extraction of seed oil from Asclepias, and that addition of methanol as a modifier was unnecessary. Results obtained for the two transesterification methods indicate that both were generally in close agreement, with some differences observed for vaccinate and linoleate content. Because milkweeds contain an unusual amount of cis-vaccenate in its oil, we compared the ability of developing milkweed (A. currasavica) seeds to desaturate palmitoyl-ACP and stearoyl-ACP seeds. The ratio of stearate to palmitate desaturation was 4.1. These results are consistent with the vaccenate arising from desaturation of palmitoyl-ACP and elongation of the palmitoleate to cis-vaccenate.
Construction and Characterization of Fusion Class III Poly(hydroxyalkanoate) Biopolymer Synthase Genes. D.K.Y. Solaiman, R.D. Ashby, J. Zerkowski, ERRC, ARS, USDA, Wyndmoor, PA, USA
Poly(hydroxyalkanoates) (PHAs) are polyesters produced by bacteria. Because these polymers arebiodegradable and produced from renewable resources, PHAs are studiedas environmentally friendly replacement for petroleum-based polymers. High production costsand limited applications impede the widespread adoption of PHAs, however.Genetic engineering of PHA-producing bacteria can help overcome this problem.Poly(hydroxyalkanoate) synthase (PHAS) is an enzyme responsible for PHA synthesis. We will discuss in this talk our study to genetically fuse the genes encoding thetwo subunits of a PHAS of Allochromatium vinosum, i.e., the phaE and phaC genes. Wehypothesize that the fused genes will facilitate the association of the two subunits toincrease product yield. Two fused genes (phaEC and phaCE) are constructed using theoverlapping-primer-extension PCR method. The overlapping primers were designed in such a waythat a stretch of 6 amino-acid sequence links the two fused pha genes in the resultant phaECand phaCE genes. The progress of the ongoing experiments to express the fused genes inPseudomonas resinovorans and Ralstonia eutropha using an expression vector, pBS29-P2, will bediscussed in detail in the presentation. The results should yield important information forimproving production yield of PHA in genetically modified organisms.
Screening of Microbial n-Alkane Degradation through Subterminal Oxidation. Eiji Sakuradani, Koji Kobayashi, Nozomu Shibata, Jun Ogawa, Sakayu Shimizu, Kyoto University, Kyoto, Japan
The terminal oxidation is well-known as a microbial oxidation of n-alkanes. Thus far, the genes and their enzymes related with the terminal oxidation have been studied in detail. However, the studied related with the microbial sub-terminal oxidation are hardly progressed. Previously, we reported that an achlorophyllous micro-alga, Prototheca zopfii, oxidizes 5th carbon of n-hexadecane through the sub-terminal oxidation to generate 5-hexadecanol and 5-hexadecanone. We have screened various microorganisms with the sub-terminal oxidation of n-hexadecane, and found that actinomycete Streptomyces griseus, basidiomycete Schizophyllum sp., and ascomycete Penicillium janthinellum oxidize mainly 4th, 6th, and 9th position of n-hexadecane, respectively, through the sub-terminal oxidation pathway. Characterization of the sub-terminal oxidation pathway in the microorganisms obtained through the screening will be discussed.
Production of Biologically Active 7, 10-dihydroxy-8(E)-octadecenoic Acid from Korean Pine Seed Oil by Pseudomonas aeruginosa PR3. H.R. Kim1, K.Y. Baek1, J.B. Ellamar1, C.T. Hou2, M.H. Kwon1, H.R. Lim1, 1Department of Animal Science and Biotechnology, Kyungpook National University, Daegu, Korea, 2Microbial Genomic and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, ARS, USDA, Peoria, IL USA
Hydroxyl group of hydroxyl fatty acid (HFA) cause HFA to carry special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. Microbial conversion of oleic acid into 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) caused DOD to carry strong antibacterial activity against several food pathogenic bacteria. However, industrial application of DOD as an antibacterial agent is limited by its high production cost. Hence we tried to use vegetable oil as a substrate for DOD production by Pseudomonas aeruginosa (PR3). Of those vegetable oils tested, we selected Korean pine seed oil as a substrate and studied about optimal culture conditions for DOD production.
Metabolism and Synthesis of Lipids in Polyunsaturated Fatty Acid-producing Fungus, Mortierella. T. Aki, W. Jermsuntiea, S. Kawamoto, K. Ono, Hiroshima University, Higashi-Hiroshima, Japan
Linoleic Acid Isomerase in Lactobacillus plantarum AKU1009a is a Multi-component Enzyme System Requiring Oxidoreduction Cofactors. Shigenobu Kishino1,2, Si-Bum Park1, Yuki Ishigaki1,2, Jun Ogawa2,3, Kenzo Yokozeki1, Sakayu Shimizu2, 1Laboratory of Industrial Microbiologys, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan, 2Division of Applied Life Sciences Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan, 3Research Division of Microbial Sciences, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
Conjugated linoleic acid (CLA) is a collective term for positional and geometric isomers of linoleic acid (LA) with conjugated double bonds. CLA has potentially beneficial effects, for example, prevention of carcinogenesis, and reduction of body fat content. Natural sources of CLA are daily products from ruminants, although their CLA contents are very low. We screened the ability to produce CLA from LA within lactic acid bacteria, and selected Lactobacillus plantarum AKU 1009a as a potential strain. This strain produced two CLA isomers, i.e., cis-9,trans-11 and trans-9,trans-11-CLA, from LA with 10-hydroxy-12-octadecenoic acids (18:1) as intermediates. These results suggested that the reaction involves hydration and dehydrating isomerization catalyzed by multiple enzymes. Then we clarified three enzymes (CLA-HY, CLA-DH, and CLA-DC) from L. plantarum AKU 1009a involved in CLA production from LA. CLA-HY was a membrane protein and catalyzed hydration reaction. LA was hydrated to 10-hydroxy-12-18:1 by CLA-HY. Further transformation of 10-hydroxy-12-18:1 to CLA and LA transformation to CLA were only detected in the presence of all three enzymes, i.e., CLA-HY, CLA-DH, and CLA-DC, and the multi-component system required NADH and FAD as the cofactors for its maximal activity.
Hydroxyl Fatty Acids and Hydroxyl Oils. C.T. Hou, Renewable Product Technology Research Unit, NCAUR, ARS, USDA, Peoria, IL 61604, USA
Soybean oil is produced domestically in large supply, averaging over 20 billion pounds per year with an annual carryover of more than one billion pounds. It is important to find new uses for this surplus soybean oil. Hydroxyl fatty acids and hydroxyl oils are platform materials for specialty chemicals and oil industry. Many years ago we started a research project to convert soybean oil and its component fatty acids to value-added products such as recinoleic acid. We successfully found many microbes that can convert fatty acids to recinoleic acid-type hydroxyl fatty acids. We also discovered many new bioactive or potentially bioactive fatty acids. Now we need a direct bioprocess to convert soybean oil to hydroxyl fatty acids. Soy polyols (hydroxyl triacylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. Currently, there are two chemical processes to produce triacylglyceride polyols, and these involve two steps: epoxidation and then opening of oxidane ring. It is important to have a non-polluting bioprocess that can directly convert soybean oil to polyol oils. Following our previous experience, we should be able to find microorganisms that can convert soybean oil directly to soy polyol oils.
BIO 1.2 / PCP 1: Process and Co-products of Biofuel and Industrial Production
Chair(s): K. Liu, USDA, ARS, PWA, USA; D. Solaiman, USDA, ARS, ERRC, USA; J. Wanasundara, Agriculture & Agri-Food Canada, Canada; and H. Wang, Iowa State University, USA
Proteins as Renewable Flocculants. G. Piazza, R. Garcia, ERRC, ARS, USDA, Wyndmoor, PA, USA
Nine billion chickens are slaughtered in the U.S. per year, but the protein rich chicken blood (CKB) is mainly treated as waste. We have investigated the use of CKB fractions as replacements for a widely used nonrenewable, polymeric flocculant, anionic polyacrylamide (PAM), which requires the addition of calcium chloride for activity. PAM is needed for several municipal and industrial operations. The CKB frozen fractions studied were (A) blood, freed of coagulated blood; (B) blood plasma (blood centrifuged to remove blood cells); and (C) heated blood, centrifuged to remove solids. These CKB fractions were dehydrated two ways: freeze drying and spray drying at elevated temperature. Flocculation activity was quantified by determining the concentration of flocculant needed to settle a constant amount of suspended clay particles in 1 h, 5 h and 24 h. Flocculation activity by the CKB fractions required the addition of buffer, citric acid, phosphoric acid or sulfuric acid to reduce the pH to 5.5, but does not require the addition of calcium chloride. The flocculation activity of frozen and freeze dried CKB fractions was superior or equivalent to that of anionic PAM. The flocculation activity of spray dried fractions A and B was nearly as good as that of anionic PAM. The results indicate that CKB fractions may be viable renewable replacements for PAM.
BIO 2: Sterols II
Chair(s): G.I. Lepesheva, Vanderbilt University, USA; and W.D. Nes, Texas Technical University, USA
Identification of Natural Ligands for the Nuclear Hormone Receptor RORγ. Fabio R. Santori1, Varun Sondhi2, Brittany Rosales3, H. Eric Xu4, W. David Nes3, David Mangelsdorf2, Dan R. Littman1, 1Skirball Institute Of Biomolecular Medicine, NYU School of Medicine, New York, New York, USA, 2Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA, 3Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, USA, 4Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, Michigan, USA
RORg is a transcription factor of the Nuclear Hormone Receptor (NHR) Superfamily. Ligands for NHRs have been identified and generally consist of small lipophilic molecules. Although, great progress has been achieved in the last years in the identification of natural ligands for NHRs, nearly half of the known human NHRs are orphan receptors. RORg plays an important role in development of lymphoid tissues like thymus, lymph nodes, gut associated lymphoid tissue (GALT) and pro-inflammatory Th17 cells.The important role played by ROR in the development and physiology of the immune system has increased the interest on the identification of natural ligands that regulate RORg activity â€œin vivoâ€. The identification of such molecules would allow the study of cellular mechanisms and pathways regulated by RORg that are required for the differentiation of Lti, Lti-like and Th17 lineages. To that end, we have designed luciferase based reporter systems as sensors for compounds that can induce RORg activity. Moreover, chemically defined synthetic media has allowed us to test the requirement of several endogenous lipids for RORg reporter activity. These experiments, supported by RNAi inhibition of lipid biosynthetic enzymes in mammalian cells have suggested that RORg ligands maybe derivatives of sterol lipids.
Genetic Dissection of AACT Paralog-function on the Phytosterol Profile of Arabidopsis. Zhihong Song1,2, Huanan Jin2, Basil J. Nikolau1,2, 1The Ames Laboratory of US DOE, Ames, IA, USA, 2Iowa State University, Ames, IA, USA
Acetoacetyl-CoA thiolase (AACT) catalyzes the condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. In the cytosol of plant cells, it is the precursor of mevalonate-derived isoprenoids. Two AACT genes, At5g47720 (AACT1) and At5g48230 (AACT2), were identified in the Arabidopsis genome by BLASTP analysis. Two T-DNA mutants at each AACT gene were characterized. These characterizations indicate that although both genes are expressed, mutations in AACT2 are embryo lethal whereas null alleles of AACT1 are viable and show no apparent growth phenotypes. Promoter::GUS fusion experiments indicate that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, leaves, stems and anthers. AACT2 RNAi knock-down lines show pleiotropic phenotypes, including dwarfing, reduced root length, early senescence, elongated flowering time, sterility, reduced cell size, early degradation of tapetum cells and loss of pollen coat. These phenotypes are rescued when the plants are grown in the presence of mevalonate. Phytosterol analysis of AACT2 RNAi lines show reduced accumulation of sitosterol, stigmasterol, campesterol, and all other sterols in the root. The accumulation of these sterols is increased to wild type levels by the application of exogenous mevalonate. In contrast, no significant phytosterol changes are detected in the aact1 mutant.
Bioengineering Strategies for Enhanced Phytosterol Accumulation in Soybean Seeds. Anjanasree Neelakandan1, Swetha Chamala2, Babu Valliyodan1, W. David Nes2, Henry Nguyen1, 1University of Missouri, Columbia, MO, USA, 2Texas Tech University, Lubbock, TX, USA
Plant sterols are an important class of nutritional and pharmaceutical biomolecules found only in minor proportions in commercial seed oils, including soybean, which are the major dietary sources. In an attempt to generate value added soybeans enriched in cholesterol lowering 4-desmethylsterols, we over-expressed soybean Sterol Methyltransferase1 (SMT1) gene, catalytic subunit of HMGR1 gene from Arabidopsis and Sterol Methyltransferase2-2 (SMT2-2) gene from soybean. We found only modest increase in total sterol levels (3-6%), which points towards a tight control of this trait in soybean seeds. However, beneficial alterations in sterol composition as evidenced by higher end product sterol accumulation (90% of total sterols, as against 65% in wild type) were noted in plants containing seed-specific promoters, whereas increased accumulation of intermediate sterols (55% of the total as opposed to 35% in wild type) was observed with constitutive HMGR1 over-expression. The intermediates abundantly present in soybean seeds are efficiently converted to sterol end-products which are important for cholesterol lowering and other therapeutic effects. Taken together, our results demonstrate the usefulness of transgenic technology for obtaining desirable sterol composition in target tissues in important oilseed crops like soybean.
Phytosterols as Novel Tools for Controlling Insect Pests. S. Behmer1, R. Grebenok2, A. Douglas3, 1Texas A&M University, College Station, TX, USA, 2Canisius College, Buffalo, NY, USA, 3Cornell University, Ithaca, NY, USA
Exploiting nutritional requirements unique to insects offers a novel, target-specific and environmentally friendly approach for controlling insects that attack agricultural crops. A key nutritional requirement for insects is the need to acquire a dietary source of sterol, which is, among other things, used in cell membranes and as a precursor to molting hormone. Cholesterol is the most abundant sterol found in plant-feeding insects, but it is rare in plants, so plant-feeding insects generate cholesterol by metabolizing ingested plant sterols. There are, however, plant sterols that plant-feeding insects cannot convert to cholesterol, and the introduction of these “bad” sterols into crop-plants might provide a novel form of plant-defense. This talk explores how two groups of plant-feeding insects (caterpillars and aphids) are affected by changes in the sterol profile of their food. This question is investigated using tobacco plants with modified sterol profiles, and artificial diets with known sterol profiles. The results from this work reveal interesting outcomes, both in terms of plant and insect physiology, and suggest that the development of transgenic crops, containing modified sterols, has real potential for managing insect crop pests.
Structural Insights into Selective Inhibition of Protozoan Sterol 14alpha-demethylase. G.I. Lepesheva, Vanderbilt University, Nashville, TN, USA
Human infections with protozoan pathogens such as trypanosomes and leishmania have been threatening lives of millions of people for hundreds of years. With human migrations and broadening of the host reservoir due to climate changes, they are now spreading all over the world as a result of HIV-coinfections, blood transfusions, organ transplantation, food contamination, and yet their treatment remains unsatisfactory. Broad search for new drug targets based on the Trypanosomatidae biology proves that sterol biosynthesis has a high potential as a drug target pathway, sterol 14alpha-demethylase being one of the most promising drug targetable enzymes. Crystal structures of sterol 14alpha-demethylases from three protozoan pathogens, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum, ligand-free and complexed with different inhibitors, solved recently in our laboratory, provide a molecular basis for better understanding the enzyme inhibition, functional conservation across the biological kingdoms, explain some cases of drug resistance, and open an excellent opportunity for rational, structure-based drug design.
New Methods for Oxysterol Synthesis: Allylic Oxidation of Steroids and Sterols. E.J. Parish, A.D. Bell, D. Lu, A.E.V. Gorden, Y. Li, X. Wu, T. Lee, Department of Chemistry and Biochemistry, Auburn University, AL, USA
Allylic oxidation of steroids, particularly at the 7-position, has attracted interest over many years. The 5-ene steroids can be oxidized to the 5-en-7-one, which are known as inhibitors of sterol biosynthesis and cell replication. They also have use in cancer chemotherapy, since they are more toxic towards tumorous than non-tumorous cells. We have recently developed two new reagents which have proven to be useful for the allylic oxidation of unsaturated steroids and sterols (steroidal alkenes) to the corresponding unsaturated ketosteroids. These are Magtrieve (chromium dioxide) and a tert-butylhydroperoxide oxidant with a modified salen-quinoxalinol Cu(II) complex catalyst. Both of these reagents have been able to replace chromium salts and chromic acid for this purpose and eliminate toxic wastes. The latter method is able to directly preform allylic oxidation of sterols to ketosterols without oxidation of the alcohol (hydroxyl) functional group of sterols.
BIO 2.1 / PRO 2.1: Processing Technologies
Chair(s): X. Xu, University of Aarhus, Denmark; and N. Dunford, Oklahoma State University, USA
Advantages and Challenges in Enzymatic Catalyzed Biodiesel Production. P.M. Nielsen, J. Brask, H. Lilbaek, M.L. Damstrup, A.R. Madsen, H.C. Holm, Novozymes, Bagsvaerd, Denmark
It is well-known that the choice of alcohol is an important factor for determining enzyme stability in a transesterification reaction. However, it further appears that enzymatic production of fatty acid ethyl ester, relative to conventional production of methyl esters, can be beneficial for both process economy, as well as from a sustainability viewpoint. The different aspects of the alcohol choice will be discussed, and the consequences of using different oil types and qualities will be illustrated.The process plant layout itself is very important for the lifetime of the enzyme and thereby the economy. A separate question is related to the enzyme product. Not only the enzyme catalyst itself but also the way it is formulated is crucial for the performance. We will discuss results from using different types of lipase, different carriers for immobilizing the enzyme, as well as the possibility of using liquid enzymes.There is not a one-fits-all solution for the enzymatic process. The enzymatic production of biodiesel will be an important opportunity for some markets with the possibility of expansion from the technology base we seek to establish for ethyl ester production into general application of enzymes with different types of oil raw materials.
Evaluation of Reaction Engineering Parameters in Enzyme-based FAEE-biodiesel Processes. M. Nordblad, Y. Xu, J.M. Woodley, Process Engineering and Technology Group, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark
Biodiesel is receiving increasing attention as a promising alternative to liquid fossil fuel for vehicles, being a direct replacement for petrochemical diesel. Compared to the conventional biodiesel production process, enzymatic synthesis offers several advantages such as lower reaction temperature, eliminated soap formation and simplified product recovery. Unfortunately, the cost of the enzyme-based catalysts is considerably higher than that of the alkaline catalysts used in the conventional process. It is thus critical that the enzyme-based catalysts can be reused.The reaction conditions greatly affect the performance of enzymatic biodiesel production. For example, the concentration of alcohol in the system affects both the reaction rate and the catalyst stability. In order to maximize the amount of biodiesel that can be produced by a given amount of catalyst, it is useful to design the process to maintain controlled substrate levels. However, the production of biodiesel is a challenging process in itself, with very strict requirements on product quality for what is essentially a low-value product, and it is essential to consider this alongside the choice of operating parameters. This study presents an evaluation of the options available for the enzymatic production of biodiesel and the consequence of these for the overall design and control of the process.
Development of Reactor Technology for Improved Catalytic Productivity in Enzymatic FAEE-biodiesel Production. Y. Xu, M. Nordblad, J.M. Woodley, Process Engineering and Technology Group, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark
Biodiesel production catalyzed by immobilized lipases is attracting considerable interest as an alternative to conventional base-catalysed biodiesel production due to its potential of improved sustainability1. However, the high cost of the immobilized lipases has limited commercial application. High process efficiency could be the key to achieving high productivities of the expensive immobilized lipases and improved economic viability for large industrial application; the reactor is the core of an efficient process design. This work has taken the stirred tank reactor and the packed bed reactor as examples of reactor technology development in order to improve the productivities of the immobilized lipases for FAEE-biodiesel production.One of the major factors causing insufficient stability of immobilized lipases is glycerol, the major by-product of biodiesel production2. A dyeing method for indicating glycerol partitioning has been developed and applied in stirred tank and packed bed reactors to assist the reactor evaluation and its effect on catalyst productivity. The mechanical stability of the immobilized lipases has also been evaluated in these reactors.  Nielsen, P.M. et al., 2008. Eur. J. Lipid Sci. Technol., 110, 692.  Dossat, V. et al., 1999. Enzyme Microb. Technol., 25, 194.
Enzymatic Biodiesel, Monitoring and Analysis of the Reaction. Sergey N. Fedosov, Xuebing Xu, Agrobiology Group, Dept. Molecular Biology, Århus University, Århus, Denmark
Biodiesel (BD) is produced by treatment of vegetable oil with alcohol in the presence of a catalyst. Application of the enzyme lipase for this purpose is presently debated, because the enzyme has low sensitivity to oil composition and produces minimum waste. Optimization of the complex reaction kinetics is, however, required. Multiple measurements make the well established analytical methods of gas-chromatography, HPLC and TLC-FID inconvenient in terms of time, labor and expenses. The current work describes two suitable analytical approaches. First of them is a low-cost method, where the major components of reaction (BD, tri/di/mono-glycerides, fatty acids) are separated on a standard TLC plate and stained. Absence of background in the suggested procedure simplifies quantification by densitometry or visual assessment. The second method is based on measurements of fluorescent signal from a probe sensitive to polarity of the environment. This approach is useful when investigating partial reactions accompanied by a change of polarity (e.g. MG + MeOH → BD + G). Fluorescent method requires calibration of the signal and is convenient if repeated measurements are carried out. Examples of the calibration curves and the reaction records are presented. Analysis of partial reactions is discussed as an approach to simplify reconstruction of the global scheme of BD-synthesis.
Predictive Studies on Phase Equilibria of Enzymatic Biodiesel Production. G. Güzel, X. Xu, Aarhus University, Department of Molecular Biology, Aarhus, Denmark
Synthesis of Diacylglycerols: Lipases vs. Macroreticular Strongly Acidic Cation Exchange Resins. O.M. Lai1,2, S.K. Lo3, 1Universiti Putra Malaysia, Serdang, Selangor, Malaysia, 2Institute of Bioscience, Serdang, Selangor, Malaysia, 3Sime Darby Research Centre, Banting, Selangor, Malaysia
Much has been published about the use of lipases for the production of structured lipids such as diacylglycerols. We now report of a chemical catalyst for the synthesis of of dietary 1,3(2)-diacylglycerol. Lipases and macroreticular strongly acidic cation exchange resin were used to catalyze the esterification of stearic and oleic acids with glycerol to synthesize 1-stearoyl-3(2)-oleoyl glycerol. A dual response surface approach was used to optimize both the esterification reaction variables and the optimized conditions for both the catalysts were compared and reported.
Enzyme-Catalyzed Production of Structured Lipids under High-Pressure Conditions. S. Ferreira-Dias1, N.M. Osório2,1, C. Tecelão3,1, V. Perrier4, E. Dubreucq4, M.H. Ribeiro5, 1Instituto Superior de Agronomia, Technical University of Lisbon, CEER, Lisbon, Portugal, 2Instituto Piaget, Núcleo de Investigação em Engª Alimentar e Biotecnologia, ISEIT de Almada, 2800-305 Almada, Portugal, 3Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, 2520-641 Peniche, Portugal, 4Montpellier SupAgro, UMR 1208 IATE, F-34060 Montpellier cedex, France, 5Faculdade de Farmácia, Research Institute for Medicines and Pharmaceutical Sciences (i-Med.UL), University of Lisbon, 1649-003 Lisbon, Portugal
BIO 3: Plant Lipid Biotechnology
Chair(s): R. Weselake, University of Alberta, Canada; and R.F. Wilson, Oilseeds and Biosciences Consulting, USA
Biogenesis of ER Subdomains Containing DGAT2, an Enzyme Involved in Industrial Oil Biosynthesis. S.K. Gidda1, J.M. Shockey2, R.T. Mullen1, J.M. Dyer3, 1Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, 2USDA-ARS, Southern Regional Research Center, New Orleans, New Orleans, LA, USA, 3USDA-ARS, US Arid-Land Agricultural Research Center, Maricopa, AZ, USA
Diacylglycerol acyltransferases (DGATs) are enzymes that catalyze the committed step in triacylglycerol (TAG) biosynthesis by transferring a fatty acyl group from the acyl-CoA pool to the sn-3 position of diacylglycerol. The substrate specificity and overall activity of these enzymes play a key role in determining the quantitative and qualitative properties of seed storage oils. Plants contain two unrelated types of DGAT enzyme called DGAT1 and DGAT2, and we have recently shown that these enzymes have different substrate preferences, that they are located in different regions or “subdomains” of the endoplasmic reticulum (ER), and that DGAT2 plays an important role in the channeling of either conjugated fatty acids or hydroxyl fatty acids into storage oils. The knowledge of DGAT2 subdomain biogenesis, therefore, will likely increase our ability to rationally engineer oilseed crops to produce industrially important oils. Here we describe the identification of two glycerol-3-phosphate acyltransferases (GPATs) from tung (Vernicia fordii) and demonstrate that both proteins target to the same subdomain of ER that is shared by DGAT2. Using a combination of mutational analyses and protein-protein interaction assays, we demonstrate that the GPAT's first transmembrane-spanning domain, which contains a leucine-zipper-like motif, is responsible for targeting to ER subdomains. These results demonstrate that the enrichment of lipid-biosynthetic enzymes into specific regions of the ER is dependent in part on formation of higher-ordered, oligomeric structures via protein-protein interactions. Implications for oilseed engineering are discussed.
FADX, DGAT, CPT, PDAT and More: a Vegetable Soup of Enzymes that Influence Industrial Oil Production in Transgenic Oilseeds. J.M. Shockey, USDA-ARS, Southern Regional Research Center, New Orleans, LA, USA
Our laboratories study tung tree (Vernicia fordii) and bitter gourd (Momordica charantia) as model systems for the production of industrially useful oils. Seed oils of bitter gourd and tung contain approximately 60% and 80%, respectively, α–eleostearic acid, a valuable conjugated fatty acid. Successful reconstitution of the biosynthetic pathway for drying oils in agronomically amenable transgenic systems may require introduction of several genes. Some important components of these pathways, such as the fatty acid conjugases (FADXs) and diacylglycerol acyltransferases (DGATs) have been identified. We are currently focused on the optimization of the FADX and DGAT transgenes currently in use. The effects of alterations in transgene construct design, including changes that affect transcriptional timing and maintenance, translational efficiency, and protein stability will be presented. Other necessary pieces of the TAG biosynthetic puzzle remain elusive. Much effort has been focused on finding other enzymes in the pathway that contribute to efficient channeling of eleostearate into TAG. Recent results from the study of co-expression of genes located in the central part of the pathway (between FADX and DGAT) will also be presented.
Producing DHA in Canola Oil Via Algal PUFA Synthases. T. Walsh1, J. Metz2, 1Dow AgroSciences, Indianapolis, IN, USA, 2Martek BioSciences, Boulder, CO, USA
The unique human health benefits of the omega-3 long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA) are well established and continue to be expanded. With increasing demand for convenient, low-cost sources of DHA, plant oils can potentially offer a healthy and sustainable method of production of this important omega-3 fatty acid. Canola (Brassica napus) is particularly suited as a production vehicle for DHA-containing plant oils because of its convenient scale, established grain identity-preservation channels and healthy oil attributes. In parallel, the use of genes encoding algal PUFA synthases offer a new and simplified mechanism for DHA synthesis in crop plants, and an alternative to strategies involving multiple elongation/desaturation steps of native fatty acids. Progress toward engineering canola oil to contain DHA using algal PUFA synthase genes will be described.
Enhancing Carotenoid Production in Brassica napus Seeds. Abdelali Hannoufa, Agriculture and Agri-Food Canada, London, Ontario, Canada
The accumulation of carotenoids in B. napus seed varies during development with beta-carotene and lutein accounting for more than 90% of the total content. The highest levels are detected at ~35-45 days post anthesis, and then drop in the latter stages of seed development. Our research is aimed at the biotechnological production of carotenoids in seeds of B. napus. To this end, we increased the level of carotenoids 48-fold by RNAi-mediated suppression of lycopene epsilon cyclase. In addition to enhanced carotenoid content, transgenic seeds showed reduced levels of fatty acids, but minimal changes in their ratios. We also increased carotenoid levels in B. napus seed by suppressing DET1, a gene involved in light regulation. Importantly, suppression of DET1 also resulted in a reduction in the level of the antinutritional factor 1,2-di-O-sinapoylglucose, thus the overall nutritional value of the seed was enhanced. In addition, we identified an Arabidopsis mutant with enhanced miRNA156b expression and a 150% increase in seed carotenoid levels. Overexpression of miRNA156 in B. napus resulted in enhanced seed carotenoid content and increased branching. Ehancing the carotenoid content of B. napus seeds is an important step towards engineering the production of astaxanthin; a pigment used in fish feed formulations, that is not normally produced in B. napus.
Quantifying the Control of Lipid Accumulation in Oil Crops. U.S. Ramli1, M. Tang1, I. Guschina1, P.A. Quant2, J.L. Harwood1, 1Cardiff University, UK, 2Oxford University, UK
We have applied the technique of top-down control analysis in order to quantify the control exerted by different parts of the triacylglycerol biosynthetic pathway in oil crops. The pathway was divided into fatty acid biosynthesis (Block A) and lipid assembly (Block B) with the acyl-CoA pool acting as the chosen intermediate. We then carried out single manipulation (using added exogenous oleate) or double manipulation with specific inhibitors of each of the two blocks.By these means we have quantified the metabolic control in a number of the major oil crops - soya bean, oilseed rape, olive and oil palm. For most crops Block A exerted about 65% of the total control but in oilseed rape (Canola) the position was reversed with Block B (lipid assembly) exerting 69% of the control. These data provide important information in order to guide future genetic manipulation of oil crops to increase yields.
The Use of Biotechnology to Alter Agronomic Properties of Camelina sativa: From Oil Production to Herbicide Resistance. Eric J. Murphy1, 1Agragen, LLC, USA, 2Unicrop, Oy, Finland
Camelina sativa is an emerging oil seed crop that has significant potential as source for biofuel feedstock. Because Camelina grows on marginal lands, it minimizes the food for fuel debate, offering farmers an additional cropping choice. For instance this is especially important in the Pacific Northwest, where class-2 herbicide contamination is prevalent and limits cropping choices. To address this issue, we used a biotechnology approach to design Camelina lines with single or multiple mutations in acetolactate synthase, the key enzyme inhibited by class-2 herbicides. Preliminary results indicated nearly a 10,000-fold increased tolerance to these herbicides. Using a similar approach and a proprietary seed promoter, we have transformed Camelina to express lauric acid thioesterase, thereby increasing the level of lauric acid in the oil seed triglycerides (TAG). Modifications of other enzymes involved in oil deposition are in process as well, as we have expressed flax diacylglycerol acyl transferase in Camelina, with an anticipated increase in total TAG content in the oil seed. Thus, using our patent-pending high efficiency transformation system, coupled with our vast experience of Camelina genetics, we have engineered a number of key plants lines in which oil content, oil composition, or herbicide tolerance has been significantly altered.
Comparative Deep Transcript Profiling Reveals a Novel DGAT with sn-3 Acetyltransferase Activity that Synthesizes Unusual, Reduced-Viscosity Oils in Euonymus and Transgenic Seeds. T. Durrett, Department of Plant Biology, Michigan State University, Michigan, USA
Euonymus alatus (Burning Bush) synthesizes high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) in the seed endosperm and normal, long-chain TAGs (lcTAGs) in the aril tissue surrounding the seed. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis using 454 pyrosequencing technology revealed an abundant acyltransferase in the endosperm which was absent from the aril. Expression of this acyltransferase in yeast resulted in the accumulation of acTAGs but not lcTAG, hence the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a seed-specific promoter in Arabidopsis resulted in the accumulation of up to 40 mole % acTAGs in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy. They also show that EaDAcT is the acetyltransferase required for the synthesis of acTAGs in Euonymus seeds, and that this activity can be transferred to other plants, allowing the evaluation of these unusual TAGs for biofuel, nutritional and other applications.
Branched-chain Fatty Acid Accumulation in Plant Oil. H. Fukushige1, A. Lewis1, T. Davenport2, D. Hildebrand1, 1University of Kentucky, Lexington, KY, USA, 2University of Florida, Homestead, FL, USA
Plant oils can currently be used as automobile engine oils but only for short periods due to the poor oxidative stability of unsaturated fatty acids. Solutions include converting the double bonds commonly found in plant oils such as soybean oil into branched-chain groups such as cyclopropyl groups. Such groups can have adequate low temperature fluidity and high oxidative stability. Various organisms are capable of synthesizing and in some cases accumulating cyclopropyl groups in seed oil triacylglyceride (TAG). In developing Litchi chinensis seeds, we found that dihydrosterculic acid (cy18:0), a cyclopropane fatty acid, accumulates in TAG as seed develop. Cy18:0 was also found in other major lipid classes such as phosphatidyl choline, but at lesser levels compared to the level in TAG. We have successfully transformed soybean with an E. coli cyclopropane fatty acid synthase gene to produce TAG with a cyclopropyl group. This initial attempt resulted in the accumulation of cy18:0 up to 15% total fatty acids in soybean seeds. In developing transgenic soybean seeds, the level of cy18:0 in TAG was lower than those in other lipid classes in early stages of seed development and reached similar levels at later stages of development indicating random distribution of cy18:0 in seed lipid classes in contrast to the selective accumulation in L. chinensis TAG.
Probing the Role of Cysteine Residues in Acyl-CoA:Diacylglycerol Acyltransferase 2 from Yeast. Q. Liu, R.M.P. Siloto, R.J. Weselake, University of Alberta, Edmonton, Alberta, Canada
The increasing demand for tailor-made seed oil has been arousing considerable interest from academic researchers and biotechnology industry in elucidating triacylglycerol (TAG) biosynthesis in oil-bearing crops. Acyl-CoA:diacylglycerol acyltransferase (DGAT), which catalyzes the final step in triacylglycerol biosynthesis, is a vital target for engineering TAG production in plants. Insight into structure/function in DGAT family members (DGAT1 and DGAT2), however, has been limited due to the difficulties in the purification of these membrane proteins. In the current study, the local functional and structural role of cysteine residues in DGAT2 from Saccharomyces cerevisiae (ScDGAT2) was studied using a combination of chemical modification with site-directed mutagenesis. Although DGAT2 was susceptible to different thiol-modifying reagents, none of the cysteines were essential for the catalytic activity. Accessibility analysis of each sulfhydryl group indicated that cysteines were also not involved in the formation of intramolecular disulfide linkages.Cysteine 314, which is close to a highly conserved motif of DGAT2, was the major target of N-ethylmaleimide-mediated inhibition of enzyme activity. Our data indicated that this cysteine residue may residue in a critical position which is near a possible active site or related to proper folding of DGAT2.
BIO 3.1: Biocatalysis II
Chair(s): C. Hou, Renewable Product Technology Research Unit, NCAUR, ARS, USDA, USA; and K. Takahashi, Hokkaido University, Japan
Suppression and Promotion of Cachexia with n-3 Fatty Acid Bounded Phospholipids. K. Ikegame1, T. Tsushima1, H. Arai2, K. Takahashi1, 1Hokkaido University, Hakodate, Hokkaido, Japan, 2Kitami Institute of Technology, Kitami, Hokkaido, Japan
Cachexia has a substantial impact on the quality of life. It is said that 10 to 20% of cancer patients die from the consequence of cachexia rather than tumor burden. We observe through couple of animal experiments that supplementing n-3 diacyl phospholipids in the diets suppress loss of body weight which is a typical symptom of tumor bearing animals. We predicted that sn-2 DHA bounded lysophosphatidylcholine may have higher potential than the n-3 diacyl phospholipids in suppressing cachexia. Because fatty acid moieties other than the DHA are cleaved off already in the lyso form. For this reason, we prepared the sn-2 DHA bounded lysophosphatidylcholine through Lipozyme RMIM mediated partial hydrolysis using squid phospholipid as substrate. Twenty five mg/day/mouse of sn-2 DHA bounded phosphatidylcholine and fifteen mg/day/mouse of sn-2 DHA bounded lysophosphatidylcholine were served in the form of liposome for the former, and micelle for the latter for 2-3 weeks to the B16 melanoma tumor bearing C57BL/6Cr mice. Unlike the previous study results carried on sp2 and sarcoma 180 tumor bearing mice, no clear evidence in suppressing cachexia was observed on the B16 melanoma tumor bearing mice served with n-3 diacyl phospholipids. And which was more unexpected, the sn-2 DHA bounded lysophosphatidylcholine rather promoted the cachexia.
Enzymatic Modification of Phospholipids Using Lipase and Phospholipase. S.H. Yoon1, S.K. Kim2, 1Korea Food Research Institute, Seongnam-Si, Kyunggi-Do, Korea, 2Doo San, Sung Bok-Ri, Kyunggi-Do, Korea
To determine phospholipase activity of enzymes, TLC-FID method was used. Chromarod S III was used as TLC rod, and the developing solvent was a mixture of chloroform : methanol : acetic acid : water (25:15:4:2, v/v/v/v). Hydrolytic activities of phospholipid by several lipases from microorganisms and porcine pancreatic phospholipase were tested in non-aqueous system. Among the enzymes tested, lipases from Aspergillus niger, Mucor miehei, Mucor javanicus, Rhizopus nieveus, and phospholipase A2 from porcine pancreace showed the hydrolytic activities in organic solvent like diethyl ether, and phospholipase showed the highest activity among them. Hydrolysis yield of soy lecithin to produce lysophospholipids in non-aqueous system was two-times higher than that in emulsion system. Among eighteen solvents tested, five solvents (diethyl ether, isopropyl ether, dioxane, tetrahydrofuran, petroleum ether) were shown to be suitable for the reaction. Reaction conditions to hydrolyze phospholipids in organic solvent by lipase were partially optimized as follows: temperature, 10-50°C; reaction time, 1-10hrs; agitation, 50-150 rpm; enzymes, lipases of Aspergillus niger, Mucor miehei, Mucor javanicus, Rhizopus niveus, Lecitase; solvent, diethyl ether, isopropyl ether, dioxane, tetrahydrofuran, petroleum ether; water content, 0.01-2%.
Novel Functionality of Glycolipids from Brown Seaweeds. A. Sho, Y. Sugiyama, T. Takahashi, M. Hosokawa, K. Miyashita, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan
In contrast to the abundance of the phospholipids in membranes of animals and yeast, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) are mainly found in photosynthetic membranes of higher plants and algae. Recently, these glycoglycerolipids have been shown to have specific biological activities, including anti-tumor and anti-inflammatory effects. In this presentation, we report the effective separation procedure of glycoglycerolipids from brown seaweeds along with special emphasis on their good resources of DHA synthesis in vivo. Lipid extraction from brown seaweeds was carried out with organic solvent and enzymatic process. Alginase decomposed polysaccharide network of the brown seaweed to enable the effective extraction of the lipids. The main component of the extracted seaweed lipids was glycoglycerolipids containing omega-3 polyunsaturated fatty acids such as 18:4n-3 and 20:5n-3 as major fatty acids. The brown seaweed glycoglycerolipids were fed to animals and then analyzed fatty acid composition of liver and other tissues. DHA content significantly increased in brown seaweed glycoglycerolipids fed group as compared with the control group, although the seaweed lipids contained no DHA.
Fractionation of Conjugated Linoleic Acid Isomers. Y. Yamauchi-Sato1, H. Uehara1, Y. Watanabe2, T. Nagao2, S. Negishi1, Y. Shimada2, 1The Nisshin OilliO Group, Ltd., Yokosuka, Kanagawa, Japan, 2Osaka Municipal Technical Institute, Osaka, Japan
Conjugated linoleic acid (CLA) is industrially produced by alkali conjugation of linoleic acid-rich oils, in the presence of propylenglycol. The product consists of almost same amounts of the isomers, 9-cis,11-trans-CLA (c9t11) and 10-trans,12-cis-CLA (t10c12), which exhibits various physiological properties, such as reduction of cancer incidence, beneficial effects in atherosclerosis, decrease in body fat content, and improvement of immune functions. In addition, the two isomers have been reported to show different activities: c9t11 isomer exhibits anti-tumor activity, and t10c12 isomer decreases body fat, increases energy expenditure, and suppresses the development of hypertension. To purify the two isomers, we developed a process using a lipase: A mixture of the two isomers was selectively esterified with an equimolar amount of lauryl alcohol (LauOH) using Candida rugosa lipase which acted more strongly on c9t11 than t10c12. A series of procedure increased the purities of c9t11-CLA to 96%, and of t10c12-CLA to 97%. The process required LauOH as a substrate in the selective esterification, but LauOH cannot be used for food processing. In this paper, we report the purification of the two isomers by selective esterification with L-menthol and by selective hydrolysis of tri-conjugated-linoleoylglycerol (CLA-TAG) using C. rugosa lipase.
Enzymatic Synthesis of Novel Phosphatidyl-pantenol by Phospholipase D-mediated Transphosphatidylation. Masashi Hosokawa, Kounosuke Suzuri, Yukihiro Yamamoto, Kazuo Miyashita, Hokkaido University, Hakodate, Hokkaido, Japan
Phospholipase D (PLD) is a lipolytic enzyme to hydrolyze terminal phosphodiester bond of phospholipids. Due to transfer phosphatidyl moiety of PLs to alcohols, PLD is a useful enzyme to synthesize novel PLs with various functional molecules which are poorly accessible on chemical route. In previous studies, we have synthesized phosphatidyl-geraniol and phosphatidyl-tyrosol with terpene and phenolic moieties in the phospholipid molecule. In this study, we further attempted PLD-mediated transphosphatidylation of soy PC and provitamin B5, panthenol (2,4-dihydroxy-3,3-dimetylbutyric 3 hydroxy propylamide) which has skin care functions. After reaction in biphasic system, a novel lipid was detected by TLC analysis. By NMR and MS analysis, the synthesized lipid was identified to phosphatidyl-panthenol, which 3-hydroxypropyl moiety in panthenol was transphosphatidylated. The yield of phosphatidyl-panthenol reached to 90% under the optimum reaction conditions of 50 μmol PC, 1 mmol panthenol, 1.6 ml of 0.2M acetate buffer (pH 5.6), 1.6 ml ethyl acetate, and 1.6 U PLD at 37°C for 24 h. Synthesized phosphatidyl-panthenol showed suppressive effects on inflammatory cytokines production in activated macrophages. The anti-inflammatory effects of phosphatidyl-panthenol was stronger than those of free panthenol and PC.
Microbial Conversion of Vegetable Oil to Rare Unsaturated Fatty Acids and Fatty Alcohols and Their Culture Conditions. T. Nagao1, Y. Watanabe1, N. Kishimoto2, Y. Shimada1, 1Osaka Municipal Technical Research Institute, Osaka, Japan, 2Kinki University, Nara, Japan
We isolated a new microorganism, Aeromonas hydrophia, capable of converting vegetable oil to several rare unsaturated fatty acids and unsaturated fatty alcohols. They were accumulated as a wax ester form. The strain converted rapeseed, safflower, and linseed oils into rare fatty acids, such as 7-C16:1, 5-C14:1, 7,10-C16:2, and 7,10,13-C16:3. Furthermore, the strain converted the resulting fatty acids to rare fatty alcohols, such as 7-C16:1, 5-C14:1, 9,12-C18:2, 7,10-C16:2, 9,12,15-C18:3, and 7,10,13-C16:3. Since shortening fatty acid carbon chain lengths from the carboxyl end and reducing unsaturated fatty acids to unsaturated fatty alcohols are both difficult reactions by chemical means, we suggest that the strain may facilitate introduction of new bioprocess for producing rare unsaturated fatty acids and unsaturated fatty alcohols, especially fatty alcohols harboring more than two double bonds. Culture conditions of the strain were also examined using rapeseed oil as a substrate. We found that aeration (>3 vvm) was important for effective wax esters production. Time course of the cultivation showed that content of C16 and C14 fatty acids, which were generated by shortening of carbon chain lengths of rapeseed oil, was significantly increased after 126 h.
Shortenings Prepared with Palm Stearin and Rice Bran Oil-based Structured Lipid. Casimir C. Akoh, Brenda H. Jennings, University of Georgia, Athens, GA, USA
We prepared rice bran oil structured lipid (RBOSL) using rice bran oil (RBO) and the medium chain fatty acid (MCFA), caprylic acid, with Lipozyme RM IM as biocatalyst. RBOSL and RBO were mixed with palm stearin (PS) in ratios of 30:70, 40:60, 50:50, 60:40 and 70:30 v/v (RBOSL to PS) to formulate trans-free shortenings. Fatty acid profiles, solid fat content (SFC), melting and crystallization curves and crystal morphology were determined. The content of caprylic acid in RBOSL ranged from 9.92 to 22.14 mol%. Shortening blends containing 30:70 and 60:40 RBOSL or RBO and PS had fatty acid profiles similar to a commercial shortening (CS). SFCs for blends were within the desired range for CS of 10-50% at 10-40 °C. Shortening blends containing higher amounts of RBOSL or RBO had melting and crystallization curves similar to CS. All shortening blends contained primarily β' crystals. RBOSL blended with PS was comparable to RBO in producing shortenings with fatty acid profiles, SFC, melting and crystallization profiles and crystal morphologies that were similar. RBOSL blended with PS can possibly provide healthier alternative to some oils currently blended with PS and commercial shortening to produce trans-free shortening because of the health benefits of the MCFA in RBOSL.
Improving Selectivity and Productivity of a Biocatalystic Reaction by Engineering Binary Media System. Zheng Guo, Derya Kahveci, Xuebing Xu, Aarhus University, Aarhus, Denmark
Improving selectivity and volumetric productivity of a biocatalystic reaction could be chaieved by tuning property of reaction media. Unfortunately this effort has been restrained by limited types and numbers of available conventional solvents. The tunable property and endless combination possibilities of ionic liquids (ILs) offers tremendous opportunity to rethink the strategy of current efforts to resolve technical challenges occurred in many production approaches. In this work, two sucessful examples in our group will be presented to demonstrate a new concept that employing binary medium system to improve productivity and slectivity, namely, using IL-organic solvent system to speed reaction and enhance volumetric productivity; and employing binary IL system to improve selectivity and productivity. A theorectical understanding is also presented, which may be useful to design advanced reaction protocols.
One Pot Production of Biodiesel from Jatropha curcas Oil. Y. Watanabe1, T. Nagao1, T. Kakihira2, H. Miyasaka2, Y. Shimada1, 1Osaka Municipal Technical Research Institute, Osaka, Japan, 2Kansai Electric Power Co., Kyoto, Japan
The oil from Jatropha curcas is non-edible, and thus evaluated to be a good source of biodiesel. Jatropha oil contains large amount of FFA and thus not be able to be converted to FAME by alkaline method solely. Among several enzymatic approaches possible, hydrolysis-esterification strategy was taken in this study. Jatropha oil was hydrolyzed in the presence of 30% water and 30 U/g Candida rugosa lipase. The degree of hydrolysis reached 95% at 44 h, but ca. 5% of acyl glycerols were remained. The addition of 1 mol of methanol at 20 h reduced the amount of residual acyl glycerols. The resulting oil contained 55% FAME and 45% FFA. Esterification in the presence of 2 mol of methanol to residual FFA and immobilized C. antarctica lipase increased FAME content to 97% for 50 continuous cycles. In the meantime, addition of 20 U/g Burkholderia cepacia lipase and 3 mol of methanol after 20 h of hydrolysis by C. rugosa lipase produced 97% FAME in one pot.
BIO 4.1: Designing Soybeans for 21st Century Markets
Chair(s): R. Wilson, United Soybean Board, USA
Economic Outlook for Soybeans and Soybean Products in Global Markets. R.F. Wilson, United Soybean Board, Raleigh, NC, USA
The outlook for global soybean supply is encouraging. Current rates of soybean, oil and meal production should be maintained. Demand for these products is expected to be strong. In the US, arable land is limited for corn and soybeans. Thus, greater yields of both crops will be required to achieve the projected supply levels for US food and fuel applications. Foreign demand for soybean meal will continue to escalate, dominating commodities with lesser protein concentration. However, greater production of DDGS, a by product of ethanol production from corn, will negatively impact the soybean meal feed market as US livestock producers experiment with cheaper sources of protein and amino acids. Future global veg-oil markets will become dominated by soybean and palm. However, US markets will be consumer driven toward more healthy oil and vegetable protein-based food products. QUALISOY, a coalition of all aspects of the US soybean industry, encourages opportunities to address these constraints on soybean markets through genetic technologies that enhance targeted quality traits. Soybeans with improved oil quality traits already are emerging from the QUALISOY pipeline. Commercial production of elite varieties with enhanced oil and meal quality is a committed step by the US soybean value-chain toward meeting the needs of 21st century markets with superior US soybeans and soybean products.
Implementing a Uniform Process for Measurement of Soybean Quality Traits. R. Cantrill, G. Clapper, A. Johnson, AOCS, Urbana, IL, USA
There has been a rapid proliferation of novel soybean lines with enhanced compositional characteristics due to the efforts of the United Soybean Board's (USB) Better Bean Initiative (BBI). The commercialization of these new lines meant that it was necessary to implement a system for easy identification of the advanced traits. The Soybean Quality Traits Analytical Standards Program (SQT) was initiated in 2002 after ad hoc discussions between AOCS and USB. SQT, supported by USB, researches the primary and secondary analytical methodologies needed to identify the new traits and assures that laboratories are able to successfully measure the traits. The goal of the program is to provide a continuum of analytical excellence incorporating all aspects of the soybean industry from seed developers to food manufacturers and consumers. In particular, SQT addresses the reliability of the analytical data supporting the development, marketing, and post-harvest processing of soybeans with enhanced traits. A five-phase program was developed to cover method identification, method development and validation, proficiency testing, laboratory certification, and laboratory accreditation. To date, AOCS has identified primary methods for protein, oil, and fatty acid composition that are regularly tested by analytical laboratories throughout the soy continuum in successive rounds of proficiency testing. These laboratories also collaborate in the development of improved, standardized methods for amino acid composition, sugar composition, and phytate content. A central theme of the project is the identification and support of robust easy-to use secondary technology. This effort is initially focused on the use of NIR instrumentation from a number of manufacturers. The proficiency samples are also determined by NIR or other secondary technologies. AOCS has assembled a library of soybean samples with valid wet chemistry results to allow the development of new calibrations and drive calibrations closer to agreement. The library is available to instrument manufacturers and analytical laboratories. Following a four year study, positive results have been seen in the development of NIR calibrations for linolenic acid levels. Additional details are available at www.SoybeanQualityTraits.org.
Modern Breeding Methods for Enhancing Soy Protein Quality. V.R. Pantalone, University of Tennessee, Knoxville, TN, USA
Collaborative research is accelerating development of soybean with superior meal attributes. This is being accomplished by increasing levels of essential amino acids, and by improving digestibility of meal to enhance feeding efficiency and reduce environmental impacts. Basic and applied research has been conducted to increase the knowledge base needed to reach targeted goals. One of those goals is to reduce seed phytate, an anti-nutritional compound that decreases soy digestibility in livestock and that contributes to phosphorous loads in animal manure. We have now confirmed quantitative trait loci (QTL) cqPha-001 and cqPha-002 for phytate concentration, allowing molecular breeders to accelerate plant selections. A major limitation of soybean seed storage proteins is their low levels of the sulfur-containing amino acids, methionine and cysteine, which are important nutritional components of soy meal. We detected QTL for glycinin and for conglycinin, leading to discovery of individual amino acid QTL in soybean meal. We developed and registered a new soybean line with enhanced cysteine concentration, and made it available to breeders. The recent advance by the USDA-ARS of the soybean universal linkage panel of single nucleotide polymorphisms (SNPs) will enable fine mapping and confirmation of amino acid QTL to guide plant breeders in accelerated selections for further improvement of soybean meal quality.
Biological Mechanisms that Influence Soy Protein Concentration and Composition. H. Nguyen, University of Missouri-Columbia, Columbia, MO, USA
With the highest protein content (40%) and moderately high oil (20%), soybean seed is the main source of vegetable oil and protein on earth. Environmental stresses including temperature variations influence soybean seed composition, mainly the protein-oil ratio. High temperatures in the Southern region or cooler conditions in the Northern area of the USA during soybean seed development and maturity have been reported to have a considerable effect on soybean yields, protein concentration and composition. Our major research goal is to understand the regulation of metabolic network at the genome and proteome level during the reserve deposition and seed development in soybean under normal and abiotic stress conditions. It will lay a foundation for understanding the genetic basis of phenotypic variation and selection divergence for protein and oil composition in soybean. The effects of temperature on the proteome and lipids were investigated using developing (R5 and R7) and mature seeds (R8) harvested from plants grown in different environments. We have identified several functional proteins that were differentially expressed in developing and mature seeds and further investigations on the changes in specific protein components are in progress. The presentation will highlight the global research strategies and key findings to date.
Enhancing Oil Seed Value by Modifying Protein Co-product Composition. Eliot Herman, Monica Schmidt, Donald Danforth Plant Science Center, St. Louis, MO USA
Oil seeds have the capacity to produce potentially valuable co-products in other storage substances as either intrinsic proteins and/or carbohydrates. These value added products increase the economic viability of oil seed production. Oil seeds, such as soybean and Camelina, accumulate 40 and 30% respectively of the seed mass as seed storage proteins. Our laboratory is developing protocols to exchange the production/accumulation of low value intrinsic seed storage protein for high value industrially useful proteins such as enzymes. We have discovered that seeds possess biological mechanisms that maintain seed protein content at a specific level without impacting the seed's oil content. We have termed this a protein content box where the seed's protein composition possesses plasticity that can be manipulated to produce foreign proteins. By manipulating the composition of seed proteins within a fixed protein content box it is feasible to engineer oil seeds to produce high value proteins that can be recovered from the meal remaining from the crush. The economic implications are that after seed oil is recovered the remaining protein is fractioned to recover industrially valuable enzymes and other biologics thereby increasing the crop's value.
Improved Model and Tools for Study of Soybean Mediated Food and Feed Allergy. N.C. Nielsen, North Carolina State University, Crop Science Department, Raleigh, NC, USA
Food hypersensitivity (allergy) is an immunological reaction to the ingestion of a food or food additive. There is a genetic predisposition for food allergy and it is characterized by reactions that involve immunoglobulin E (IgE). Modern advances in immunology are dominated by experiments with rodent models. Use of these models has resulted in identification of cellular and molecular components of the immune system and insight into how these components function in a coordinated fashion. However, substantial differences exist in the immune and digestive systems of rodents vs. humans that will complicate or preclude translating basic discoveries into useful clinical outcomes. The similarities in digestive and immunologic function in pigs vs. humans make it a more relevant model to explore food allergens and gut hypersensitivity. A swine breeding project was initiated to obtain 2 pig populations, one producing piglets hypersensitive to soy and the other insensitive. Foundation gilts chosen for this purpose were bred artificially with semen from selected boars and maintained on soy-free rations throughout gestation. Offspring were sensitized orally with soy at 21 days, and intraperitoneal skin test challenged with soy extract 14 days later. The prevalence of diarrhea was also monitored. Subsequent generations were produced with gilts and boars from litters having the greatest and least hypersensitivity. After three cycles of breeding and selection nearly all piglets in litters from the high sensitivity population respond to intraperitoneal skin test challenge by soy or peanut extracts. ELISA measurement of soybean specific swine IgE in sera collected an hour following challenge revealed an increase compared to controls. Most piglets from litters selected for lack of hypersensitivity do not respond to challenge with soy after 3 generations, although occasionally a piglet or two in a litter will respond to challenge as strongly as those in litters from the hypersensitive population. Piglets were also challenged with soybean extracts that contain different concentrations of known seed allergens. Interestingly, the magnitude of the response of hypersensitive piglets to skin test challenge by extracts from the two types of soybeans was different, which means the model appears capable of distinguishing degrees of antigenicity. These results justify continued effort to develop a swine model to study food allergy.
Next Generation Innovations for Market Integration of Soybean Quality. T. Ulmasov, Monsanto, USA
Most industrialized countries recognizing the risks of trans-fat have adopted policies strictly regulating its presence in food supply. Today, more than 4 years after the introduction of regulation in US, food companies are still trying to find a cost-effective solution to the trans-fat problem. Using combination of biotechnology and traditional breeding, Monsanto developed a soybean containing increased (>70%) oleic acid, <3% of linolenic acid, and reduced (5-7%) saturated fat. This oil is ideal for frying applications, providing an abundant and inexpensive supply with saturated fat content lower than in most vegetable oils. It will also find its use in industrial applications, making it a preferred feedstock for biodiesel, lubricants and hydraulic fluids. Also, to meet intake needs for long-chain omega-3 fatty acids, plant-based sources are being developed that will augment and provide an alternative to fish sources that are limited in sustainable supply. A soybean that produces 20-30% stearidonic acid (SDA) has been developed through biotechnology. SDA is the delta-6 desaturase product of α-linolenic acid and clinical data show that it readily enriches blood tissues with EPA. SDA soybean oil is processed similar to commodity oil, is similarly bland in flavor and has been successfully added to a range of foods resulting in acceptable flavor and shelf life.
The Use of Biotechnology to Enhance Soy Protein Ingredients Quality. P. Kerr1, N. Deak1, S. Knowlton2, T. Wong1, N. Shah1, D. Staerk1, 1Solae Co., St. Louis, MO, USA, 2DuPont Company, Wilmington, DE, USA
The main focus of the soy protein ingredient industry is two fold: to deliver good tasting and health enhancing ingredients. Biotechnology has now been used to enhance soy protein ingredient quality. Two major approaches for enhancing ingredient quality will be discussed: the first is the use of genetically modified soybeans, specifically Plenish™ soybeans with High Oleic acid (HO) oil profile, and the second, is the use of enzymes to enhance physical and sensory properties. This presentation will deal with both approaches and examples of each will be discussed. Through the use of a lysine or arginine specific Trypsin Like (TL1) endopeptidase enzyme, soy protein isolates with improved solubility, viscosity, and sensory properties have been created and are now commercially available. In addition soy protein isolates from HO Plenish™ soybeans have been produced with superior flavor volatile profile, reduced viscosity, and whiteness index. Collectively soy protein isolates made using biotechnology tools yielded refined protein ingredients which display superior performance attributes in beverage (dry blended and ready to drink) and meat products (injected whole muscle poultry and surimi).
BIO 4 / S&D 4.1: Biobased Surfactants and Ingredients
Chair(s): D. Hayes, University of Tennessee, USA; G. Smith, Huntsman Performance Products, USA; and D. Solaiman, USDA, ARS, ERRC, USA
Chemical Modification of Sophorolipids for Improved Water Solubility. J.A. Zerkowski, D.K.Y. Solaiman, ERRC, USDA, ARS, Wyndmoor, PA USA
Sophorolipids (SL) are biosurfactants produced from fats or oils by yeasts. They can be produced in good yields (approx. 100 g/L) and have attracted attention for commercial materials, but they have the drawback of being poorly soluble in water (roughly 50 – 100 mg/L). This talk will present our recent work on attempting to prepare sophorolipid derivatives with improved water solubility. Our first approach is to append charged units to the carbohydrate headgroups. The goal is to preserve the macrolactone version of the sophorolipid, since this structure appears to be the better surfactant. The groups we are attaching via esterification are simple amino acids, which permit positive, negative, or zwitterionic groups to be introduced. Preliminary results indicate that in some cases, solubility of the sophorolipids is much improved, while surfactancy, at least as measured by minimum surface tension, is little affected. A second route to increasing water solubility that we are investigating is to apply olefin metathesis chemistry to SL. This route involves use of the open chain form: the fatty acid side chain is shortened from its native C18 length. New functional groups can simultaneously be included in the hydrophobic chain.
The Use of Biosurfactants in Detergents. Dirk Develter, Ecover Belgium N.V., Industrieweg 3, 2390 Malle, Belgium
Glycolipids can be produced by bioconversion of native and renewable feedstocks such as rapeseed oil. Sophorolipids and rhamnolipids for example are currently finding their way to the detergent market. These attractive surfactants combine green chemistry and a lower carbon footprint without the undesirable side products or environmental downsides found in many market reference surfactants. Sophorolipids are reported to be fully aerobically and anaerobically biodegradable and to have a low acute toxicity. They furthermore do not affect Daphnia reproduction, resulting in a chronic toxicity of an order of magnitude lower than reference surfactants with a no observed effect concentration (NOEC) of an order of magnitude higher than reference surfactants. Their minimum surface tension was found to be 32.1-34.2 mN.m-1 depending on the method used. Sophorolipids are shown to be useful in hard surface cleaning and automatic dishwashing rinse aid formulations. This is attributed in part to their low foaming profile combined with their surface activity properties, which are of potential interest in additional applications. They thus combine an outstanding environmental profile with excellent performance, which resulted in their use in commercial household products.
Alkyl Polyglyceride Surfactants. G.A. Smith, H. Zhao, Huntsman Corporation, The Woodlands, TX USA
There is a growing demand for green surfactants based on sustainable raw materials. In an effort to generate surfactants from sustainable raw materials, we have concentrated on the use of polyglycerin due to the availability of cheap glycerin and its favorable environmental profile. Products derived from polyglycerin produced by conventional base catalyzed polymerization don't produce water soluble materials due to the low degree of polymerization and the presence of cyclic compounds. To increase the degree of polymerization and reduce the amount of cyclic species, we have developed a novel synthesis scheme based on glycerin carbonate. Polyglycerin produced by the present invention can be reacted with aliphatic epoxies to produce water soluble surfactants.Water soluble alkyl polyglycerides have been prepared in good yields using sustainable raw materials. The green score for these materials is around 92% based on biobased carbon. A series of products with different alkyl chain distributions were prepared and the surface properties determined. In general, alkyl polyglycerides surfactants have good surface properties including low equilibrium surface and interfacial tension, low dynamic surface tension, short wetting times and a low to moderate foam profile. In detergent applications, APG surfactants show good cleaning performance in textile cleaning and hard surface applications.
Control of Water Activity of Solvent-Free Lipase-Catalyzed Synthesis of Saccharide-Fatty Acid Ester Biobased Surfactants. R. Ye, D.G. Hayes, University of Tennessee, Knoxville, TN, USA
Saccharide-fatty acid esters, biodegradable, environmentally-friendly, and biobased nonionic biobased surfactants that are utilized in foods, pharmaceuticals and cosmetics, have been synthesized using lipases and 10-200 micron-sized suspensions of saccharide in solvent-free medium. The reaction by-product water plays several significant roles. High concentrations of it promote hydrolysis, leading to the limitation of final conversion. However, excessively low liquid-phase water concentration promotes the diffusional loss of water from lipase, reducing the enzyme activity. Therefore, it is of vital importance to identify the optimal liquid-phase water concentration during the time course of lipase-catalyzed esterification. The highest conversion yield (90%) was achieved by employing a means of water removal when the conversion reached ~65%, yielding a water content of ~0.4 wt %. In comparing 4 four means of water removal: molecular sieves (12% being optimal), vacuum, nitrogen gas, and a combination of the latter two, the nitrogen/vacuum combination was the most efficient, with the removal rate of 0.039 % per hour achieved.
Technical Criteria of Enzyme Systems for Cleaning. E. Concar, V. Huang, O. Stoica, C. Adams, Genencor, A Danisco Division, Palo Alto, CA, USA
Enzyme systems are key components in high performing detergent cleaning formulations. Enzyme performance in the wash enables cleaning at cold temperatures and short wash times while enhancing the soil removal performance of surfactants and other detergent ingredients. Assessing biochemical activity and cleaning performance of highly engineered enzyme systems involves tools and techniques ranging from the cell to the washing machine. Furthermore, using these tools to improve cleaning performance of enzymes and surfactants requires coformulation of surfactants and enzymes. This talk will explore the technical criteria for assessing the biochemical activity and cleaning performance of enzyme systems and improvements to detergent cleaning performance at cold temperatures and short wash times.
Use of Biobased Surfactants to Stabilize Emulsions Relevant for Industrial Lubrication. Kenneth Doll1, B. K. Sharma1,2, 1BOR-NCAUR, ARS, USDA, Peoria, IL, USA, 2ISTC-University of Illinois, Urbana-Champaign, Champaign, IL, USA
Emulsion based lubricants are used widely in metalworking, mining, fire-resistant hydraulic applications and more due to their low cost high performance. Key emulsification factors, such as appearance, particle size distribution and stability, are very important to lubricant applications. Water based metalworking fluids traditionally employ mineral oil emulsions however vegetable oil use is gaining in popularity. It has been shown that the chemical modification of vegetable oils can improve physical properties in base oils, such as surface adsorption, oxidative stability, and viscosity. However, it has not yet been reported if these oils are suitable for emulsification based lubrication. This presentation will show the emulsion stability of several biobased lubricant formulations where both traditional alcohol ethoxylate surfactants and biobased glyceride surfactants were used. The friction and wear reducing performance of some of these emulsions will also be reported.
Application of Response Surface Methodology in the Optimization of Inoculum Age and Size for Enhanced Biosurfactant Production from Pseudomonas aeruginosa Isolated from Petroleum Contaminated Soil. Chukwudi O. Onwosi, Fidelis J.C. Odibo, Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Anambra State, Nigeria
Biosurfactants are surface-active compounds produced by a wide range of microorganisms and they are of great industrial and commercial interests. In this work, optimizing inoculum age and size was used as a primary strategy to improve biosurfactant production from Pseudomonas aeruginosa isolated from petroleum-contaminated soil. Statistical experimental designs (Response surface methodology (RSM) and full factorial design) were used for the purpose of identifying optimal inoculum age and size in the medium. The growth media used in this study was mineral salt medium (MSM). Out of the four variables (primary inoculum age (X1), primary inoculum size (X2), secondary inoculum age (X3) and secondary inoculum size (X4)), the best biosurfactant production came from interaction from secondary inoculum age and secondary inoculum size. The overall results also suggested that the interaction of X1 (90h), X2 (1%v/v), X3 (28h) and X4 (20%v/v) gave an optimal biosurfactant yield of 7.94g/L. This is an improvement on the original yield of 2.45g/L.
A Green Alternative to Polyacrylates in Home Autodish Detergents. Geneviève Bonnechère1, Jeffrey Kolpa2, 1Dequest AG, Louvain-La-Neuve, Belgium, 2Thermphos USA Corporation, Anniston, Alabama
The detergents business in the US is facing tremendous change with more stringent regulations and increased focus on sustainable raw materials. One example is that starting in July 2010, many States will have laws going into effect that will limit the total phosphorous content allowed in home autodish products. This new trend coupled with economic recession has impacted the raw materials available to detergent formulators.To fit this future needs in detergent applications, we developed an alternative to traditional raw materials. This new green ingredient, the carboxymethylinulin (CMI) is derived from renewable chicory root and it contains neither phosphorous nor nitrogen which can lead to eutrophication. It also has an excellent Eco-tox and environmental profile.CMI offers value added performance in detergents as a co-builder. Evidence from both the laboratory and commercial products find it excellent replacement for less sustainable raw materials such as polyacrylate polymers, especially in non phosphate builder systems. Suprior cleaning performance with regard to stain removal allows formulators to reduce the total amount of chemicals used in a detergent system. In applications such as autodish washing, CMI was observed to have superior benefits including stain removal, anti-filming and anti-spotting, making it an ingredient of choice to replace polyacrylates in detergents.
Synthesis and Properties of Novel Cationics Containing Carbonate Linkages Directed Towards Green and Sustainable Chemistry. T. Banno1, K. Kawada2, S. Matsumura1, 1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama-shi 223-8522, Japan, 2Department of Chemistry, School of Science, Kitasato University, 1-15-1, Kitasato, Sagamihara-shi 228-8555, Japan
Quaternary-ammonium cationics showed strong antimicrobial activities in addition to good surfactant properties. However, they are generally highly resistant to biodegradation due to the lack of a primary degradation site in the molecule. As a next generation cationics, biodegradability is required in terms of green and sustainable chemistry. In this report, cationics containing carbonate linkages as biodegradable segments were designed and synthesized by a green method. Also, their surfactant properties, antimicrobial activities and biodegradabilities were evaluated.The gemini-type cationics containing carbonate linkages showed higher surface activities when compared to the corresponding single-type cationics. It was found that single-type cationics containing the carbonate linkage showed strong antimicrobial activities and excellent biodegradabilities. Also, the gemini-type cationics containing the carbonate linkage in the linker moiety showed higher antimicrobial activities and biodegradabilities when compared to the gemini-type cationics containing carbonate linkages in the hydrophobic moiety. It was confirmed that the antimicrobial activities and biodegradabilities were improved by the introduction of the carbonate linkage into the linker moiety.
Protein-Based Enhancement of Surfactancy. Carl Podella, Jack Baldridge, Michael Goldfeld, Advanced BioCatalytics Corp., Irvine, CA, USA
Low molecular weight proteins released by heat-shocked bakers yeast form tightly bonded complexes with a broad range of surfactants: anionic, cationic and non-ionic. These protein-surfactant complexes (PSC) are heat-resistant, retaining their high surface activity upon heating up to 100°C for 96 hours, and increasing the stability of the synthetic surfactants involved. They are stable in a pH range from 1 to 12 and are compatible with certain strong oxidants, such as hydrogen peroxide, for extended time periods (a year or more). PSC display enhanced surface activity as compared to their synthetic surfactant ingredients, allowing reduction of interfacial tension (IFT) between mineral oil and water down to 0.0002 mN/m. Mechanistically, they appear to constitute a class of versatile extended hybrid synthetic-biosurfactants. They provide the core for wetting agents, industrial and consumer-oriented cleaners, agricultural adjuvants, EOR chemicals, skin-care products and alike. Exposure of PSC to the non-sterile conditions results in a further development of surface activity in the course of time, due to a synergistic enhancement of bacterial transformation of grease into surface-active material. PSC also increase the permeability of model hydrophobic membranes to hydrogen ions, similar to the known, but highly toxic uncouplers of oxidative phosphorylation. The latter effect is related to their ability to enhance the rate of bacterial bio-oxidation of nutritious organic contaminants without a concomitant growth of bacterial biomass, thus resulting in a reduced volume of solid sludge in wastewater treatment systems. PSC-based products are safe, non-toxic and environmentally friendly. PSC-based products have been certified by the National Sanitary Foundation (NSF) for processing potable water, and by the International Maritime organization (IMO) for chemical cargo tank cleaning.
BIO 5: General Biotechnology
Chair(s): R. Patel, SLRP Associates, USA; and J. Ogawa, Kyoto University, Japan
Eicosapentaenoic Acid Production by Molecular Breeding of Filamentous Fungus Mortierella alpina. Akinori Ando1,2, Yosuke Sumida2, Hiroaki Negoro2, Dian Anggraini Suroto2, Jun Ogawa1,2, Eiji Sakuradani2, Sakayu Shimizu2, 1Research Division of Microbial Sciences, Kyoto University, Kyoto, Japan, 2Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
A filamentous fungus, Mortierella alpina 1S-4, belonging to the Zygomycetes, has been isolated from soil as a potent producer of polyunsaturated fatty acids (PUFAs) in our laboratory and utilized for commercial production of arachidonic acid (AA, 20:4n-6). First of all, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil auxotrophic mutant (ura5- strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. As a result, we revealed that the frequency of transformation surpassed 400 transformants/108 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60-80%) showed mitotic stability. Subsequently, the ω3-desaturase gene catalyzing conversion of n-6 fatty acids to n-3 fatty acids was homologous overexpressed in M. alpina 1S-4 by employing ATMT system, and we evaluated EPA production, which was an end product of n-3 fatty acids synthesized in M. alpina 1S-4. As a result, the accumulation of n-3 fatty acids in transformants was observed. In particular, the content of EPA reached 40% of the total fatty acids. We revealed that molecular breeding of M. alpina 1S-4 by ATMT achieved the high EPA production.
Arabitol Production from Glycerol by Yeast Fermentation. Srujana Koganti1, Lu-Kwang Ju1, Tsung-Min Kuo2, 1Department of Chemical and Biomolecular Engineering, University of Akron, Akron, OH, USA, 2USDA, NCAUR, Peoria, IL, USA
Biodiesel is a major alternative renewable fuel. Biodiesel production processes typically produce glycerol as a major byproduct. Developing new industrial/commercial uses of glycerol is important to economics and sustainability of biodiesel industry. Xylitol and arabitol are sugar alcohol isomers with current markets as high-value products and future potential as major building blocks for various chemicals through biorefinery. We conducted extensive culture screening of more than 300 strains in the USDA ARS culture collection (NRRL). While no strains were found to produce xylitol as the dominant metabolite, several osmophilic yeast strains produced arabitol as the dominant metabolite. We subsequently studied the optimal arabitol production conditions for the most promising strain, and developed the fermentation process for consistent production of arabitol at more than 50% yield from glycerol. Various medium composition and culture conditions were evaluated; the latter included temperature, pH, dissolved oxygen concentration, nitrogen-to-phosphorus ratio, and glycerol concentration. We are currently evaluating the downstream arabitol collection and purification methods to complete the preparation for industrial arabitol production. Results of these studies will be presented.
Off-Flavor Problem in Soy Proteins: a Process for the Removal of Phospholipids in SPI. A. Aurora, S. Damodaran, Dept. of Food Science, University of Wisconsin-Madison, Madison, WI, USA
The off-flavor problem in soy protein isolate (SPI) is mainly due to the presence of residual amounts of phospholipids, which undergo autoxidation during storage. Thus, removal of phospholipids from SPI is a necessary first step to improve its flavor stability and enhanced utilization. A β-cyclodextrin-based process has been developed to remove soy protein-bound phospholipids. Treatment of SPI solution (8%) with 10 mM βCD alone at pH 8.0 reduced the phospholipid content of SPI by 36.64%. Greater than 99% removal of phospholipids from SPI was achieved by using a combination of treatments involving sonication of the SPI solution for 5 min at 50°C followed by treatment with phospholipase A2 and βCD. SPI prepared by this method was very white in color. The concentration of headspace volatile compounds after accelerated storage for 90 days at 40°C in the β-cyclodextrin-treated SPI sample was much lower than in the untreated control.
The Extraction of Rhodotorula glutinis from Aqueous Media Using the Solvent Extraction Technique. M. Hetrick, C. Zumbro, B. Holmes, J. Donaldson, R. Hernandez, W. French, Mississippi State University, Mississippi State, MS, USA
Oleaginous microorganisms produce triacylglycerol (TAG), a metabolic by-product that is synthesized when organisms are cultivated on media that has high sugar content. TAG can be used to generate biodiesel however; the current method for the recovery of TAG from the oleaginous microorganisms is costly, thereby generating an economic barrier for implementing biodiesel production by microorganisms into an industrial atmosphere. The removal of water induces an elevation of cost, therefore, if the amount of water present in the system is minimized, the cost of TAG preparation for biodiesel can be successfully lowered. Using a two phase system comprised of an aqueous phase and a solvent phase, we can simulate a gradient, where those cells that have the highest TAG content are located at the surface of the aqueous phase and those cells that have a lower TAG content are located at the bottom of the aqueous phase. Cells that have high TAG content will be buoyant, and this will cause them to rise to the surface of the aqueous phase. When the solvent layer is added to the aqueous phase, buoyancy of â€œTAG-fatâ€ cells should aid in propelling these cells into and trapping them within solvent layer. The solvent layer can be removed, and evaporated off thus leaving behind a concentrated mass of cells that contain a high TAG content.
Enzymatic Production of Healthier Lard-based Fats. L.Z. Cheong1, L. Nersting2, X. Xu1, 1Aarhus University, Aarhus, Denmark, 2Danish Meat Research Institute, Roskilde, Denmark
Lard is the key ingredient contributing significantly to the flavour and texture of many food products. Despite its exceptional sensory properties, lard is notorious for negative nutritional values namely low digestibility, high calories and saturated fatty acids. This work attempted to produce healthier lard-based fats at higher production scale through various enzymatic approaches. One of the approaches used was enzymatic interesterification of lard with fish oil to produce fat with higher degree of unsaturation. Considering the fact that lard had high melting point while fish oil are heat sensitive, enzymatic interesterification was conducted using packed bed bioreactor. The key factor in this process is the residence time between the blend and enzyme. The process was optimized wherein the fat blend was subjected to high temperature at the shortest residence time which kept the lard in liquid form while minimized the oxidation of fish oil. More than 80% interesterification degree was achieved. Another approach used was enzymatic glycerolysis of lard to produce lard diacylglycerols (lard-DAG) fat. Enzymatic glycerolysis was conducted using batch reactor which promoted formation of a homogenous reaction substrate. More than 50% of lard-DAG yield can be achieved. These new healthy lard-based fats are determined in terms of feasibility for applications in meat products.
Enzymatic Conversion of Castor Bean Oil to Biodiesel. S. Basheer, E. Masoud, Transbiodiesel Ltd., Shfar-Am, Israel
Biodiesel is produced by transesterification reaction between oil and fat triglycerides with methanol. The reaction is typically catalyzed by either an alkali metal strong base or a strong acid. These processes are characterized by numerous disadvantages, resulting in the formation of environmental hazards and cost-ineffectiveness.During the last two decades lipase-catalyzed transesterification of plant oils and animal fats with short-chain alcohols for the production of biodiesel has been extensively studied. Because of inhibition effect of short-chain alcohols and the high prices of lipases the production of biodiesel at industrial scales with a cost-effective lipase remains unresolved issue. TransBiodiesel has developed new modified-immobilized lipase preparations capable of tolerating high concentrations of short-chain alcohols for the transesterification reaction of oils and fats particularly with methanol. This presentation will cover the use of newly developed lipase preparations for the production of biodiesel form castor bean oil.
Metabolic Engineering of Yarrowia lipolytica: Production of Eicosapentaenoic Acid-Rich Oil for Commercialization. N. Yadav, Q. Zhu, B. Tyreus, P. Sharpe, H. Damude, D. Xie, D. Short, R. Hong, A. Kinney, Z. Xue, DuPont Co., Wilmington, DE19898
Omega-3 fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), mainly found in fish oil, have proven human health benefits. Concerns over the quality and sustainability of the fish oil supply have generated interest in alternative sources for them. We report the development of a clean and sustainable source of omega-3 fatty acids by fermentation, using a metabolically engineered strain of the oleaginous yeast, Yarrowia lipolytica. While Y. lipolytica can accumulate oil up to 40% of the dry cell weight, the only PUFA it synthesizes is linoleic acid (18:2n-6). High level expression of desaturases and elongases comprising omega-3 fatty acid biosynthesis pathway, enhanced acyl-exchange, and improved fermentation process resulted in the generation of oil with greater than 55% EPA, and less than 8% saturated fatty acids. We will also describe the importance of the peroxisome in regulating fatty acid metabolism to achieve high EPA titer in engineered Yarrowia strains. Our land-based production of omega-3 fatty acids is a superior source for these essential molecules for various applications from nutritional supplement to animal feed.
Chair(s): R. Ashby, USDA, USA
Comparison of 1H NMR, HPLC, GOPOD and UV Absorbance Procedures to Estimate Brassica Seed Myrosinase Activity.
Lawrence W Thomson, Bonnie Li, Martin JT Reaney, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Brassica seed meal and seed meal extracts are being used as commercial biopesticides and as condiments. Both biocidal and flavour properties are attributed to the ability of the oilseed meal to release isothiocyanates after contact with water. Exposing meal to moisture provides the environment for enzyme (myrosinase) cleavage of glucosinolates and consequent release of isothiocyanates. Processors of mustard for condiment and biocide industries require reliable analytical methods to determine myrosinase levels. Myrosinase isoforms occur as both soluble and membrane associated complexes. An estimate of both isoforms will be presented. Methods for measuring myrosinase activity (1H NMR, HPLC, GOPOD and UV absorbance) using both crude and enriched extracts of mustard will be discussed.
Enzymatic Interesterification: Confirmation Experiments Following a Fractional Factorial Design.
C. Pacheco, G.H. Crapiste, M.E. Carrín, Plapiqui (UNS - CONICET), Bahía Blanca, Buenos Aires, Argentina
In the present work the effect of certain reaction parameters over the enzymatic interesterification of fully hydrogenated soybean oil and liquid soybean oil was studied. The experiments were performed with a 27-2IV fractional factorial design with two replicates. The design factors considered were: enzyme concentration (A), immobilized enzyme type (B), reaction time (C), substrate mass ratio (D), temperature (E), agitation level (F) and moisture content (G). The reaction products were analyzed with respect to melting point (m.p.), byproducts (free fatty acids, mono- and diglycerides) and triglyceride content and seven models were obtained. It was determined that the reaction conditions to obtain minimal m.p. and minimal byproducts generation should be: 5% enzyme (w/wsubst), Lipozyme RM IM, 24 h, 70% oil (w/w), 65ºC, low agitation level and absence of molecular sieves. A confirmation assay to test the accuracy of the developed models was performed at this point of the design space obtaining that the measured responses fell inside the corresponding prediction intervals, validating the models.
Efficient Production of Multiple Functional Trehalose Catalyzed by Picrophilus torridus Trehalose Synthase in Permeabilized Cells.
Shu-Ming Liou1, Jei-Fu Shaw2, Shu-Wei Chang3, 1Department of Bioindustry Technology, Dayeh University, 168 University Rd., Dacun, Changhua 51591, Taiwan, R.O.C., 2Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd. Taichung, 402 Taiwan, R.O.C., 3Department of Medicinal Botanicals and Health, Dayeh University, 168 University Rd., Dacun, Changhua 51591, Taiwan, R.O.C.
Trehalose is a natural multiple functional disaccharide and has been widely used in food, cosmetics, pharmaceutical and agriculture etc. Generally, it can be produced by intracellular enzymes which required protein expression and purification procedure to increase its production cost. The permeabilization of microbial cells accompany with trehalose synthase activity were used to simplify trehalose production steps, increase its reusability and reduce the production cost for further industrial applications. For these reasons, the permeabilized cells of Picrophilus torridus trehalose synthase were therefore developed by using different organic solvents and detergents in this study. The result showed that up to 50% of trehalose molar conversion was obtained by toluene-treated permeabilized cell at 45°C for 4 h.
Enzymatic Modification of Corn Oil with Conjugated Linolenic Acid Originated Momordica charantia (Bitter Gourd) Seed Oil.
H. Funda Suzen, Begum Elibal, H. Ayse Aksoy, Istanbul Technical University Chemical Engineering Department, Istanbul, 34469 Maslak, Turkey
Conjugated linolenic acid (CLNA) is a mixture of positional and geometric isomers of octadecatrienoic acid with three double bonds in conjugation. CLNA is an effective antioxidant, slows down the tumor growth and enriches the immunity system. Bitter gourd (Momordica charantia) seed oil (CLNA content: 55-60%; major fatty acid: α-eleostearic acid) is one of the most important available sources of CLNA. Corn oil (CO) is benefical for health due to its high unsaturated fatty acids content. The aim of this study was enrichment of corn oil with CLNA using bitter gourd seed oil fatty acids (BGFA) by enzymatic acidolysis. The total CLNA content of BGFA was 57.2% (α-eleostearic acid, 50.3%). Acidolysis reactions were conducted using immobilized sn-1,3 specific lipase from Thermomyces lanuginosa (Lipozyme TL IM) in an orbital shaker at 200 rpm. Experiments were carried out to determine the effects of enzyme amount, reaction time, temperature, and CO: BGFA molar ratio on the reaction. The highest CLNA incorporation (46.3%) was observed at 60 ºC, 1/5 CO: BGFA molar ratio, 10% enzyme based on total substrate weight after 3h reaction.Thus obtained CLNA rich structured lipid originated from corn oil could be used as a source of dietary CLNA structured lipid in the formulation of food products.
Enzymatic Esterification of PUFA Isolated from Rapeseed Oil.
A. Onofre-Sestiaga, C. Marquez-Beltrán, R.E. Lugo-Sepúlveda, J.A. Noriega-Rodríguez, Universidad de Sonora Unidad Regional Norte., H. Caborca Sonora, Mexico.
In this work, structured lipids with n-6 and n-3 from rapeseed oil were prepared. Canola oil was subjected to alkaline hydrolysis to release fatty acids (FA), obtaining 89% yield. Polyunsaturated fatty acids (PUFA) n-6 and n-3 were purified by urea complexation. The esterification of PUFA to glycerol was conducted in batch reactors using Candida antarctica lipase (NV435). Glycerides produced were analyzed by HPTLC and quantified by densitometric evaluation. The optimum conditions of temperature, time and molar proportion were determined by response surface analysis. A maximum of global esterification was obtained (83%) for 2.0 mol PUFA/mol glycerol, 62°C y 200 min. The formation of monoacylglycerols was favored when the molar proportion was increased, while at lower temperature a higher production of diacilglycerols was obtained.
Enzymatic Synthesis of Lysophosphatidylcholine Containing CLA from Glycerophosphatidylcholine Under Vacuum System.
Seung In Hong1, Mi-Hwa Chae1, Jong-Hun Choi1, TingTing Zhao1, Inhwan Kim2, Jong-Wook Kim3, Eun Ju Lee3, Kwang-Il Kwon3, In-Hwan Kim1, 1Department of Food and Nutrition, Korea University, Seoul, 136-703, Republic of Korea, 2R&BD Oils & Fats Development Team, NONG SHIM CO., LTD., 370-1, Shindaebang-Dong, Dongjak-Gu, Seoul, Republic of Korea, 3Korea Food and Drug Administration (KFDA), Seoul, 122-704, Republic of Korea
Esterification by enzyme is related to water but no research using vacuum system has performed to control water content for synthesis of structured lysophosphatidylcholine (LPC). In this study LPC was successively synthesized by lipase-catalyzed esterification of glycerol-sn-3-phosphatidylcholine (GPC) with conjugated linoleic acid (CLA) under vacuum. The GPC used as substrate was prepared from soybean phosphatidylcholine by alkaline deacylation using tetrabutylammonium hydroxide as a catalyst. Five immobilized enzymes, namely Novozym 435, Lipozyme TL IM, Lipozyme RM IM, phospholipase A1, and phospholipase A2, were screened for their activity to catalyze the esterification reaction. Novozym 435 gave the highest yield of structured LPC. Thus, this enzyme was chosen for subsequent experiments to investigate optimal conditions on the vacuum system. The effects of reaction parameters, namely temperature, vacuum, and reaction time were monitored by combination of TLC and GC-FID analyses.
Using a Biotechnology Approach to Increase Oil Content of Camelina sativa.
Kimmo Koivu1, Viktor Kuvshinov1, Seppo Kaijalainen1, Randal Weselake3, Murphy Eric 1,2, 1Unicrop, Oy, Helsinki, Finland, 2Agragen, LLC, Cincinnati, Ohio, USA, 3University of Alberta, Edmonton, Alberta, Canada
Camelina sativa is an emerging oil seed crop that is ideally suited for production on marginal lands and requires minimal inputs. Because of the limited biodiversity in Camelina germplasm, we have taken a biotechnology approach to enhance seed triacylglycerol (TAG) content over a conventional breeding approach. Because diacylglycerol acyl transferase-1 (DGAT-1) is the rate limiting step in oil seed triacylglycerol (TAG) biosynthesis, we transformed Camelina with the DGAT-1 from flax. Using a transgenic approach rather than over expression of the endogenous DGAT found in Camelina, avoids the potential of gene silencing. While our current plants are awaiting TAG analysis, previous studies have demonstrated that DGAT over expression can increase TAG content up to 5%. We look for a similar increase in Camelina and by using our patent-pending non-selection marker system, we look for rapid introduction of these new Camelina varieties into commercial use.
Enzymatically Production of Structured Lipids from Sunflower Oil in a Packed Bed Reactor.
C. Palla, M.E. Carrin, PLAPIQUI (UNS-CONICET), Bahía Blanca, Buenos Aires, Argentina
The production of structured lipids by acidolysis with immobilized enzymes is generally performed in batch reactors. Using packed bed reactors (PBR) to confine the immobilized enzyme may be promising for further scale-up of the process. The aim of this work was to produce trans-free structured lipids in a PBR by acidolysis of sunflower oil and palmitic/stearic acids testing the catalytic activity of lipases from Rhizomucor miehei immobilized on modified chitosan microspheres. Different operational conditions in continuous mode (mass flow rate, temperature) were tested with hexane as the solvent. Saturated fatty acid incorporation was favored by a longer residence time, and a higher reaction temperature.
Probing the Limits of Very Long Chain Polyunsaturated Fatty Acid Accumulation in Transgenic Brassica napus.
C.L. Snyder1, R. Hryniuk1, J. Bauer2, T. Senger2, R.J. Weselake1, 1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada, 2BASF Plant Science GmbH, Limburgerhof, Germany
Very long chain polyunsaturated fatty acids (VLCPUFA), such as arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential for human health. Inadequate consumption of VLCPUFA, particularly n-3 PUFAs in fish oil, has been associated with a variety of chronic diseases. Over the past decade, there have been efforts to develop transgenic oilseeds producing high levels of VLCPUFA, as an alternative to marine oils. Introduction of the minimal set of genes required for EPA and ARA synthesis by the Δ6-desaturase pathway revealed a “bottleneck” in the elongation of Δ6-desaturated C18 PUFA. Further analysis suggested this was due to the absence of Δ6-C18 PUFA-CoA substrates for elongation. This could be explained by rapid removal of C18-PUFA-CoA by acyltransferases or by inefficient release of newly desaturated PUFA from phosphatidylcholine (PC), which is the site of desaturation. Introduction of an acyl-CoA dependent Δ6-desaturase in an attempt to bypass this bottleneck resulted in lower overall VLCPUFA levels due to inefficient Δ6-desaturation. In this study, we further characterized transgenic B. napus lines which accumulate VLCPUFA, in order to identify new targets for overcoming substrate dichotomy in VLCPUFA biosynthesis in transgenic plants.
Production of Class-2 Herbicide Tolerant Camelina sativa via Introduction of Specific Mutations in the Acetolactate synthase gene from Arabidopsis thaliana.
Kimmo Koivu1, Viktor Kuvshinov1, Seppo Kaijalainen1, Eric Murphy1,2, 1Unicrop, Oy, Helsinki, Finland, 2Agragen, LLC, Cincinnati, Ohio, USA
Camelina sativa is an emerging oil crop that offers farmers in the Pacific Northwest an alternative cropping option as a rotational plant with wheat. However, high soil levels of residual class-2 herbicides limits cropping choices by these farmers, including the planting of Camelina, which is highly susceptible to these herbicides. To circumvent this problem, we produced a Camelina that is more tolerant to class-2 herbicides, which inhibit acetolactate synthase (ALS), the rate limiting enzyme in plant branched chain amino acid synthesis. We cloned the ALS gene from A. thaliana and introduced either a Ala122>Thr, Pro197>Ser, or a Trp573>Leu mutation into the gene or a combination of these mutations. Using our patent-pending, non-selection marker high efficiency transformation system, we transformed Camelina with the a.t. ALS gene under control of the endogenous promoter from A. thaliana. We observed a significant increase in tolerance in excess of 10,000-fold compared to control plants using our proprietary in vitro testing protocol. Greenhouse testing is currently underway to further substantiate class-2 herbicide tolerance, but it is our expectation to rapidly move these plants into a field testing phase, followed by rapid introduction into field production.
Isolation of Fungi from Diesel Oil Polluted Soil In Mbaise Nigeria.
Chinedu William Agbakwuru1, Joachin O. Anyanwanu1, 1Imo State University Owerri, Owerri, Imo, Nigeria, 2Imo State University Owerri, Owerri, Imo, Nigeria
This research was designed to determine the influence of NPK fertilizer, palm from ash and poultry waste treatments on the rate of fungal biodegradation of diesel oil in soil. The total heterotrophic fungal count revealed that 3.2X105 cfu/ml, 1.35X105 cfu/ml, 2.65X105 cfu/ml and 0.85X104 cfu/ml were isolated from sample A, B, C and D respectively. While the hydrocarbon utilizing fungal counts were found to be 1.55X104 cfu/ml (for 0 hour), 1.80X104 cfu/ml (for 24th hour), 2.05X104 cfu/ml (for 72nd hour) and 2.15X104 cfu/ml (for 6th day) for sample A; that of sample B were found to be 0.45X104 cfu/ml (for 0 hour) and 0.65X104 cfu/ml (for 24th hour), 0.70X104 cfu/ml (for 72nd hour) and 0.85X104 cfu/ml (for 6th day). Sample C were recorded to be 1.05X104 cfu/ml (for 0 hour), 1.40X104 cfu/ml (for 24th hour), 1.50X104 cfu/ml (for 0 hour), 1.40X104 cfu/ml (for 24th hour), 1.50X104 cfu/ml (for 72nd hour) and 1.70X104 cfu/ml (for 6th day) whereas sample D gave 0.25X105 cfu/ml (for 0 hour), 0.25X105 cfu/ml (for 24th hour), 025X105 cfu/ml (for 72nd hour) and 0.30X105 cfu/ml (for 6th day). Objectively, this research project was designed to isolate hydrocarbon utilizing fungi to estimate their growth rate in both treated and untreated diesel-polluted soils with respect to the time of sample collection and analysis.
Analyses of Glucosinolate Content in Brassica Seeds.
B. Li1, L.W. Thomson1, D.P.O. Owiti1, K. Thoms2, M.J.T. Reaney1, 1University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 2Saskatchewan Structural Science Centre, Saskatoon, Saskatchewan, Canada
Mustard seed is increasingly being used as a source of pesticide and processors require analytical methods to determine the levels of active ingredient (glucosinolate) prior to purchase as well as during and after processing. A method for rapid HPLC estimate of glucosinolate type and concentration is described that effectively determines glucosinolates in Brassica juncea, Brassica carinata, Brassica napus and Sinapis alba. The method was validated by further analyses preformed on HPLC coupled with Hybrid Quadrupole-TOF LC/MS/MS with a turbo spray ESI source. Parent ion mapping allowed rapid assessment of the glucosinolate and the anion fragmentation pattern confirmed the presence of specific glucosinolates. We present our rapid seed sample preparation and glucosinolate estimates on Sinapus alba, Brassica juncea (mustard and canola quality) and B. napus (canola quality) and blends of mustard and canola. This test provides processors with an estimate of glucosinolate levels in seed and meal samples. The results from these analyses will be presented.
Isolation of Segetalins A and B from Saponaria vaccaria.
P.G. Burnett1, I. Ramirez-Erosa2, J. Balsevich2, P. Arnison3, M. Reaney1, 1Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, SK, Canada, 2National Research Council - Plant Biotechnology Institute, Saskatoon, SK, Canada, 3Botanical Alternatives Inc., Saskatoon, SK, Canada
Cyclic peptides are naturally occurring molecules that are known to exhibit a wide range of biological activity. Segetalins A and B are a cyclic hexapeptide and pentapeptide, respectively, found in Saponaria vaccaria. Segetalins A and B have displayed estrogen-like activity, in addition to vasorelaxant and contractile activity. We have found these peptides useful as internal standards for the analysis of similar cyclic peptides. The extraction of small quantities of these cyclopeptides from Segetalis vaccaria has been documented in the scientific literature. The goal of our research is to obtain pure cyclopeptides in high yield. Cyclopeptide fractionation from the germ extract of Saponaria vaccaria and the isolation, recovery and purification of segetalins A and B will be presented.
Systems Biology of Fatty Acid Biosynthesis in Marine Microorganisms.
J. Zhang, G. Burgess, Dove Marine Laboratory, School of Marine Science and Technology, Newcastle University, UK
Monitoring Enzymatic Interesterification: Quantification of Mono-, Di- and Triglycerides.
C. Pacheco, G.H. Crapiste, M.E. Carrín, PLAPIQUI (UNS - CONICET), Bahía Blanca, Buenos Aires, Argentina
During enzymatic interesterification two mixtures of triglicerides are initially put together and the addition of an enzyme determines the beginning of a fatty acids exchange between them. The final product results in a new mixture of triglycerides, different from the substrates ones, and an amount of byproducts of the reaction. These byproducts are free fatty acids, monoglycerides and diglycerides. The main objective of this work was to quantify the changes of these compounds at initial time in order to start understanding the kinetic behavior of this complex enzymatic reaction.
Lipase-catalyzed Synthesis of Triacylglycerols Enriched in Pinolenic acid at sn-2 Position from Pine Nut Oil.
Jong-Hun Choi1, Seung In Hong1, Bo-Mi Lee1, Young-Gil Pyo1, Jong-Wook Kim2, Eun Ju Lee2, Kwang-Il Kwon2, In-Hwan Kim1, 1Department of Food and Nutrition, Korea University, Seoul, 136-703, Republic of Korea, 2Korea Food and Drug Administration (KFDA), Seoul, 122-704, Republic of Korea
Pine nut oil contains about 14 wt% pinolenic acid (PLA) of total fatty acids. PLA was esterified predominantly at sn-3 positions (>90% of total PLA) of triacylglycerol (TAG) of pine nut oil. Meanwhile, the PLA was scarcely at sn-1,2 positions. The aim of this study was to synthesize efficiently TAG enriched in PLA at sn-2 position by lipase-catalyzed acyl migration. Hence optimal condition, which occurs fast acyl migration, was investigated to synthesize efficiently the modified TAG. The activities of eight commercial lipases were screened to catalyze the acyl migration. Two immobilized lipases namely, Novozym 435 and Lipozyme TL IM were selected for subsequent experimental trials. The effects of parameters, such as temperature, water content and reaction time and their correlation were observed. Lipozyme TL IM was more effective than Novozym 435 without water. However, the yield of TAG modified with Lipozyme TL IM was lower than that with Novozym 435.
'Designer Liquids' for Sustainable Development of Lipid Processing.
S.C. Chua, Z. Guo, X. Xu, Department of Molecular Biology, University of Aarhus, Denmark
‘Designer liquids' are made up of different anion and cation combinations, they can be designed to fix a particular purpose and possess certain set of properties for special lipid reactions. The common term for this ‘designer liquids' is ionic liquids (IL). ILs are the alternative to replace harmful organic solvents and having special properties, for instance no measurable vapor pressure, non-flammability, and a wide temperature range for liquid phase. Recently, ILs are getting popular in enzymatic reaction because of the enormous advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. All the advantages create the opportunity for a truly sustainable development of this fascinating group of chemicals substances to the researches and industrials. This review highlighted the latest technology developments of biocatalyst reactions in IL and its sustainable development for reaction, separation and processing in the lipid area.
Vegetable Oil Hydrolysis in Submerged Membrane Bioreactor for Innovative Production Systems.
Sudip Chakraborty1,2, Lidietta Giorno1, Enrico Drioli1,2, 1Research Institute on Membrane Technology, 17C, Rende (CS), Italy, 2Dept. of Chemical and Materials Engineering ,University of Calabria, 44A, Rende (CS), Italy
Continuous hydrolysis of olive oil by Candida Rugosa (CRL) lipase was studied in a two configuration of microporous hydrophilic membrane bioreactor. This two confuguration of module side stream and submerged membrane bioreactors is based on the separation of enzyme and products (or substrates) by a semipermeable membrane that creates a selective barrier. Permeable solutes can be separated from the reaction mixture by the action of a driving force (chemical potential, pressure, electric field) that is present across the membrane.A complete retention of the enzyme within the system is the first and most important requirementfor a successful continuous operation of a membrane bioreactor. Upon this retention, the enzyme becomesconfined to a defined region of the membrane reactor, where reaction with the substrate occurs. Theenzyme was immobilized on the shell side of the membrane and is usually entrapped inside the pores ormembrane matrix. Olive oil and buffer solution, fed continuously through two compartments partitionedby membrane, caused reaction at the interface of lipase-adsorbed membrane and buffer solution. Thehydraulic pressure in the oil phase was enough to maintain phase separation, an accurate pressure controlwas not necessary.Fatty acid was obtained in a single phase without being mixed with components ofother phases for different production systems.
Enzymatic Production of Omega-3 Specialty Oils: Emerging Process Technologies.
Derya Kahveci1, Kjartan Sandnes2, Xuebing Xu1, 1Department of Molecular Biology, Aarhus University, Aarhus, Denmark, 2Marine Bioproducts AS, Storebo, Norway
Omega 3 fatty acids are known to have many beneficial effects on human health, such as treatment and/or prevention of cancer and cardiovascular diseases; development of nervous tissues, especially retina and brain; improvement of immune system; treatment of mental diseases; etc. Concentrated form of such fatty acids is a commonly preferred dietary supplement, since it highly reduces the oil intake. There have been a number of methods used in the industry for the purpose of concentrating omega 3 fatty acids in marine oils, including chromatography, extraction, fractional distillation, and urea complexation, all of which has some drawbacks in terms of feasibility and environmental issues. Enzymatic processes, on the other hand, are considered to be superior in fish oil industry, since lipases are effective under mild reaction conditions, and thus, do not initiate lipid oxidation; and they have fatty acid selectivity, which improves the omega 3 yield. This review aims to cover the emerging process technologies in the field of enzymatic processing of fish oil in order to produce omega 3 specialty oils.
Enzymatic Synthesis of Novel Phytosteryl Caffeates and their Antioxidant Activity.
Z. Tan and F. Shahidi, Department of Biochemistry, Memorial University of Newfoundland, St John's, NL, Canada.
FADX, DGAT, CPT, PDAT and More: a Vegetable Soup of Enzymes that Influence Industrial Oil Production in Transgenic Oilseeds.
J.M. Shockey, USDA-ARS, Southern Regional Research Center, New Orleans, LA, USA
Our laboratories study tung tree (Vernicia fordii) and bitter gourd (Momordica charantia) as model systems for the production of industrially useful oils. Seed oils of bitter gourd and tung contain approximately 60% and 80%, respectively, α–eleostearic acid, a valuable conjugated fatty acid. Successful reconstitution of the biosynthetic pathway for drying oils in agronomically amenable transgenic systems may require introduction of several genes. Some important components of these pathways, such as the fatty acid conjugases (FADXs) and diacylglycerol acyltransferases (DGATs) have been identified. We are currently focused on the optimization of the FADX and DGAT transgenes currently in use. The effects of alterations in transgene construct design, including changes that affect transcriptional timing and maintenance, translational efficiency, and protein stability will be presented. Other necessary pieces of the TAG biosynthetic puzzle remain elusive. Much effort has been focused on finding other enzymes in the pathway that contribute to efficient channeling of eleostearate into TAG. Recent results from the study of co-expression of genes located in the central part of the pathway (between FADX and DGAT) will also be presented.
Production of a High Lauric Acid Containing Camelina sativa Using Biotechnology.
Kimmo Koivu1, Viktor Kuvshinov1, Seppo Kaijalainen1, Eric Murphy1,2, 1Unicrop, Oy, Helsinki, Finland, 2Agragen, LLC, Cincinnati, Ohio, USA
Camelina sativa is a oil seed crop that is well suited for use as a biofuel feedstock. However, the oil composition of Camelina offers unique challenges for conventional biodiesel production as well as emerging technologies to make alternative biofuels such as biojet. Because of the limited biodiversity of the Camelina germ plasm, we have used a biotechnology approach to modify its fatty acid composition. We transformed Camelina using a synthetic gene encoding for lauric acid-acyl carrier protein (ACP) thioesterase from the California bay plant to truncate fatty acid synthesis from ACP at 12:0 carbons. Initial analysis of lines from Camelina demonstrate 12:0 content up to 23%, as compared to no 12:0 detectable in control seeds grown under the same greenhouse conditions. In the highest 12:0 producing lines, 14:0 was also increased from none detected to 4%, thus short chain saturated fatty acids were increased to 27%, while 18:0 was reduced 50%, 18:1n-9 reduced >60%, and 18:2n-6 reduced 25%, while there was minimal reduction in 18:3n-3. These data suggest that Camelina will continue to desaturate 18 carbon fatty acids to 18:3n-3 at the expense of less unsaturated 18 carbon fatty acids under these condition. Further transformations are on-going to optimize 12:0/14:0 content to produce Camelina lines with up to 60% of these two fatty acids.
Effect of Nutrients on Growth and Lipids Accumulation in Dunaliella tertiolecta.
Meng Chen1, Hongzhi Ma1, Haiying Tang1, Thomas Holland2, Steven Salley1, Simon Ng1, 1Wayne State University Department of Chemical Engineering, Detroit, MI, USA, 2Wayne State University Department of Microbiology and Immunology, Detroit, MI, USA
Microalgal based biofuels production has the potential to significantly supplement, if not completely replace, petroleum-derived transport fuels without adversely affecting the supply of food and other crop products. The production of biofuel from algae is dependent on to the microalgal biomass production rate and lipid content. Both biomass production and lipid accumulation are limited by several factors, of which nutrients plays a key role. In this research, we used: Dunaliella tertiolecta as the model and identified inorganic phosphate and trace elements such as cobalt, iron, molybdenum and manganese as required for cell optimum growth. Inorganic nitrogen as nitrate instead of ammonium is required for maximal biomass production. Lipids were accumulated under nitrogen starvation growth condition and are time-dependent. In addition, fatty acid composition is more suitable for biodiesel production in the nitrogen starvation growth condition. Results of this research can be applied to obtain maximal production of microalgal lipids by appropriate photobioreator design.
Acidolysis of Sunflower Oil with Rhizomucor miehei Lipases Immobilized on Modified Chitosan Microspheres.
C. Palla, C. Pacheco, M.E. Carrin, PLAPIQUI (UNS-CONICET), Bahía Blanca, Buenos Aires, Argentina
Enzymatic lipid modification is of current interest to scientists and industrials, particularly to obtain trans-free fats and oils. An extra advantage is obtained when immobilized enzymes are used. Nowadays different lipases are commercially available immobilized on different supports but economical or operational problems could happen with them. The aim of this work was to obtain a proper biocatalyst, cheaper and difficult to be broken, in order to use it, in the future, into a fix-bed reactor. Chitosan microspheres were prepared for using as immobilization support. The hydrophilic nature of them was modified to a more hydrophobic one by reductive amination, and lipases from Rhizomucor miehei were adsorbed onto them by physical adsorption. Different variables in the immobilization protocol were analyzed: an initial soak of the support with ethanol or phosphate buffer and the addition or not of chilled acetone at the end of the process. The catalytic behavior of the obtained enzyme derivatives was evaluated in the acidolysis of sunflower oil with a mixture of palmitic—stearic acids at standardized conditions. The best results were obtained when the immobilization was performed using buffer media alone, achieving palmitic and stearic acid incorporations of 274.4 % and 320.7 %, respectively.
Unmasking the Molecular Libraries of the Trypanosoma cruzi Sterol Metabolome.
C.R. Nes1, W.D. Nes1, F. Villalta2, Y. Kleshchenko2, 1Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA, 2Department of Microbial Pathogenesis and Immune Response, Meharry Medical College, Nashville, TN, USA
In order to devise methods to control the rate-limiting steps in sterol biosynthesis that affect phytosterol homeostasis in Trypanosoma cruzi (TC), we have analyzed the sterol composition of control and inhibitor-treated TC cultures by GC-MS, UV and NMR. Eight sterols were detected in epimastogotes, including 24-alkyl sterols with one or two methyl groups attached to C24 that formed the end products. Cells treated with 25-azalanosterol (IC50 = 100 nM) accumulated zymosterol with a corresponding loss in 24-alkyl sterol production. The cloned TC sterol methyltransferase (SMT) was shown to favor zymosterol over lanosterol as the optimal substrate. Product profiling of the TCSMT catalyzed reaction assayed with zymosterol revealed that the catalyst can form multiple products. The formation of 24-alkyl sterols as end products and the significant potency of inhibition of growth by 25-azalanosterol and coordinate accumulation of zymosterol (natural substrate of the SMT) indicated that the SMT is a crucial enzyme in sterol biosynthesis, and therefore, can be targeted in rational drug design.
Probing Hydrogen-bonding in Sterol Catalysis.
A. Howard, W.D. Nes, K.S. Ganatra, C.A. Nwogwugwu, E. Collins, K. Brooks, B. Rosales, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
A group of enzymes known as sterol methyltransferases (SMT), which fail to share amino acid sequence homology with other sterol biosynthetic enzymes but share similar substrate requirements, are currently being studied in our laboratory as model sterol catalysts. We hypothesized that the ground-state hydrogen-bonding strength of the bound intermediate depends upon the kinetic specificity of the C3-hydroxyl feature recognized by the SMT. Lanosterol (3Î²-OH) and five analogs containing a modified C3 group of oxo, methyl ether, amino, fluorine or hydrogen were assayed with a cloned Paracoccidiodes brasiliensis (PB) enzyme that converts lanosterol to euboricol. Catalytic competence as measured in steady-state kinetics and product profiling by GC-MS of the six test compounds (Nes, W. D. et. al., J. Biol. Chem. 277, 42549 (2002), afforded similar affinity constants and product conversions (formation of 24(28)-methylene side chain) yet differed as follows (relative activity; Vmax/Km): 3Î²-OH (100%; 31 pmol/min/mg/21 ÂµM = 100%) > 3-oxo (48%) > 3b-amino (33%) = 3-OMe (31%) > 3F (17%) > 3H (not bound productively). The results indicate that the C3-hydroxyl group of the natural substrate is bound to a contact amino acid in the active site as a hydrogen acceptor and that this substrate-enzyme interaction contributes to the potential transition state stabilizations in the activated complex utilized in the bond-making and breaking steps.
Manipulation of Fungal Sterol Biosynthesis to Form Substrates and Inhibitors.
S. Chamala, D. Carpenter, A. Shiva, W.D. Nes, Department of Chemistry and Biochemistry Texas Tech University, Lubbock, TX, USA
As an approach to generate difficult to obtain and non-commercially available sterol substrates for enzyme assay, fungal cultures were manipulated to form them. In the first trials, Candida albicans was cultured with 25-azalanosterol (AL) in the presence and absence of itraconazole (IC), sterol biosynthesis inhibitors that block the sterol methyltransferase and sterol demethylase activities involved with the C24- methylation and C32-demethylation reactions, respectively; AL plus IC treatment led to the accumulation of 31-norlanosterol and 14-methylzymosterol and IC treatment led to formation of obtusifoliol. These sterols replaced the normal end product ergosterol in stoichiometric fashion. Secondly, using the sterol auxotroph GL7 that was sterol adapted, 26, 27-difluorolanosterol, a potential suicide substrate, was converted to 26, 27-difluorocholesta-5, 7-dienol in high yield. Additional examples of novel compounds formed using these cultures techniques will be presented. Confirmation of sterol structure is based on GC-MS, UV and NMR analyses.
Candida Sterol Methyltransferase: Properties and Inhibition.
Kulothungan Ganapathy, Ragu Kanagasabai, W.D. Nes, Texas Tech University, Lubbock,Texas,USA
The S-adenosyl-L-methionine: Δ24-sterol methyltransferase (SMT) from Candida albicans has been solubilized with polyoxyethylene 10 tridecyl ether and kinetic constants determined. Zymosterol was found to be the optimal substrate over lanosterol for the microsome-bound enzyme, yielding an apparent Km of 50 µM and Vmax of 35 pmol/min/mg total protein. The apparent molecular weight of the SMT enzyme is estimated to be 178,000 Da as measured by gel permeation chromatography. Assay of the transition state analogs of the C-methylation reaction, 24(R,S),25-epiminolanosterol and 25-azalanosterol inhibited the activity of the enzyme in a non-competitive manner relative to zymosterol generating Ki values of 11 nM and 54 nM, respectively. In addition, the product analogues ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited SMT activity affording Ki values of 20 µM and 72 µM, respectively. The suicide substrate-26,27 dehydrozymosterol (DHZ) inactivated the enzyme generating Ki and Kinact values of 9 µM and 0.032 min-1.
Synthesis of Substrate Analogs for Study in Sterol Biosynthesis.
Gamal A.E. Aly, Jialin Liu, W. David Nes, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
Sterol biosynthesis in parasites proceeds from the first tetrayclic intermediate lanosterol formed by the cyclization of squalene oxide. In order to probe sterol catalysis of isolated enzymes, perturb flux in these parasites and provide substrate analogs of lanosterol a series of modified lanosterols have been constructed. The following replacements at C3 have been made: 3β-OH to 3α-OH, 3α-fluorine, 3-acetate, 3β-amino, 3β-amino salt, 3β-fluorine, 3β-dlfluoro, 3-Omethyl, 3-oxo, and 3-hydrogen. The preparative routes, yields and characterization of the products by GC-MS and NMR are described.