BIO 1: Sterols I
Chair(s): W.D. Nes, Texas Technical University, USA; and G. Lepesheva, Vanderbilt University, USA
Biochemical and Biophysical Studies of Cytochrome P450 46A1, the Principal Cholesterol Hydroxylase in the Brain. I. Pikuleva, University of Texas Medical Branch, Galveston, TX, USA
By converting cholesterol to 24S-hydroxycholesterol, cytochrome P450 46A1 (CYP46A1) initiates the major pathway for cholesterol removal from the brain. This laboratory has pioneered biochemical characterization of CYP46A1 followed by determination of the enzyme's two crystal structures. First is the 1.9-Å structure of CYP46A1 complexed with a high affinity substrate cholesterol sulfate. The second structure is that of the substrate-free CYP46A1 at 2.4 Å resolution. The structures provided important information about cholesterol binding and catalysis and prompted evaluation of CYP46A1 as a drug target. A number of compounds, some naturally occurring steroids and some marketed drugs, were found to alter (inhibit or stimulate) the enzyme's activity in vitro. The data obtained may aid in design of new therapeutic agents and more selective drugs.
Mechanisms Regulating Substrate Delivery in Adrenocortical Steroid Hormone Biosynthesis. D. Li, M.B. Sewer, Georgia Institute of Technology, Atlanta, GA, USA
In the adrenal cortex, cortisol is synthesized in response to signaling cascades initiated by the trophic peptide hormone adrenocorticotropin (ACTH). The mechanisms by which ACTH directs cortisol production are multifaceted and include controlling the transcription steroidogenic genes, regulating cholesterol (substrate) uptake and transport, modulating steroidogenic enzyme activity, and controlling electron availability. Moreover, steroidogenesis is a multi-step process that occurs in two organelles, the endoplasmic reticulum (ER) and the mitochondrion. However, the precise mechanism by which substrates are delivered back and forth between these two organelles is unclear. Thus, we examined the mechanism by which ACTH directs inter-organelle substrate delivery. We show that ACTH promotes the rapid increase in the rate of mitochondrial movement. Increased mitochondrial trafficking and cortisol production require microtubule polymerization. ACTH signaling increases the phosphorylation of the small GTPase RhoA. Finally, interaction between RhoA and the Rho effector mDia1 are essential for mitochondrial movement and cortisol secretion. These studies identify a role for RhoA and mDia1 in directing the trafficking of mitochondria during ACTH-stimulated cortisol biosynthesis in the human adrenal cortex.
Cytochrome P19: Sex, Fat and Cancer. E. Simpson, Prince Henry's Institute of Medical Research, Clayton Vic, Australia
Cytochrome P450-Dependent Steroid Hydroxylases. R. Bernhardt, Universität des Saarlandes, Saarbrücken, Germany
Steroids play an important role as hormones in mammals. In addition to this, many steroids are interesting pharmaceutical target substances for the production of different types of drugs. CYP106A2 from Bacillus megaterium ATCC 13368 is one of the few known bacterial steroid converting cytochromes P450 and hydroxylates many 3-oxo-delta-4-steroids mainly in 15beta-position. Here we report on the creation of mutants of this enzyme with improved activity and changed selectivity of hydroxylation. Basing on a computer model, amino acids in the putative substrate recognition site 6 of CYP106A2 were converted to the corresponding ones found in the human steroid 11beta-hydroxylase CYP11B1 (S394I, A395L, T396R, G397P and Q398S). The selectivity of hydroxylation was shifted from the 15 to the 11-position. Mutations that were not associated with substrate recognition in the model did not change catalytic activity whereas mutations predicted by the model to participate in active site formation, dramatically changed catalytic activity and region-specificity. To check mutants produced by directed evolution, a whole-cell screening system, where Adx and AdR were co-expressed in E. coli, has been developed. We were able to select mutants with considerably improved activity and with changed selectivity of hydroxylation.
Sterol Biosynthesis, Ancient Cytochrome P450 Activities and Drug Action. S.L. Kelly, D.C. Lamb, D.E. Kelly, Swansea University, Swansea, Wales, UK
The presentation will focus on the role of CYP51 in sterol biosynthesis and as a target of drugs in fungal ergosterol biosynthesis. As an ancestral activity in the cytochrome P450 superfamily this form deserves special attention and the state of understanding of CYP51 forms, orthologues and homologues with different functions and mutant azole resistant proteins will be reviewed together with presentation of recent data.
CYP701A: A Family of Multifunctional Cytochromes P450 in Plant Diterpenoid Natural Products Biosynthesis. D. Morrone, R. Peters, Iowa State University, Ames, IA, USA
Members of the CYP701A sub-family of cytochromes P450 are typically kaurene oxidases involved in biosynthesis of the diterpenoid gibberellin plant growth hormones. These P450s catalyze three consecutive reactions to convert an extra-cyclic methyl from its tetracyclic olefin substrate, kaurene, into the carboxylic acid group that defines its final product, kaurenoic acid. A major focus of our work is elucidation of the mechanistic features underlying the multiply reactive nature of this enzyme-substrate pair. In addition, we are also interested in elucidating the varied metabolic functions of the five members of the CYP701A family found in rice. Our preliminary evidence indicates that at least some of the rice CYP701A family members are involved in biosynthesis of additional gibberellin related diterpenoids that serve as natural antibiotics against fungal infection in rice (and potentially other cereal crop plants). This suggests that the closely related, yet functionally distinct, P450s from the CYP701A sub-family will provide an ideal model system for enzymatic structure-function studies.
Reconstructing the Biosynthetic History of Divergent Plant Sesquiterpene Synthases. J.P. Noel, The Salk Institute for Biological Studies, La Jolla, CA, USA
CYP51, A Drug Target Enzyme in the Cytochrome P450 Superfamily. G.I. Lepesheva1, W.D. Nes2, F. Villalta3, M.R. Waterman1, 1Vanderbilt University, Nashville, TN, USA, 2Texas Tech University, Lubbock, TX, USA, 3Meharry Medical College, Nashville, TN, USA
The cytochrome P540 superfamily currently consists of about 8,000 proteins joined into 866 families. The enzymes catalyze monooxygenation of a vast variety of compounds and yet play essentially two roles in living species. They provide biodefense (detoxification of xenobiotics, antibiotic production) and/or participate in biosynthesis of important endogenous molecules, particularly lipids. Based on these two roles, sterol 14-alpha demethylases (CYP51) belong to the second group of P450s. The family, however, is very special as its members, being found in all biological kingdoms preserve strict functional conservation. Being regarded as the possible ancestor to all eukaryotic P450s, the CYP51 family can serve as an excellent subject for fundamental research on P450 evolution. Its practical importance is based on the fact that while vertebra can accumulate cholesterol from the diet, blocking of sterol production in fungi is lethal. CYP51 inhibitors are currently amongst the most widely used clinical and agricultural antifungals. We have recently shown that specific inhibition of protozoan CYP51 can provide treatment for human trypanosomaises. The sets of compounds tested in vitro and in trypanosomal cells include clinical and experimental azoles, non-azole inhibitors found via high-throughput screening and substrate analogs
BIO 1.1: Biocatalysis I
Chair(s): C.T. Hou, USDA,, ARS, NCAUR, USA; and R. Patel, Bristol-Myers Squibb, USA
Chemo-Enzymatic Synthesis of Chiral Pharmaceutical Intermediates for Development of Drugs. R. Patel, Bristol-Myers Squibb, New Brunswick, NJ, USA
The production of single enantiomers of chiral intermediates has become increasingly important in the pharmaceutical industry. Biocatalysis often offers advantages over chemical synthesis as enzyme-catalyzed reactions are often highly enantioselective and regioselective. They can be carried out at ambient temperature and atmospheric pressure, thus avoiding the use of more extreme conditions which may cause problems with isomerization, racemization, epimerization of molecules. Microbial enzymes can be immobilized and reused. In addition, enzymes can be over expressed to make biocatalytic processes economically efficient. Enzymes with modified activity can be tailor-made by directed evolution to increased enzyme activity, selectivity and stability. This presentation will provide the use of enzymes for the synthesis of single enantiomers of intermediates used in the development of drugs. Synthesis of key intermediates for the antiviral, antidiabetic, anticancer, antianxiety and antialzheimers, and anticholesterol drugs will be presented.
New Uses of Bioglycerin: Production of Arachidonic Acid. C.T. Hou, National Center for Agricultural Utilization Research, ARS, USDA, Peoria, IL. USA
Filamentous fungi of the genus Mortierella are known to produce arachidonic acid from glucose and M. alpina is currently used in industrial scale production of arachidonic acid in Japan. In anticipation of a large excess of co-product bioglycerin from the national biodiesel program, we would like to find a new uses for bioglycerin. We screened twelve Mortierella species for their production of arachidonic acid (AA) and dihomo-Î³-linolenic acid (DGLA) from glycerol. We use glycerol as substrate and found that all species tested grew on glycerol and produced AA and DGLA except M. nantahalensis NRRL 5216, which could not grow on glycerol. The amount of AA and DGLA produced are comparable with those obtained with glucose-grown mycelium. The top five AA producers (mg AA/CDW) from glycerol were in the following order: M. parvispora > M. claussenii > M. alpina > M. zychae > M. minutissima. The top five dry mycelia weights productions were: M. zychae > M. epigama > M. hygrophila > M. humilis > M. minutissima. Time course studies showed that the maximum cell growth and AA production were at 6 days of incubation. Therefore, glycerol can be considered for industrial use in the production of AA and DGLA.
Engineering of Bacterial Fatty Acid Monooxygenase, Cytochrome P450 Bm-3, from Bacillus Megaterium. J. Ogawa1, A. Kudo1, Y. Yano1, V. Urlacher2, R.D. Schmid2, S. Shimizu1, 1Div. Appl. Life Sci., Grad. Sch. Agric., Kyoto University, Sakyo-ku, Kyoto, Japan, 2Institut für Technische Biochemie, Universität Stuttgart, Allmandring, Stuttgart, Germany
Cytochrome P450 BM-3 from Bacillus megaterium is a fatty acid monooxygenase. Its substrate specificity was expanded to aromatic hydrocarbons and phenolic and arylalkyl compounds by means of crystal structure-based directed mutation. For example, the single mutant (F87V) catalyzed regioselective hydroxylation at the para position of various phenolic compounds. In particular, the mutant enzyme showed high activity as to the hydroxylation of 2-(benzyloxy)phenol to 2-(benzyloxy)hydroquinone, a pharmaceutical intermediate. To enhance the productivity, P450-stabilizing/activating factors were screened in the cell-free extracts of various microorganisms. The addition of the cell-free extracts of Escherichia coli DH5α resulted in about eight-fold increase in the specific activity. One of the factors was isolated and identified as iron-containing superoxide dismutase. Addition of superoxide dismutase to the P450 BM-3 reaction mixture greatly increased NADPH-consumption rate and slightly increased coupling efficiency and resulted in acceleration of the reaction rate. This effect was observed with various P450 BM-3 mutant enzymes. 2-(Benzyloxy)phenol conversion to 2-(benzyloxy)hydroquinone catalyzed by a P450 BM-3 mutant enzyme was optimized in the presence of superoxide dismutase.
Elucidation of the Metabolic Pathways of n-Alkanes in the Microalga Prototheca sp. E. Sakuradani1, Y. Natsume1, Y. Takimura2, N. Shibata1, S. Shimizu1, 1Kyoto University, Kyoto, Japan, 2Kao Corporation, Tokyo, Japan
The achlorophyllous microalgal genus Prototheca was found to degrade hydrocarbons in 1960′s. Especially, Prototheca zopfii was shown to be a significant utilizer of crude-oil hydrocarbons as well as n-alkanes. Aliphatic hydrocarbons are assimilated by various microorganisms such as bacteria, yeasts, fungi, and algae. In general, two major degradation pathways of acyclic hydrocarbons are well-known. The terminal oxidation pathway is widely found, whereas the sub-terminal oxidation pathway is just reported in limited microorganisms. Regardless of the notable capacity in degradation of hydrocarbons, the metabolic pathway of hydrocarbons in Prototheca sp. remained unclear.To elucidate the metabolic pathway, unknown compounds derived from n-hexadecane in P. zopfii were identified. P. zopfii was cultivated in the medium containing 2% glucose, 1% yeast extract, and 4% n-hexadecane at 28°C with shaking. After cultivation, total lipids were extracted with methanol-chloroform and analyzed by GLC. Two unknown peaks were detected on the GLC chromatogram. These compounds were independently purified by HPLC and identified as 5-hexadecanol and 5-hexadecanone with GC-MS. P. zopfii was supposed to utilize n-hexadecane through the sub-terminal oxidation mainly at 5th carbon.
The Role of Diacylglycerol Acyltransferases in Castor Oil Biosynthesis. T.A. McKeon, X. He, J.T. Lin, G. Chen, USDA-ARS WRRC, Albany, CA, USA
Castor oil contains the hydroxy fatty acid ricinoleate as its major (90%) component. We have cloned and expressed the diacylglycerol acyltransferase type 1 (DGAT1) from castor (RcDGAT1) since the final step in castor oil biosynthesis appears to be a key to producing a high ricinoleate triacylglycerol fraction. The DGAT carries out the final reaction step in the biosynthesis of triacylglycerol, the principal constituent of seed oil, and is considered to be the step that controls the oil content of seeds. We also report the cloning of a gene sequence that encodes a putative castor diacylglycerol acyltransferase type 2 (DGAT2), and discuss the role of the different types of DGATs in castor oil biosynthesis. Our results suggest a comparative biochemical approach will be invaluable in elucidating the means by which castor produces such an unusual oil.
Genetic Modification of Microorganisms for Improved Production of Glycolipid Biosurfactants. D.K.Y. Solaiman, K.K. Aneja, N.W. Gunther IV, R.D. Ashby, J.A. Zerkowski, USDA/ARS/Eastern Regional Research Center, Wyndmoor, PA, USA
Sophorolipid (SL) and rhamnolipid (RL) are microbial glycolipids with surfactant properties. They are useful in applications including cleaning, cosmetic formulation, biocontrol and enhanced oil recovery. Commercial uses of SL and RL are hampered by certain physical properties (e.g., solubility) and high production costs. We have taken a genetic engineering approach to address these needs. We have cloned and sequenced the genes (rhlABPx) responsible for the biosynthesis of RL in a patented nonpathogenic bacterium. We subsequently inserted the rhlABPx genes into the genomes of P. corrugata and P. chlororaphis by using a transposon-based chromosomal integration system. Preliminary assay study did not show an increased production of RL. We subsequently investigated the use of a strong promoter to express the integrated rhlABPx. Separately, we have cloned and sequenced a gene potentially involved in the biosynthesis of SL in C. bombicola. This gene is subsequently cloned into another yeast system for high-level expression and the characterization of its gene product. The results of these studies lay important groundwork for subsequent genetic engineering of RL- and SL-producing microbes to achieve high-level production of natural or modified biosurfactants.
Enzymatic Modification of Antioxidants for Different Applications. X. Xu, The University of Aarhus, Aarhus, Denmark
The main barrier against a successful launch of foods containing omega 3 oils is the high susceptibility to lipid oxidation, which will lead to the formation of unpleasant off-flavours and reactive free radicals and aldehydes. The latter compounds have been suggested to promote the development of cardiovascular disease. Lipid oxidation can be prevented by antioxidants. Studies have shown that many of the commercial free radical scavenging antioxidants are not efficient in preventing lipid oxidation in omega-3 oil enriched foods. Moreover, the efficacy of antioxidants seems to be influenced by their localisation in the food system, which is dependent on the polarity of the antioxidants and on the emulsifiers used. Therefore, there is a need to develop new antioxidants based on natural sources but with improved physical properties. Bt this way, they can be designed tolocate where they are needed and to have the right antioxidative properties required in the particular food system (e.g. free radical scavenging and metal chelating properties). In this talk, a few antioxidants will be modified using enzymatic synthesis targeted for better antioxidative properties for omega-3 oils.
Structural Modification of Phospholipids Using Enzymes. S.H. Yoon, Korea Food Research Institute, Seongnam-Si, Kyunggi-Do, Korea
Lysophospholipids are well known to have strong emulsification activity which can be utilized in food, pharmaceutical, and cosmetic industries, etc. Lysophospholipids have also been manufactured by chemical and/or enzymatic modification of phospholipids. For the production of lysophospholipids through enzymatic modification of phospholipids using enzymes, several phospholipases and lipases were tested. Purified soybean oil and soy lecithin were used as substrates for enzyme reaction. Lecitase (porcine pancreatic phospholipase A2) showed no activity on soybean oil hydrolysis, whereas other free and immobilized lipases revealed various degrees of activity. When soy lecithin was used as substrate, Lecitase showed the activity of 19.7 unit at pH 8, while 4 lipases showed substantial activities on lecithin. Maximum activity was shown at around 50°C and pH 7-9. Maximum velocity and Km value of Lecitase were determined through kinetic analysis as 21.88mmole and 89.29mmole, respectively. Optimum concentrations of sodium deoxycholate and calcium ion, which are considered critical for Lecitase and lipases activities, were also determined. In conclusion, lipase showed significant hydrolytic activity on phospholipids, however, no direct relationships were observed between enzyme activities on soybean oil and soy lecithin.
Preparation of Novel Phospholipids via Phospholipase D-Mediated Transphosphatidylation. M. Hosokawa, Y. Yamamoto, K. Miyashita, Hokkaido University, Hakodate, Hokkaido, Japan
Phospholipase D (PLD) is a lipolytic enzyme that hydrolyzes the terminal phosphodiester bond in phospholipid molecule. Due to an ability to transfer the phosphatidyl moiety of glycerophospholipids to alcohols, it is also used to synthesize phospholipids with desired head groups. On the other hand, terpene and phenolic compounds are known to show pharmacological effects such as anti-inflammatory and anti-cancer activities. To extend utilization of terpene and phenolic compounds in various fields, PLD-mediated transphosphatidylation was conducted in an aqueous system. At the optimum conditions determined in this study, the yield of phosphatidyl-geraniol, phosphatidy-lnerol and phosphatidyl-perillyl alcohol from soy PC and substrate terpenes were 90 mol%, 91 mol% and 78 mol%. The synthesis of phosphatidyl-tyrosol was also observed by NMR and MS analysis.
HPLC Analysis of Composition of Phosphatidylinositol Isomers Synthesized by Mutant Phospholipase D. Y. Iwasaki, A. Masayama, K. Tsukada, H. Nakano, Nagoya University, Nagoya, Japan
Phospholipase D (PLD) is an enzyme that hydrolyzes phospholipids to phosphatidic acid and the corresponding alcohols. PLD also catalyzes transphosphatidylation by which various phospholipids can be synthesized. Synthesis of phosphatidylinositol (PI) by PLD was difficult in the past, due to the enzyme's substrate specificity. But we have recently succeeded the synthesis of PI by using mutated PLD with altered substrate specificity. Myo-inositol has six hydroxy groups, which are not chemically equivalent. Therefore, in the enzymatically synthesized PI, six isomers (1-, 2-, 3-, 4-, 5- and 6-PI) are possible. The purpose of this study is to establish an HPLC method for separation of PI isomers, and to analyze the composition of PI isomers synthesized by the mutant PLD enzymes. An HPLC method was established by using standard PI isomers, which were chemically synthesized from protected myo-inositols. Analysis of the enzymatically synthesized PIs using the HPLC method revealed that mutant enzymes showed regio-preference on certain positions of the inositol ring, depending on the mutations. Among the 90 mutant PLDs investigated, some mutants generated exclusively 1(3)-PI, which was equivalent to natural-type PI isomer. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN).
PHO 1 / BIO 1.2: Structured Lipids
Chair(s): X. Xu, University of Aarhus, Denmark; and M. Ahmad, Jina Pharmaceuticals Inc., USA
Scope and Limitations of Enzymatic Synthesis of Structured Glycerolipids. Patrick Adlercreutz, Lund University, Lund, Sweden
Nutritional and/or physico chemical properties of lipids can be tailored by the synthesis of structured lipids. Due to their selectivity enzymes are excellent tools in the preparation of such lipids. In this presentation enzymatic synthesis of structured glycerolipids will be reviewed. In the enzymatic steps either lipases or phospholipases are used as catalysts. Water activity, solvent, equilibrium constants and fatty acid selectivity of the enzymes are some factors of importance for the reaction rate and yield in the reactions. The scope and limitations of the enzymatic conversions will be presented and chemoenzymatic synthesis routes will be discussed. In the quality control of structured lipids, analysis of molecular species is important. Recent advances in analysis of molecular species of phospholipids and other lipids will be reviewed.
Solubilization of Nutraueuticals into Typical and Low Viscosity Reversed Hexagonal Mesophases. Nissim Garti, Idit Amar-Yuli, Casali Institute of Applied Chemistry, Institute of Chemistry, Faculty of Science, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
This study reports on the formation of a low-viscosity HII mesophase at room temperature upon addition of Transcutol (diethylene glycol mono ethyl ether) or ethanol to the ternary mixture of GMO (glycerol monooleate)/TAG (tricaprylin)/water. The microstructure and bulk properties were characterized in comparison with those of the low viscosity HII mesophase formed in the ternary GMO/TAG/water mixtures at elevated temperatures (35-40°C).The typical and two low-viscosity reverse hexagonal systems were examined as solubilization reservoirs for four bioactive molecules with different polarity.The bioactive guest molecules include: a) Ascorbic acid - an hydrophilic molecule, which additionally to its well-defined antioxidation properties, it can support the myoelectrical activity of the guts, b) A lipophilic derivative of ascorbic acid known as ascorbyl-6-palmitate, c-d) The hydrophobic D-α-tocopherol and D-α-tocopherol acetate that are known to prevent free radical damage in polyunsaturated fatty acids. The nature of the guest molecule, the phase behavior, swelling or structural transformations and its location in the different HII structures (typical and fluids) were explored using a combination of polarized light microscopy, DSC, X-ray diffraction and ATR FTIR spectroscopy.
Trans-Free Margarine Prepared with Canola Oil/Palm Stearin/Palm Kernel Oil-Based Structured Lipids. B.H. Kim, S.E. Lumor, C.C. Akoh, University of Georgia, USA
Trans-free margarines were prepared with structured lipids (SLs) as a healthy substitute for traditional margarine made with partially hydrogenated vegetable oils. Their fatty acid (FA) profiles and physical and textural properties were compared to those of a traditional margarine. Six kinds of SLs were produced by Lipozyme TL IM-catalyzed transesterification of oil blends of canola oil (CO), palm stearin (PS), and palm kernel oil (PKO), based on the weight ratios (CO:PS:PKO) of 40:60:0, 40:50:10, 40:40:20, 40:30:30, 50:30:20, and 60:25:15, respectively, in a stirred tank batch reactor. No trans FAs were detected in all SLs. SLs prepared from the oil blends with higher content of PKO tended to have more desirable crystal forms (β′) for margarine formulation but show higher contents of hypercholesterolemic FAs (lauric and myristic). All SLs displayed different melting and crystallization behaviors and solid fat contents from the traditional margarine fat. However, the margarines made with SLs prepared from the 50:30:20 and 60:25:15 blends have similar textural properties, such as hardness, adhesiveness, or cohesiveness to the traditional margarine. Based on properties and FA profiles, SLs prepared from the 50:30:20 or 60:25:15 blends were considered most suitable for the preparation of trans-free margarine.
Oral Delivery of Nutraceuticals with Lecithin-Based Microemulsions. E. Acosta, J. Chu, S. Yuan, University of Toronto, Toronto, Ontario, Canada
In this work, lecithin-based microemulsions for oral delivery of cholesterol reducing agents obtained from plant extracts is reported. The formulation of these microemulsions is inspired on the combined linker concept, which involves a lipophilic and a hydrophilic amphiphile accompanying the lecithin phospholipids. GRAS linkers were chosen for this application. The phase behavior studies were carried out using simulated intestinal fluids in order to produce a formulation preconcentrate containing lecithin, linkers, and the active ingredient. Such preconcentrate mixture was used as a self-microemulsifying drug delivery system (SMEDDS). To test its performance, the preconcentrate was dissolved in simulated intestinal fluid, then neutralized with simulated pancreatic release, and finally contacted with pig intestine tissue to determine the kinetics of uptake. The results from the phase behavior studies along with the dissolution/absorption tests are presented and discussed.
Solvent, Catalyst or Surfactant? Ionic Liquids may Reshape Enzymatic Reaction System. Zheng Guo, Xuebing Xu, BioCentrum-DTU, Technical University of Denmark, Lyngby, Copenhagen, Denmark
Emergence of Ionic Liquids (ILs) not only is capable of resolving those reactions that remained unsolved or even impossible in conventional solvent, but also show some interesting and different behaviors. This work presents our researches on ILs-mediated glycerolysis of oils and fats and new progress in enzymatic esterification of some physiologically active componds, e.g. flavonoids, Vc. The results showed that, unlike conventional solvents, only small amount of ILs can significantly increase the productivity and the yield of desired products, which remained to be a big challenge in conventional solvents due to lower solubilities. This raised a question: what is the role of the ILs played in this enzymatic reaction system? This work, therefore, also reports our efforts to clarify this question using model (COSMO-RS) and other characterizations. Part of the results has been published in Chen, et al., Bioeng. Biotechnol, 2007, OI 10.1002/bit. 21520 in press; Guo, et al., Green Chem, 2007, DOI:10.1039/b709786g, in press; Guo, et al., Inform, 2007, 18 (2), 78-82. 2-3 papers are in preparation to further address this issue.
Effects of Structured Lipids Containing Linoleic Acid or EPA or DHA and Medium-Chain Fatty Acids on Lipid Profiles in Rats. Junichi Nagata1, Michio Kasai2, Satoshi Negishi2, Toshiaki Aoyama2, Kazuhiko Yamada1, Morio Saito1,3, 1National Institute of Health and Nutrition, Tokyo, Japan, 2The Nisshin OilliO Group, Ltd., Kanagawa, Japan, 3Research & Development Institute, MIKI Corporation, Hyougo, Japan
To examine the effects of some kinds of structured lipids (SLs) on lipid profiles in rats, we prepared the SLs containing linoleic or eicosapentaenoic or docosahexaenoic acid and caprylic acid. Male 4-wk-old Wistar rats were fed the experimental diets containing SLs for 4 weeks. In the case of SLs containing linoleic acid and medium-chain fatty acids, serum cholesterol levels were significantly lower in the L8L, 10L10 and L10L groups than control group. Serum TG levels were significantly lower in rats fed LML types than those in the other groups. Serum b-hydroxybutylate levels were significantly higher in rats fed MLM types than those of the other groups. Whereas, in the case of SLs containing EPA or DHA and caprylic acid, the serum lipid concentrations of rats fed SLs were significantly lower than those of the control diet. The liver TG concentration in rats fed E-8-8 diet was significantly lower than that of soybean oil group. These results indicate that the feeding of highly purified LML types could improve effectively on serum and liver lipid profiles undre various dietary conditions.
Synthesis of Oleoyl Triglycerol Products Containing DHA at Alpha or Beta Positions by Lipase-Catalyzed Reactions. H. Zhang1, C. Wijesundera2, X. Xu1, 1BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark, 2CSIRO Food Futures National Research Flagship and Food Science Australia, Werribee, Victoria 3030, Australia
The potential routes for the lipase catalyzed production of 1,2- oleoyl 3-docosahexaenoyl (OOD) and 1,3-oleoyl 2-docosahexaenoyl (ODO) were studied. OOD was produced with triolein and docosahexaenoyl/eicosapentaeonyl ethyl ester (DHA/EPA-EE) with 70% of yield. However, for the production of ODO, three pathways were tried. First, the production of 1,3-diolein by the reaction of glycerol and oleic acid followed by incorporation of DHA/EPA-EE at sn-2 position was studied. Second, the direct acidolysis was performed between single cell oil (SCO) and oleic acid (1/12, mole/mole) in both solvent and solvent-free systems. Finally, ethanolysis of SCO to produce DHA monoacylglycerol followed by esterification with oleic acid or ethyl oleate was investigated. Their yields and lipase specificity related to the reaction systems were discussed.
Application of Structured Lipids. C. Jacobsen, M . Timm-Heinrich, N.S. Nielsen, Danish Institute for Fisheries Research, Technical University of Denmark, Kgs. Lyngby, Denmark
During the last decade there has been an increasing interest in the use of enzyme technology to produce lipids with a specific structure and specific fatty acid composition for nutritional/clinical purposes. If such structured lipids are to be successfully employed in foods the sensory quality of these lipids has to be similar to or better than the sensory quality of traditional fats. Moreover, the shelf life of foods enriched with these lipids must also be acceptable. Our research has shown that the enzymatic interesterification process as well as the process to purify the structured lipid may significantly reduce the quality of structured lipids depending on the fatty acid composition of the lipids. The quality of the structured lipids may affect the oxidative stability of food products enriched with these lipids. Therefore, special precautions need to be taken during production and purification of structured lipids and it may also be necessary to add antioxidants to the structured lipids during their production or to foods containing such lipids. The presentation will review the current knowledge within this area.
Enzymatic Production of Ceramide from Sphingomyelin. L. Zhang, L.I. Hellgren, X. Xu, BioCentrum-Technical University of Denmark, Lyngby, Denmark
Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide has great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. Chemical synthesis of ceramide is a costly and time consuming process, and development of alternative cost-efficient, high yield production methods is of great interest. The potential of producing ceramide through the enzymatic hydrolysis of sphingomyelin (SM) has been studied. SM is a ubiquitous membrane lipid and enriched in dairy products or by-products.SM hydrolysis has been improved by system evaluation and the optimization of several factors. The reaction is more efficient in two-phase (water: organic solvent) system than in one-phase (water-saturated organic solvent) system. Phospholipase C from C. perfringens is the selected enzyme which has the most advantageous properties. For reusing the enzyme, enzyme immobilization and its properties after immobilization have been studied. Nine carriers have been tested, and the enzyme immobilized on Lewatit (Bayer AG) has the highest catalytic activity. After seven cycles, immobilized enzyme retains around 70% of its initial activity. The hydrolysis catalyzed by both the soluble and immobilized enzyme follow Michaelis-Menten kinetics. Ceramide as product activate the reaction. The results from a membrane reactor study will also be presented.
Physical and Nutritional Properties of Some Structured Soy Phospholipids. D.K. Bhattacharyya, Bengal Engineering & Science University, India
The present study is concerned with evaluation of physical properties such as the surface active properties of some structured soy phospholipids, prepared by modifying the fatty acid composition of soy phospholipids by enzymatic interesterification reaction catalysed by lipase.Soy phospholipids have been modified by incorporating acetic acid, hydroxy oleic, hydroxystearic and epoxy oleic and dibasic acids like adipic and sebacic, by interestification reaction with TLIM as biocatalyst. The surface-active properties such as interfacial tension, critical micelle concentrations and free energy of micellization of original soy phospholipids and structured soy phospholipids exhibit distinct changes. Soy phospholipids containing Capric acid (C10: O) and Eicosapentaenoic acid (EPA, C20:5) have been also prepared by lipase catalysed interesterification reaction.Nutritional properties of these two structured soy phospholipids, when evaluated in comparison with original soy phospholipids as dietary components along with dietary soybean oil in growing rats, reveal that soy phospholipids with C10:O or C20:5 as constituent fatty acids are significantly more hypolipidemic in nature. The content of total cholesterol, LDL-and VLDL cholesterol and triacylglycerol decreases in serum of rats while increasing HDL level pronouncedly when ingested at a particular dosage in dietary soybean oil.
BIO 2: Sterols II
Chair(s): R. Moreau, USDA, ARS, ERRC, USA; T. Bach, University Louis Pasteur, France; and J. Zawistowski, Forbes Medi-Tech Inc., Canada
A Breakthrough in Isoprenoid Biochemistry: Discovery and Elucidation of the Mevalonate-Independent Methylerythritol Phosphate Pathway for the Formation of Isoprene Units. M. Rohmer, Université Louis Pasteur / CNRS, Institut de Chimie, Blaise Pascal, Strasbourg Cedex, France
Incorporation of 13C labelled precursors into the bacterial triterpenoids of the hopane series and into isoprenoids from plant plastids disclosed a novel biosynthetic pathway towards isoprene units, representing an alternative to the classical mevalonate pathway. Complete elucidation required labelling experiments and molecular biology techniques, including gene identification and characterization of the enzymes.Starting from pyruvate and glyceraldehyde phosphate, isopentenyl diphosphate and dimethylallyl diphosphate, the universal isoprenoid precursors, are synthesized via deoxyxylulose phosphate, four methylerythritol derivatives and 2-methylbutene-1,4-diol diphosphate. Incorporation of deuterium labelled precursors threw light on the stereochemical course of the last two reactions catalysed by Fe/S cluster enzymes. This pathway occurs in most eubacteria, in the Plasmodium spp. responsible for malaria and in the plastids of higher plants, where it responsible for the formation of hemi-, mono- and diterpenes, carotenoids and the prenyl chain of plastoquinone. Striking aspects of the elucidation of this long overlooked biosynthetic route will be discussed.
Dissecting the Sterol C4-Demethylation Complex in Plants. A. Rahier1, S. Darnet1, F. Bouvier1, B. Camara1, M. Bard2, 1Institut de Biologie Moléculaire des Plantes,, Strasbourg, France, 2Indiana Purdue University, Indianapolis, Indiana, USA
In plants, the sterol molecule becomes functional only after removal of the two methyl groups at C4. The process of C4-demethylation is complex and involves a sterol-C4 methyl oxidase (SMO), a 3β-hydroxysteroid dehydrogenase/C4 decarboxylase (3βHSD/D) and a 3-ketosteroid reductase. Two distinct families of SMO isoforms termed SMO1 and SMO2, and two 3βHSD/D isoforms have been identified and the corresponding cDNAs cloned from Arabidopsis thaliana. We report in vivo functional expression in the relevant yeast deletion mutants, in vitro enzymological characterization of the recombinant isoenzymes and in planta down regulation by VIGS. Homology modeling of the three-dimensional structure of 3βHSD/D was carried out, and validity of the model was tested biochemically by mutations studies. It allowed deciphering of the catalytical residues and mechanism of this unique decarboxylating HSD which constitutes the first HSD genes of the SDR superfamily to be molecularly and enzymatically characterized in plants.The present combination of molecular, biological chemical and structural approaches allowed a thorough functional characterization of SMO and 3βHSD/D as two further steps in the completion of the molecular inventory of sterols synthesis in higher plants. It provides important clues for the study of the physiological roles of C4-substituted sterols in photosynthetic eukaryotes.
Cholesterol Hardens Arteries by Quashing TGF-β Signaling. J.S. Huang, Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, USA
TGF-β is a protective cytokine for atherogenesis. Increasing evidence indicates that suppression of TGF-β responsiveness in vascular wall cells leads to atherosclerosis. Recent studies have revealed that TGF-β responsiveness is determined by TGF-β partitioning between lipid raft-and non-lipid raft-mediated endocytosis pathways. We hypothesized that cholesterol suppresses TGF-β responsiveness by increasing formation of, or stabilizing, lipid rafts. We have shown that cholesterol, alone or complexed in lipoproteins (LDL and VLDL), increases accumulation of TGF-β receptors in lipid rafts. This leads to rapid degradation of TGF-β and suppression of TGF-β signaling and responsiveness in all cell types studied. Among the cholesterol derivatives and analogs studied, cholesterol is the most potent agent for suppressing TGF-β responsiveness. Conversely, cholesterol-lowering agents and cholesterol-depleting agents, and HDL increase accumulation of TGF-β receptors in non-lipid raft microdomains. This results in enhanced endosomal signaling and TGF-β responsiveness in these cells. These cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE-null mice with hypercholesterolemia. These results indicate that cholesterol initiates and/or facilitates atherogenesis, at least in part, by suppressing TGF-β responsiveness in vascular wall cells.
Plant Sterols and Cholesterol Lowering: Future Opportunities as Functional Food Ingredients. P.J.H. Jones, Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Manitoba, Canada
Clinical trials attest to plant sterols/stanols lowering cholesterol absorption by 20-40%, an extent beyond which cholesterogenesis can compensate to restore normal circulatory cholesterol levels. As such, meta- analyses show that consumption of 2 g/d of plant sterols/stanols effectively lowers LDL-C by 8-12%. Future opportunities for plant sterols include combination intervention with complementary bioactives such as fibers. Data demonstrate additive actions of plant sterols and fermentable fibers such as glucomannans which may act simultaneously in blunting cholesterol absorption and synthesis. Alternatively, additional agents esterified to the hydroxyl group of plant sterols or stanols can provide additional health benefit. For instance, esterification of long chain omega-3 fatty acids to plant sterols results in demonstrated reductions in triacylglycerol (TAG), as well as LDL-C, levels in humans consuming this novel sterol ester. When plant stanols were esterified as a disodium-ascorbyl-sitostanol-phosphate (DASP), marked reductions in TAG and LDL-C were observed in animal models, well beyond those seen with free stanols. Moreover, DASP administration also reduced fat absorption resulting in weight loss. In summary, future opportunities lie in both combination approaches and creation of novel sterol/stanol esters.
Serum Sterols in Patients with Different Types of Liver Failure Before and After Liver Transplantation. M.J. Nissinen1, K. Nikkilä2, H. Gylling3, H. Isoniemi2, T.A. Miettinen4, 1Department of Medicine, Division of Gastroenterology, University of Helsinki, Helsinki, Finland, 2Transplantation and Liver Surgery Clinic, Helsinki University Hospital, Helsinki, Finland, 3Department of Clinical Nutrition, University of Kuopio and Kuopio University Hospital, Kuopio, Finland, 4Department of Medicine, Division of Internal Medicine, University of Helsinki, Helsinki, Finland
Liver diseases modify sterol metabolism. Liver transplantation (LTX) provides a model to evaluate impact of disease-affected liver itself on this respect. We studied serum non-cholesterol sterols with GLC in consecutive patients with acute liver failure (ALF, n=39) and end-stage primary biliary cirrhosis (PBC, n=67) before and 27 d after LTX. Generally, of non-cholesterol sterols lathosterol/cholesterol ratio is surrogate marker of cholesterol synthesis, whereas ratios of cholestanol, sitosterol and campesterol are markers of dietary cholesterol absorption. Before LTX, cholesterol concentration and ratios of lathosterol and cholestanol were 18% - 41% lower in ALF than in PBC, but those of phytosterols were equal. Interrelation of surrogate sterols showed that homeostasis of cholesterol metabolism prevailed in lowest cholestanol tertile of ALF patients before LTX. After LTX, cholesterol levels and lathosterol ratios increased in both groups and phytosterol ratios decreased. Cholestanol decreased profoundly in PBC, but it remained 26% higher than in ALF. In conclusion, non-cholesterol sterols depicted differences in cholesterol metabolism in two different types of liver disease, and its changes before and after LTX. Cholesterol synthesis was extremely low in ALF, cholestanol values were high in PBC, whereas phytosterol metabolism was equal in study groups.
Effects of Plant Sterol/Stanol Ester Enriched Foods on Macular Pigmentation and Serum TAG Concentrations. J. Plat1, T.T.J.M. Berendschot2, A. de Jong1, M. R.P.1, 1Maastricht University, Maastricht, The Netherlands, 2University Eye Clinic Maastricht, Maastricht, The Netherlands
Foods enriched with plant stanol esters lower serum LDL-C. In a recent meta-analysis, we found that in subjects with high baseline TAG concentrations, plant stanol esters may lower serum TAG. An additional placebo-controlled study with a plant stanol ester yogurt drink (2.0 g/d) indeed showed a 28% reduction in serum TAG concentrations (P=0.044) in metabolic syndrome subjects. This, plus the 12% reduction in non-HDL cholesterol (P=0.012) illustrates their value in CHD risk-management. Plant sterol and stanol esters may lower serum carotenoid concentrations. So far no long-term implications of these carotenoid reductions have been monitored. Epidemiology has suggested that low carotenoid blood levels may increase the risk of age related macular degeneration. Therefore in a 18 month placebo controlled study we evaluated the effects of plant stanol/sterol esters (2.5 g/d) on serum lutein and ?-carotene concentrations iand macular pigment optical density (MPOD). By ANCOVA (taking baseline carotenoid concentrations into account), we found that although there was a significant contribution of diet to the reduction in cholesterol standardized serum ?-carotene and lutein concentrations, there were no changes in the MPOD between the 3 groups. This illustrates that the observed carotenoid reduction during 18 months consumption of these functional foods does not affect MPOD.
Clinical Evidence for the Cholesterol-Lowering Efficacy of Plant Sterol-Enriched Foods and New Insights into Their Mechanism of Action. E.A. Trautwein, Unilever Food and Health Research Institute, Vlaardingen, The Netherlands
A vast number of human studies have shown the LDL-cholesterol (LDL-c) lowering efficacy of dietary interventions with phytosterol (PS)-enriched foods. The effect of PS is dose-dependent with an optimal intake around 2 g/d resulting in a 10% LDL-c reduction. A recent meta-analysis including 84 studies found a reduction in LDL-c of -0.34 mmol/l (95%CI:-0.36, -0.31) for an average PS dose of 2.15 g/d. Inhibition of intestinal cholesterol absorption is the key mechanism of action responsible for the PS-induced LDL-c lowering effect. With a PS intake of 2 g/d, cholesterol absorption is reduced by 30-40%. Although not all details are fully elucidated, several mechanisms are thought to contribute to the overall inhibition of intestinal cholesterol absorption. The key mechanism of action is displacement of cholesterol by PS from the micellar phase. Stimulation of bile flow prompted by food intake is a crucial step for the formation of mixed micelles and plays an important role for the optimal cholesterol-lowering efficacy of PS. There is also emerging evidence that PS interfere with transporter-mediated processes of cholesterol uptake. Recent insights into the role of influx/efflux sterol transporters in the gut, like NPC1L1 and ABCG5/G8 have shown that PS and cholesterol share the same transport processes.
The Formulation and Shelf Life Extension of Phytosterol-Containing Dairy Products. J. Zawistowski, M. Laza, Forbes Medi-Tech Inc., Vancouver, BC, Canada
Plant sterols reduce total and LDL-cholesterol by inhibiting intestinal cholesterol absorption from the diet. Food products enriched with sterols have been proven to lower the plasma LDL-cholesterol levels by 10-15%. Therefore, these bioactives have applications in functional foods for cardiovascular risk management, and may provide significant benefits to the health care system. However, phytosterols are very difficult to formulate into foods, due to their insolubility in water, and limited solubility in fats and oils. To increase fat-solubility, phytosterols can be esterified with unsaturated fatty acids. This however, does not provide a good solution for formulation of certain food matrices such as fat-free milk or yogurt. It also impairs the shelf life of food products due to relatively fast oxidation of unsaturated fatty acids. A number of stabilizing systems containing various gums and high HLB value emulsifiers had been developed in order to enrich milk and milk-based products with sterols. The results indicated that phytosterols have the potential to impart other benefits to foods, in addition to hypocholesterolemic properties. They exhibit antioxidant and antimicrobial activities, with the potential for shelf life extension of food products
ANA 2.1/BIO 2.1: Analytical Methodology in Functional Genomics
Chair(s): R. Weselake, University of Alberta, Canada; and J.T. Lin, USDA, USA
A Method of Regiospecific Analysis of Triacylglycerols by ESI-MS3 and Its Use for Olive Oil Analysis. J.T. Lin, United States Department of Agriculture, Albany, CA, USA
A method of regiospecific analysis of triacylglycerols (TAG) in vegetable oils and animal fats is reported here using electrospray ionization MS3 of TAG lithiated adducts. The fragment ions of the MS3 from the loss of fatty acids at the sn-2 position as α,β-unsaturated fatty acids were used for the regiospecific identification and quantification. The ratio of the regiospecific TAG, ABA and AAB, in a oil sample usually fraction collected by HPLC can be determined by the ratio of the abundances of the fragment ions of [ABA + Li – ACOOH – BCH=CHCOOH]+ and [ABA + Li – ACOOH – ACH=CHCOOH]+. The method was used to analyze regiospecific TAG in extra virgin olive oil. The results showed that the saturated fatty acids, palmitic acid and stearic acid, were mostly located at the sn-1,3 positions and unsaturated fatty acids, oleic acid and linoleic acid, were mostly located at the sn-2 position.
Tilling in the Botanical Garden: a Reverse Genetic Technique Feasible for all Plant Species. G.W. Haughn, E. Gilchrist, University of British Columbia, Vancouver, BC, Canada
TILLING is a reverse genetics technique that can identify a series of alleles or SNPs in a target gene. It can be adapted for use in a high-throughput facility, and has been successfully applied to all organisms tested in our laboratory. To date we have established TILLING in Arabidopsis thaliana, Caenorhabditis elegans, and Brassica oleracea, and we are currently testing this technique in Brassica napus. We have also used the technique to look at natural variation in Populus trichocarpa (here known as Ecotilling).The Brassica project on which we are currently working, is part of an oilseed initiative funded by Genome Canada/Genome Alberta project entitled Designing Oilseeds for Tomorrow's Markets and the Alberta Value-Added Corporation-sponsored project, Bioactive Oils Program. The goals of the project are to develop Canola with optimal seed coat characteristics and minimal levels of anti-nutritional factors. It is anticipated that the results of this research will enhance the overall usefulness of canola seed leading to improved meal for new food and feed applications, and increased seed oil content. We are generating a population of 6000 EMS-mutagenised B. napus lines for TILLING, and have begun to use TILLING to identify mutations in several genes involved in oil biosynthesis and or modification. Our results to date indicate that TILLING will be a powerful addition to the reverse genetic techniques available to Brassica napus researchers.
Chemical and Flavor Profile of Genetically Modified Peanut Varieties. N.T. Dunford1, R. Jonnala1, E. Ng1, K. Chenault2, 1Oklahoma State University, Stillwater, OK, USA, 2USDA, ARS, Stillwater, OK, USA
Peanut (Arachis hypogaea L.) is an economically important crop throughout the world. It is susceptible to many types of fungal pathogens. Genetic engineering offers great potential for developing peanut cultivars resistant to a broad spectrum of pathogens that pose a recurring threat to peanut health. In an effort to improve the disease resistance of peanuts, transgenic peanut lines were developed. Somatic embryos of the peanut cultivar Okrun were transformed with a chitinase gene from rice and/or a -1-3- glucanase from alfalfa via microprojectile bombardment. Modified peanut lines have been tested for S. minor resistance under greenhouse and field conditions Three transgenic peanut lines, 188, 540 and 654 showed increased resistance to fungal diseases, as compared to the parent line.The main objective of this research project was to assess the “substantial equivalence” and nutritional safety of biotechnology-derived peanut lines. The three transgenic peanut lines were analyzed for their oil, protein, ash, moisture, sugar, total dietary fiber, mineral and fatty acid, tocopherol, phytosterol and phospholipid compositions. The flavor analysis was performed using a gas chromatograph/mass spectrometer (GC/MS) equipped with an olfactory detector. The compositions of transgenic lines were compared to those of the parent cultivar Okrun.
Improved Resolution of Reverse Isomers of 1, 2-Diacylglycerols as 3, 5-Dinitrophenylurethanes by Reversed-Phase HPLC on Polymeric Octadecyl Silica Columns. Y. Itabashi1, C. Aizawa1, A. Kuksis2, 1Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan, 2Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada
The resolution of reverse isomers still remains a major unsolved problem in glycerolipid chromatography. We have investigated HPLC behavior of the reverse isomers of 1,2-diacylglycerols (DAG) on different types of octadecyl silica (ODS) columns. The reverse isomers of DAG having various pairs of acyl groups including highly unsaturated chains were chromatographed as 3,5-dinitrophenylurethanes (DNPU). Complete resolution of the reverse isomers possessing minor differences in chain lengths and degree of unsaturation, such as16:0-18:0 and 18:0-18:1, which had not been resolved previously by any chromatography techniques (Itabashi et al., J. Chromatogr. A 893, 261-279. 2000), was achieved within 60 min on a polymeric ODS column (150 x 4.6 mm i.d., 3 μm particles) using methanol as the mobile phase. Excellent resolution was also obtained for the reverse isomers of very different pairs of acyl groups, such as 16:0-22:6. The less polar isomers with the acyl chains of the higher degree of unsaturation or shorter chain in the sn-2 position were retained more strongly than the respective sn-1 isomers. The highly dense packing of octadecyl groups in polymeric ODS and the planar structure of the DNPU moiety in the derivatives may contribute to the separation.
Plant Hormone Profiling—A Targeted Metabolomics Technology for Oilseed Crop Improvement. S.R. Abrams, National Research Council of Canada, Saskatoon, SK Canada
Plant hormones regulate gene expression, control development, and regulate plants' responses to the environment through complex signaling networks. In particular, the plant hormone abscisic acid (ABA) regulates synthesis of genes involved in lipid synthesis and modification. For functional genomics research programs, we are developing and implementing technology for measuring multiple plant hormones in plant tissue samples (Chiwocha et al. 2005 Plant Journal 42: 35-48). We measure biologically active forms of the hormones and also inactive catabolites, which together afford an overview of hormone metabolism. Our approach is based on liquid chromatography coupled with tandem mass spectrometry, using specific deuterated internal standards to quantify more than twenty hormones and metabolite of interest in a single experiment, without derivatization and with minimal sample work up. Examples to be discussed include hormone profiling of developing seeds of Brassica napus, correlating with, oil content and gene expression information obtained through microarray analysis; determining the roles of ABA catabolites in regulating oil synthesis; hormone profiles of developing Brassica napus seeds and Arabidopsis seedlings subjected to freezing, drought, and biotic stresses.
Detection and Quantification of TAG Accumulation in Recombinant Yeast Systems. R.M.P. Siloto, M. Truksa, R.J. Weselake, Dept. of Agricultural, Food and Nutritional Science; University of Alberta, Edmonton, AB, Canada
Metabolomic Approach in Studying Biochemical Effects of Alfa-Tocopherol Using a Rat Model. A. Moazzami1, C. Sandström2, A. Kamal-Eldin1, 1Dept. of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2Dept. of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden
Metabolomics is a new approach in which profiles of metabolites in different tissues and/or biofluids are investigated to understand the changes, which is induced in the profile of metabolites, following an induced modulation. We have used this approach to investigate the biochemical activities of α-tocopherol in the liver using a rat model. 21-day Rats were either fed a sufficient control (n=10) or a deficient diet (n=12) for two month before sacrifice. Their livers were collected and extracted with chloroform-methanol-water. The extracts were analyzed using 1H-NMR and GC-MS to profile the metabolites and the data was analyzed using multivariate statistical method (PCA and PLS). The statistical analysis revealed that α-tocopherol modulates the contents of some amino acids, amino acid intermediate and lactate suggesting the possible impact of α-tocopherol on energy metabolism in rat liver.
BIO 3: Biocatalysis II
Chair(s): C.T. Hou, USDA, ARS, NCAUR, USA; and Y. Shimada. Osaka Municipal Technical Research Institute, Japan
Enzymatic Purification of Tocopherols and Sterols from Soybean Oil Deodorizer Distillate. Y. Shimada, Osaka Municipal Technical Research Institute, Osaka, Japan
Soybean oil deodorizer distillate (SODD) was distilled, and tocopherols and sterols were enriched. The preparation was named SODDTSC, which mainly contained free fatty acids (FFAs), sterols, and tocopherols. If sterols are converted to steryl esters (FAStEs) and FFAs are converted to fatty acid methyl esters (FAMEs), relatively easy purification of tocopherols and FAStEs can be achieved because the boiling points of FAMEs, tocopherols, and FAStEs are different significantly. Hence, the development of a two-step in situ reaction system was tried out for esterification of sterols with FFAs (first step) and esterification of FFAs with methanol (MeOH) (second step). The first-step reaction was conducted with dehydration in a mixture of SODDTSC/water (95:5, wt/wt) and Candida rugosa lipase (esterification of sterols, 95%). To the reaction mixture were added MeOH, water, and Alcaligenes sp. lipase, and the second-step reaction was conducted. Consequently, 95% FFAs were converted to FAMEs, and FAStEs synthesized by the first-step reaction were not reconverted to free sterols. Finally, SODDTSC (1.5 kg) was subjected to this two-step in situ reaction, and tocopherols and FAStEs were purified from the reaction mixture by distillation. Tocopherols were purified to 72% (yield, 88%) and FAStEs were purified to 97% (yield, 90%).
Synthesis of Tri-Conjugated-Linoleoylglycerol by Using Powdered Lipase Reaction System. S. Negishi1, Y. Yamauchi1, Y. Shimada2, 1The Nisshin Oillio Group, Ltd., Yokosuka, Kanagawa, Japan, 2Osaka Municipal Technical Research Institute, Osaka, Japan
Lipase catalyzed trans-esterification is one of the excellent methods to obtain functional foods oils. Nevertheless, this techniques has been put into practice only in limited areas. It is necessary to design the reaction system as simply as possible to develop it for versatile use such as hydrogenation, chemical esterification and fractionation.For the immobilization processing, other substances are required as a carrier, by which the specific activity of the enzyme is inevitably decreased. Thus, we investigated the trans-esterification reaction in consideration of the point that a compact reaction system can be designed by using powdered lipase.We applied of the reaction system to synthesis of ester not only trans-esterification, such as synthesis of CLA-TAG. CLA-TAG could be synthesized smoothly by eliminating water from the reaction. The rate of synthesis of CLA-TAG was increased when the different lipases powder which have different characteristic were mixed and used for synthesis of CLA-TAG. The method developed by us has been put into practice in the industrial production of CLA-TAG.
Transportation of Bioconverted Lipid Classes through Small Intestinal Epithelial Cell Model. H. Naito1, T. Ohkubo2, K. Takahashi1, 1Graduate School of Fisheries Sciences, Hokkaido University, Japan, 2NOF Corporation, Japan
Squid meal phosphatidylcholine, a typical marine phospholipid rich in DHA exclusively in sn-2 position of the glycerol backbone, and as comparison, soybean phosphatidylcholine rich in linoleic acid in both sn positions, were subjected to lipozyme RMIM (NOVO A/S Bagsvaerd, Denmark) mediated selective partial hydrolysis. The principle of this reaction was cleaving off fatty acid moiety in sn-1 position leaving the desired fatty acid moiety in the sn-2 position. Transportations under coexistence of bile acid through small intestinal epithelial cell model of the obtained lysophosphatidylcholines and the original substrate phosphatidylcholines were evaluated using DPH (1,6-diphenyl-1,3,5-hexatriene) as fluorescent probe. Tight junction opening of the small intestinal epithelial cell model was monitored by measuring TER (Transepitherial Electrical Resistance). Both the DHA enriched lysophosphatidylcholine and the soy bean lysophosphatidylcholine seemed to pass through the small intestinal epithelial cell model more rapidly than those original diacyl chemical form phospholipids. The lipid chemical form should thus influence the transportation of DHA. And for this reason, other various lipid chemical forms are under exploration, and should be defined by May.
Synthesis of trans-Free Structured Lipids for Possible Margarine Formulation. S.E. Lumor, B.H. Kim, C.C. Akoh, University of Georgia, Athens, GA, USA
This study investigated the synthesis of trans-free structured lipids (SLs) for possible margarine formulation. A series of SLs were synthesized by incorporating stearic acid (40 and 50%, w/w) into canola oil catalyzed by Lipozyme RM IM (from Rhizomucor miehei), and by transesterifying blends of canola oil, palm stearin and palm kernel oil (40:60:0, 40:50:10, 40:40:20 and 40:30:30, w/w/w) catalyzed by Lipozyme TL IM (from Thermomyces lanuginosa). After short-path distillation, the products were characterized. No trans-fatty acids were detected in the SL products. The structured lipids synthesized by incorporating stearic acid into canola oil (SL-40 and SL-50) did not have adequate solid fat content or β' crystal forms desirable for tub margarine formulation. These were improved by blending the SLs with different proportions of palm mid-fraction or a SL made by reacting equal amounts of palm mid-fraction and canola oil. For SLs made by interesterifying blends of canola oil, palm stearin and palm kernel oil, more β' crystal forms were observed in products made with higher amounts palm kernel oil. Considering solid fat contents, melting properties and crystal forming habit together, the SL prepared from the 40:40:20 blend was found to be the most suitable for tub margarine formulation.
BIO 4 / PRO 4.1: Bioprocessing-Enzymes
Chair(s): H.C. Holm, Novozymes AS, Denmark; and N. Dunford, Oklahoma State University, USA
The Use of Lipases in High Acid Animal Fat and Tropical Oils for Biodiesel Production. G. Malta2, R. Sponquiado2, V. Ferraz3, W. Artz4,1, S. Segall1,2, 1A&S Bioenergia, Belo Horizonte, MG, Brazil, 2UNIBH, Belo Horizonte, MG, Brazil, 3UFMG, Belo Horizonte, MG, Brazil, 4UIUC, Urbana, IL, USA
The objective of the work was to compare chemical and enzymatic catalysts for conversion of various oil sources to ethyl esters for use as biodiesel. Three fat sources, animal fat, castor oil and pequi oil, were used to produce biodiesel with transesterification reactions using lipases (lipolase, palatase and lipex) and NaOH. Ethanol was used with 2 molar ratios (6:1 and 12:1 ethanol/triacylglycerols). The reactions were performed at 55C and 45C with NaOH and lipases, respectively. The catalyst concentrations were 0.5, 1.0, 1.5 and 5, 7.5 and 10% for chemical and enzymatic catalysts, respectively. The fat sources performed differently with different catalysts. The animal fat used was a “low cost residue” with a high acid value (up to 14%). When used with NaOH, the conversion rates were very low or nonexistent. However, conversion rates up to 82% were obtained using lipolase with the high value acid animal fat. The conversion rates for castor oil and pequi oil did not show great differences between the biological and chemical catalysts. The enzymatic catalysts proved very useful for biodiesel production using feedstock with a high acid value but further adjustments need to be made to achieve the high industrial conversion rates needed to produce biodiesel. All the ethyl esters were analyzed using a GC with an internal standard (margaric acid).
Enzymatic Production of Mono- and Diglycerides. X. Xu, The University of Aarhus, Aarhus, Denmark
Enzymatic processing of oils and fats has been the research and development areas for 20 years. The brainstorming of application ideas has been the main concern for most previous studies. Enzyme technology has been used for numberless product development. The basic understanding of the enzymatic reactions has been highly advanced. It is now in the stage for industrial take-over. Central issues for commercial exploitation lie in benefits in economy, product quality, product nutrition, simplicity and easiness of technology, social and environmental concerns. For technologists, a big concern is to develop the efficient processes focusing on most beneficial products with as high as possible of the economical turnover. This often refers to product development and process development. In this talk, a special focus will be given on the enzymatic production of partial glycerides where the system is often complicated by the three phase media. The development is targeted on simplicity, efficiency, and practical feasibility.
Improved Enzymatic Esterification by Novel Reactor Concepts. O. Thum1, L. Hilterhaus2, A. Liese2, 1Evonik Goldschmidt GmbH, Essen, Germany, 2Technical University Hamburg-Harburg, Hamburg, Germany
Even though the enzymatic production of bulk chemicals, such as of cosmetic ingredients, has already been established on multi ton scale, there are still technical limitations existing that need to be overcome. For example fixed bed reactor systems are limited to low viscous reaction mixtures whereas classical strirred tank reactors destroy enzyme immobilisates and therefore do not allow sufficient reuse of expensive catalysts.We have developed an universal reactor concept for enzymetic esterifications that allows the use of raw materials of low and high viscosity. As model reaction served the synthesis of fatty acid esters of simple fatty alcohols as well as of high viscous polyglycerols.Furthermore the new reactor concept allows easy product separation and catalyst recovery, minimizes mechanical stress to the biocatalysts, reduces reaction times compared to known production methods and causes only low investment costs.
New Immobilized Lipases for Production of Biodiesel. S. Basheer, M. Haj, M. Kayal, TransBiodiesel, Ltd., Shfar-Am, Israel
Mono-alkyl esters of fatty acids derived from different oils and fats have been commercially prepared at industrial scales and are currently being used as biodiesel in many countries around the world. Biodiesel is prepared using either base- or acid-catalyzed conventional chemical processes.Lipases offer an attractive alternative in order to avoid the drawbacks of the chemical processes currently practiced for the conversion of oils and fats to biodiesel. This group of enzymes is capable of catalyzing the direct transesterification reaction between oil triglycerides and short-chain alcohols to yield biodiesel and glycerol. Unfortunately, the economic costs for commercially available lipases are still unaffordable for production of biodiesel. This work will present the potential of lipases modified-immobilized following different techniques for the production of biodiesel at semi-industrial scales with competitive costs compared to the conventional chemical processes.
New Enzyme Process for Biodiesel. P.M. Nielsen, H.C. Holm, Novozymes A/S, Bagsvaerd, Denmark
Research on enzymatic catalyzed biodiesel production has been carried out in several groups around the world during the last ten years. The research has mainly focused on development of a process capable of processing low quality oils with high free fatty acid content as this raw material is causing technical problems in chemically catalyzed processing. A few articles document the productivity of the enzyme catalyst and can be expressed as the amount of biodiesel produced per kg of enzyme. This productivity data can be used to calculate the catalyst cost and to compare to chemical catalyst. It becomes evident that the economy is still a problem with the enzymes availabe today. However, new developments in enzyme technology are able to provide catalysts at a price level where the biodiesel production costs become competitive to chemical catalyzed processing. This paper discusses the challenges in the biodiesel process with respect to enzyme stability and efficiency in the biodiesel process, and how they can be solved.
Enzymatic Process for Biodiesel Production and its Industralization Progress. W. Du, Department of Chemical Engineering, Tsinghua University, China
Biodiesel production with enzymatic approaches Lipase-catalyzed transesterification from renewable oils for biodiesel production has many advantages over chemical approaches though the latter has been put into the industrialization for biodiesel production. However, the low stability (poor operational life) and the high cost of the lipase have been thought to be the main hurdle to the industrialization of lipase-catalyzed biodiesel production. Tsinghua University has proposed a novel route and the operational life of the immobilized lipase could be improved over 50-fold than traditional enzymatic approaches. This novel route is thought to be very promising for the commercialization of biodiesel production since it would reduce the lipase cost dramatically by significantly improving the stability and the operational life of the lipase. The demonstrational test in a pilot plant with capacity of 100kg/d biodiesel was successful. A new plant with 20,000ton/year capacity has been constructed in Hunan, China and it was put into operation in Dec. 8, 2006. The results from the full scale operation are even better than in the pilot plant demonstration. Integrated production of 1,3-PDO from by-product glycerol As a by-product, glycerol will be yielded at about 10% of biodiesel during the process of biodiesel production. How to convert glycerol has become a common problem which has to be resolved if considering large amount of biodiesel production. Integrated production of 1,3-propanediol (PDO) from glycerol could be a promising way to improve the profit of the whole process during biodiesel production.1,3-PDO is a valuable chemical material and especially it could be copolymerizes with terephthalic acid (or methyl ester) to form polytrimethylene terephthalate (PTT). PTT has excellent properties compared to other polymers such as PET. 1,3-PDO can be synthesized from petrochemicals by chemical approaches or manufactured from renewable substrates by fermentation. However, it's well known that there are many disadvantages associated with chemical approaches for 1,3-PDO production, such as low selectivity, high temperature and high pressure needed etc. Especially the material for chemical approaches is unrenewable and some intermediates such as ethylene oxide and acrolein are explosive or high toxic. Therefore, fermentation approaches for 1,3-PDO production have drawn more and more attention by considering its advantages over chemical method such as relatively low investment, mild reaction conditions and using renewable sources as the starting materials.Tsinghua University has proposed a novel flexible process for 1,3-PDO production from glycerol or glucose, and the demonstration was finished in pilot plant at the end of 2003 and in industrial scale(50m3fermentor) in June 2006. The purity of final product 1,3-PDO is as good as 99.92%. Some 1,3-PDO sample was tested for PTT polymerization by Chinese and Japanese companies. The results showed that the key characteristics (such as inherent viscosity) of PTT polymerized with the 1,3-PDO produced by biological route are even better than that by chemical route. Currently the large-scale production of 1,3-PDO(20000tons/year) is being under construction. Based on the above technology, It could be well integrated to produce biodiesel and 1,3-PDO.
State-of-the-Art Enzymatic Processing of Fish Oils. J.A. Kralovec, W. Wang, P.F. Mugford, M.A. Potvin, C.J. Barrow, Ocean Nutrition Canada, Dartmouth, NS, Canada
Omega-3 fatty acids are important components of a balanced diet and not surprisingly they have an established position as science-backed nutritional supplements and ingredients of functional foods. Fish oils are the best sources of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the two most important omega-3 fatty acids. Naturally, the development of novel methodologies geared towards the production of EPA/DHA concentrates plays a significant role in R&D efforts at Ocean Nutrition Canada (ONC). Remarkable progress has been made in the area of enzyme driven technologies with two of the concepts being transferred in different variants to the manufacturing scale, the conversion of EPA/DHA ethyl ester (EE) concentrates to the corresponding triglycerides (TG) and the formation of DHA/EPA enriched fish oil involving hydrolysis by selective lipases. Although omega-3 TG concentrates can be prepared using enzymatic trans-esterification of the omega-3 EE concentrates with glycerol, the process involves a total split of the original TG molecules and it may lead to the re-esterified TG molecules that may structurally be very different. The second concept does not suffer from this potential drawback. It is based on the selective removal of saturated fatty acid residues from fish oil, followed by re-esterification of the generated mono- and diglycerides with EPA or DHA in the presence of Candida antarctica lipase B. The hydrolysis was tested on starting oils with various EPA and DHA levels using different lipases. The regio-specificity of the trans-esterification and the re-esterification was investigated and the structural assessment of the final products was conducted. The results were compared with the data obtained for the starting natural fish oils.
Bioprocessing from the Crop to the Bottle: What to Expect from Enzymes in the Future Oil Processing Line. M. Kellens, W. De Greyt, De Smet Ballestra Group, Brussels, Belgium
The use of enzymes in our daily food is quite established and generally accepted. Without enzymes, we even would not exist. Newer food products are introduced in the market where enzymes play a crucial role: special treated yoghurts, nutrient enzyme enriched drinks, enzyme treated nutritional minor components, meat treated with enzymes to make it tenderer and so on. Enzymes play also a key role in various household and industrial cleaning products, especially to breakdown fatty substances. Today, we see a similar evolution in the oil processing industry: more and more chemical based processes are being replaced by enzyme based treatments. Some of them are applied already today on an industrial scale. Typical examples are the enzymatic degumming, enzymatic oil recovery from gums and enzymatic interesterification. Although proven on both technical and economical level, the industry remains reluctant and hesitating. Major reason for this is the lack of knowledge which in turn results in a lack of confidence. But this is changing, mainly thanks to the continuing efforts of enzyme producing companies as Novozymes.Other potential development areas in oils and fats processing, where enzymes are tested, are in the mechanical extraction (enzyme assisted pressing), enzymatic transesterification (biodiesel production), and enzymatic fat splitting. And we may expect enzymes to be introduced in even more processes. But much will depend upon the stability and activity of these biocatalytic wonders of nature.
A New Process for Degumming: The Use of Phospholipase C to Improve Yields during Refining of High Phosphorus Vegetable Oils. N. Barton, Verenium Corporation, San Diego, CA, USA
A phospholipase C (PLC) enzyme product has been developed to increase oil yield during the degumming of high phosphorus vegetable oils. The PLC is process-compatible, requiring minimal capital investment for implementation. The application of PLC does not require the adjustment of the pH of the water in oil emulsion prior to enzyme addition. The oil yield is increased due to the conversion of hydratable phospholipids to diacylglycerol (DAG) that remains in the oil and the reduction of the amount of neutral oil lost during primary centrifugation. The use of PLC does not result in the production of additional free fatty acids. While PLC can be used with a variety of oils including canola, rapeseed, corn, and sunflower, it provides the most significant yield improvement when used to degum soybean oil due to the high percentage of hydratable phospholipids present in the crude oil (up to 70%). For example, when used to treat a soybean oil containing 900 ppm phosphorus, PLC provides a 1.2% DAG bonus and a 0.8% neutral oil gain to produce an overall yield improvement of 2%.
Enzymatic Degumming of Vegetable Oils. C.L.G. Dayton, Bunge North America, Bradley, IL, USA
The use of enzymes for vegetable oil degumming fulfills the promise for a third wave in Biotech. The enzymatic degumming is already competing with traditional manufacturing processes and has achieved industrial sustainability. A thorough examination of the process waste reduction and its economical implications will be made for the current and next generation of enzymes for vegetable oil degumming in food and biodiesel production.
State-of-the-Art Enzymatic Processing in the Oils and Fats Industry and Future Perspectives. W.D. Cowan1, H.C. Holm2, 1Novozymes UK, Chesham, Bucks, UK, 2Novozymes DK, Bagsvaerd, DK
This paper examines the latest developments in enzymatic processing as applied to the oils and fats industry with particular reference to bulk fat modification and degumming. In addition is will use results from ongoing laboratory trials to present a perspective on where the industry could usefully apply enzymatic solutions in the near future
S&D 4 / BIO 4.1: Bio based Surfactants
Chair(s): G. Smith, Huntsman Performance Products, USA; and D. Hayes, University of Tennessee, USA
Biosynthesis of Flavonoid Esters in Ionic Liquids. B.M. Lue, Z. Guo, X. Xu, BioCentrum, Technical University of Denmark, Kgs. Lyngby, Denmark
Room temperature ionic liquids (RTILs) are organic salts with low melting points and are often referred to as "green solvents" because they do not release volatiles into the atmosphere. RTILs are regarded as interesting substitutes for traditional organic media due to their high thermal stability, wide liquid range and potential for solubility modifications (through appropriate cation & anion selection).Flavonoids are widespread in foods and have been characterized by their anti-oxidative, anti-carcinogenic and antimicrobial properties. Unfortunately, flavonoids are not very soluble in hydrophobic environments and this limits their anti-oxidative efficiency. The acylation of flavonoids with fatty acids is of great interest as this would increase solubility of the flavonoids in the hydrophobic phase. These acylation products could then be employed as antioxidants or emulsifiers.Due largely to differences in solubilities, development of an efficient enzyme system for the acylation of flavonoids is problematic. The use of RTILs may potentially reduce or eliminate these difficulties through increased substrate solubility and the possible use of vacuum for removing water (i.e. due to lower vapour pressure), thereby increasing reaction efficiency, stability & perhaps specificity.This presentatoin will explore the use of various RTILs for the production of flavonoid esters.
Modified Vegetable Oils as Detergents and Emulsifiers. Raymond W. Cen, George A. Smith, Huntsman Corporation, HATC, 8600 Gosling Rd, The Woodlands, TX 77381, USA
We modified vegetable oils chemically via a proprietary G2 catalyst in Huntsman to form a group of ethoxylated vegetable oils (VEO). Several vegetable oils such as peanut oil, soy bean oil, canola oil, corn oil, and coconut oil can be easily ethoxylated through EO insertion to ester portion of triglycerides. Physiochemical properties as well as spectroscopic characterization were listed as function of EO stoichometry and oil identity. The application and efficacy for laundry detergents and other cleansing applications were shown. This group of bio-based detergents have tendency to form O/W emulsions even at oil:water ratio of 9:1 and beyond with good efficiency. Several examples of crowded O/W emulsions were also demonstrated here for their potential in different consumer goods application.
Kinetics Control of the Synthesis of Branched Hydrophobes from Oleochemicals. K.M. Doll1, S.Z. Erhan1, B.K. Sharma1,2, 1FIO, NCAUR, ARS, USDA, Peoria, IL, USA, 2Chemical Engineering, Pennsylvania State University, University Park, PA, USA
The synthesis of a series of branched hydroxy stearates from commercially available methyl oleate and common organic acids is reported. A variety of different acids, with 3 to 8 carbon atoms, and also varying in their branching and functionality, were used. The kinetics of the ring opening reaction was studied, under both pseudo 1st order conditions, and 2nd order conditions. An Arrhenius activation energy of 66.7 kJ mol-1 (16.0 kcal mol-1) was found in a system using propanoic acid as the ring opener. The resultant compounds have calculated molar volumes of ~375 cm3 making them a candidate for use as surfactant hydrophobes. The added oxygen groups and branched structures may allow the synthesis of improved surfactants from these materials as well. The general ease of the method should also allow its use with a variety of commercially available substrates, in order to obtain desired molecular structure. Finally, commercially available epoxidized soybean oil may also be modified by this method if a product with higher molecular weight is desired.
Formulation Design of Metalworking Fluid Microemulsions Using Bio-Based Surfactants. Fu Zhao1, Kim Hayes2, Steven Skerlos2, 1Purdue University, West Lafayette, IN, USA, 2University of Michigan, Ann Arbor, MI, USA
Metalworking fluids (MWFs) are ubiquitously used in the machine tool industry as coolants and lubricants. Conventional MWFs are based on petroleum feedstock, which result in high treatment and disposal costs. These factors along with increasing oil prices and the national security concerns associated with imported oil, serve as motivations to consider alternatives to petroleum-based MWFs. This paper presents a set of surfactant-selection guidelines that can be used to design bio-based MWF microemulsions. Nine classes (five anionic and four nonionic, with different head and tail structures) of bio-based surfactants are investigated for their capability to emulsify three representative vegetable base oils. Experimental results suggest that: a combination of two surfactants, one nonionic and one water soluble co-surfactant (either nonionic or anionic) is preferred over a single surfactant; the nonionic surfactant should have a carbon tail length greater or equal to the nominal carbon chain length of the fatty acids in the oil as well as a head group that is not excessively small or large (e.g., 10-20 ethylene oxide groups); the difference in tail lengths between the surfactant and the co-surfactant should be less than 6 to maximize the feasible range of oil to surfactant ratios yielding stable emulsions.
Extraction of Biosurfactants from Wastewater Biosolids: Challenges and Opportunities. F.Y. Garcia, D.G. Allen, S. Liu, E.J. Acosta, University of Toronto, Toronto, Ontario, Canada
This presentation describes the method of extraction and properties of a surface active materials extracted from activated sludge biosolids. While the composition of the extracted mixture is complex, its surface activity (CMC and surface tension after CMC) suggest that this material has similar properties to that of soaps derived from the saponification of lipids. Size exclusion chromatography, reverse phase - ion coupled chromatography, and interfacial tension data are presented in order to characterize the size, charge and hydrophobicity of the extracted material. Preliminary cleaning performance data and cytotoxicity studies will also be presented. The potential opportunities for these surface active materials and the challenges for their development into commercial products will be discussed.
Sugar Fatty Acid Esters and Alkyl Glucosides, Two Families of Enzymatically Prepared Biosurfactants in a Green Chemistry Perspective. A. Ducret, M. Trani, W. Levadoux, R. Lortie, Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada
Sugar fatty acid esters and alkyl glucosides are two families of non-ionic biosurfactants having excellent surface-active properties (emulsifying, stabilizing, conditioning effects) making them suitable as ingredients in foods, detergents, therapeutic formulations, or personal care products. Enzymatically prepared from renewable resources (vegetable oils and carbohydrates) under mild conditions by direct condensation reactions (generating no by-products except water), they are completely biodegradable, and have a low environmental impact. Sugar esters are obtained by esterification of a sugar with fatty acid using a lipase while alkyl glucosides are obtained by condensation of a sugar and an alcohol using a glucosidase. In both cases, reactants (sugar and fatty acid or alcohol) are very poorly soluble in each other, limiting the productivity. The production of alkyl glucosides faces an additional challenge, the low tolerance of glucosidases to reaction conditions (low water activity, organic media). The experience of our lab on the synthesis of both families of biosurfactants and the approaches followed to overcome the hurdles mentioned above will be presented.
Bioreactor Design for the Solvent-Free Lipase-Catalysis of Saccharide-Fatty Acid Ester Biosurfactant. Sang-Hyun Pyo, Douglas G. Hayes, University of Tennessee, Knoxville, TN, USA
Bioreactor systems have been designed for the immobilized lipase-catalyzed synthesis of fatty acid-saccharide mono- and di-esters, which are useful biosurfactants and value-added products. The reactions were operated in the absence of solvent; moreover, the major technical bottleneck to overcoming the slow rate of reaction, the poor miscibility of the saccharide and acyl donor substrates, was addressed using the saccharide monoester product as a miscibility agent and an elevated temperature of 65 C. Two main reactor designs have been assembled and are compared. The first employs a packed bed containing saccharide and an adsorbent upstream of the bioreactor. The second involves introduction of saccharide through a suspension of crystalline saccharide powders into a fatty acid/monoester liquid mixture. Both systems were operated under continuous recirculation of reaction mixture through the bioreactor system. Although System 1 yields a lower saccharide solubilization compared to System 2, the latter of which produces micron-sized suspensions of crystalline saccharide, both provide similar time courses of esterification, converting a mixture of 20% ester / 80% fatty acid into 75-90% ester. The effect of operating parameters and water content are currently under exploration
Using Biosurfactant Mixtures to Form Microemulsions: Enhanced Solubilization and Potential Applications. T. Nguyen1,2, L. Do1,2, D. Sabatini1,2, 1University of Oklahoma, Norman, OK, USA, 2Institute of Applied Surfactant Research, Norman, OK, USA
Biosurfactant mixtures and biocompatible lipophilic linkers were used to form microemulsions with a wide range of oils. The biosurfactants studied are anionic rhamnolipids produced by microorganisms and nonionic methyl ester ethoxylates made from biorenewable feedstock of plants. Three different oils were studied: limonene, diesel, and vegetable oil. Due to the molecular structure of these surfactants and linker molecules, we hypothesize that these formulations can form alcohol-free and non-toxic microemulsions while achieving high solubilization capacity which is enhanced by using mixtures of biosurfactants versus single biosurfactants. These microemulsions can be used for various applications such as cosmetics, where an oil-in-water microemulsion is used, and vegetable oil extraction, where a water-in-oil microemulsion is used. We extensively study the vegetable oil extraction using water-in-oil microemulsion of diesel, which produces a blend of vegetable and diesel after the extraction. Various parameters such as surfactant concentration, salinity, and ratio of oilseed to liquid feedstock of the extraction process were evaluated for the effects on the ratio and viscosity of this vegetable oil â€“ diesel blends and their ability to be used as an alternative fuel to diesel and biodiesel.
Vegetable Oilseed Cake: Raw Material for Protein-Based Surfactants. Adinath Mahadeo Ware1,2, Sushilkumar Dubal1, S.A. Momin1, 1Institute of Chemical Technology, Mumbai University, Mumbai, Maharahstra, India, 2Chemithon Engineers (P) Ltd., Mumbai, Maharashtra, India
Synthesis of new surfactants with special functionalities are greatly needed to fine-tune existing surfactants for specific tasks. The raw materials cost, market size, energy needs, and health and environmental concerns have been the important factors especially for discovery and introduction of new surfactants. In this regard protein from oilseed cake, an agricultural byproduct abundantly available in the world, is one of the few major and economic sources of naturally occurring amino acid and can be regarded as a versatile and valuable raw material for surfactant production.Protein-based surfactants are usually synthesized with amino acids/peptides and fatty acids as building blocks. They are mainly of two types: peptide and amino acid surfactants. Both are interesting compounds that contain an amino or a peptide as the hydrophilic part and a long hydrocarbon chain as the hydrophobic portion. The hydrocarbon chain can be introduced through acyl, ester, amide, alkyl or ether linkage. Protein-based surfactants are usually considered biodegradable, nontoxic and nonirritating and, in some cases they have antimicrobial properties therefore they have great demand in cosmetics. The review deals with the utilization of oilseed cake in preparation of protein based surfactants and their applications in cosmetics and toiletries.
Novel Rinse Aid Microemulsions with Improved Biodegradability. Andras Nagy, Saiid Mohammed, Dennis Parrish, Janet Kosiek, Georg Schick, Goldschmidt Chemical Corporation, Hopewell, VA, USA
Conventional rinse aids/drying agents consist of an emulsifier, a hydrophobe, a coemulsifier and water. Most ingredients are not biodegradable according to OECD methods and can have other undesirable effects and characteristics.This paper defines novel rinse aids with improved biodegradability and more desirable environmental and performance profiles. Traditional ingredients are replaced with more biodegradable, environmental friendly ones: dialkyl dimethyl quaternary ammonium emulsifiers are replaced with ester quaternaries, mineral seal oil hydrophobes with ester oils, flammable solvents and co-emulsifiers with non-flammable ones. The resulting microemulsion rinse aids are clear or translucent, easily dilutable even with cold water, will not go through viscous gel phases, have favorable performance characteristics in terms of beading and sheeting and are compatible with other performance and aesthetic enhancing additives like organo- modified silicones, dyes, perfumes, etc. Test methods and advantages will be presented.
Interfacial Properties of Sugar-Based Surfactants and Their Applications. P. Somasundaran, S. Lu, J. Wu, P. He, Columbia University, New York, New York, USA
The interfacial properties of surfactants play a critical role in determining their performance in many industrial applications, such as detergency, enhanced oil recovery and water treatment. Due to the increasing demand for environmental friendly reagents, green surfactants have received considerable attention recently. The unique surface activity and biodegradability make this group of surfactant become the candidates for the next generation reagents. In this work, the interfacial properties of sugar based alkyl glycosides and sophorolipid esters were investigated. These surfactants show high surface activity and salt tolerance. When mixed with conventional surfactants including anionic, nonionic and cationic, the system exhibit nonideal mixing suggesting synergistic interactions. In addition, sugar based surfactant, n-dodecyl-β-D-maltoside, was found to have high selectivity of adsorption on oxide minerals. For instance, it adsorbs on alumina, titania and hematite but not on silica. The mechanism has been successfully revealed by correlating the adsorption to the solid surface species concentrations. The wettability of the substrate surfaces was found to change significantly with the adsorption. Furthermore, this sugar based surfactant was successfully applied to separate a model pollutant, phenol, from aqueous solutions using micellar enhanced ultrafiltration. The performance of the surfactant was found to be governed by the intermolecular interaction and the nanostructure of the surfactant micelles. Undoubtedly sugar based surfactants will have a potential impact on the chemical evolution of the down-stream industries and this study will promote the application of these environmentally friendly surfactants.
BIO 5: General Biotechnology
Chair(s): D. Hildebrand, University of Kentucky, USA; and K. Takahashi, Hokkaido University, Japan
Prediction and Verification of the Solubilities of Flavonoids in Ionic Liquids: A Priori Design of the Green Routes for Efficient Enzymatic Synthesis of Flavonoid Ester. Z. Guo1, B.-M. Lue1, K. Thomasen2, A.S. Meyer2, X. Xu1, 1BioCentrum-DTU, Technical University of Denmark, Lyngby, Copenhagen, Denmark, 2Department of Chemical Engineering, Technical University of Denmark, Lyngby, Copenhagen, Denmark
Predictions of the solubility of flavonoids in a large variety of ionic liquids (ILs) with over 1800 available structures were examined based on COSMO-RS computation. Experimental measurement of the solubilities of esculin and rutin in 12 ILs with varying anions and cations show that predicted and experimental results generally have a good agreement. Based on the sound physical basis of COSMO-RS, the solubility change of flavonoids were quantitatively associated with solvation interactions and structural characteristics of ILs. COSMO-RS derived parameters, i. e. misfit, H-bonding and van der Waals interaction energy, are shown to be capable of characterizing the complicated multiple interactions in IL system effectively. COSMO sigma-moment descriptors, which roughly denote the characteristic properties of the ILs, might be of general value to have a fast estimation for the solubilities of flavonoids as well as those compounds with massive moieties as H-bonding donor. The results obtained in this work may be important for achieving an improved understanding of IL solvations and the tailoring of the desired structures of ILs used as the media for efficient enzymatic esterification of flavonoids.This work has been accepted for publication in Green Chemistry
Evaluation of Methods to Concentrate Highly Unsaturated Fatty Acids from Fish and Microbial Oils. R.E. Armenta, A.M. Burja, H. Radianingtyas, C.J. Barrow, Ocean Nutrition Canada Ltd., Dartmouth, NS, Canada
Fish and microalgal oils were winterized with a variety of solvents to assess their efficiency in concentrating highly unsaturated fatty acids (HUFA). Acetone winterization concentrated HUFA approximately 15% more in microalgal oils when compared to fish oils, potentially because saturated FA in untreated fish oil are characteristically lower. Yield recoveries of acetone winterized oils were 0.70-0.85 and 0.40-0.75 for fish and microalgal oil, respectively. Winterization changed microalgal oils from solid to liquid at room temperature, and acetone winterization was the most efficient chemical method to concentrate HUFA. However, the corrosive nature of acetone on equipment must be considered when winterization at larger scale is attempted. Pros and cons for the use of different solvent combinations are discussed.
Yeast Triacylglycerol Production from Industrial Grade Glycerol. D.R. Iassonova, S.E. Beattie, E.G. Hammond, Iowa State University, Ames, Iowa, USA
Biodiesel is produced when triacylglycerols are converted into fatty acids methyl esters. Glycerol is a co-product of biodiesel manufacture. Glycerol from biodiesel production usually contains a variety of other impurities that make it unsuitable for food or cosmetic use without first improving its quality by expensive purification processes. We found that the oleaginous yeast Cryptococcus curvatus was able to synthesize and accumulate triacylglycerols in lipid bodies when grown on industrial biodiesel-based glycerol. The amount of oil and the fatty acid profile of total yeast fat depended on glycerol concentration, fermentation time and media type. Under optimized conditions, yeast accumulated over 50% lipids on a cellular biomass basis in less than 50 h when grown on 6.4% glycerol in a synthetic media restricted in nitrogen. The fatty acid profile of total yeast oil was predominately palmitate (16:0, 27%), stearate (18:0, 8%), oleate (18:1, 51%) and linoleate (18:2, 9%). Fed-batch fermentation increased biomass production up to 54g/L. Several nitrogen sources, such as asparagine, urea and ammonium chloride, were compared. C. curvatus was able to use an inorganic nitrogen source such as urea. We have demonstrated that industrial biodiesel-based glycerol can be converted into single cell oil which could be further transformed into biodiesel.
Continuous Glycerolysis in an Immobilized Enzyme Packed Reactor for Industrial Monoacylglycerol Production. M.L. Damstrup1, S. Kiil2, A.D. Jensen2, F.V. Sparsø3, X. Xu1, 1BioCentrum, Technical University of Denmark, Lyngby, Denmark, 2Department of Chemical Engineering, Technical University of Denmark, Lyngby, Denmark, 3Danisco A/S, Brabrand, Denmark
Lipase catalyzed glycerolysis processing is of industrial interest since it can be carried out at ambient temperatures and atmospheric pressures. Previously, glycerolysis conducted in a column packed with immobilized lipase were highly potential to ensure a high MAG formation in a short time. In spite of optimal reaction conditions a complex heterogeneous reactant mixture with a glycerol in oil emulsion occurs. Hence, the movement of material from phase to phase as well as through the catalyst pores becomes important since it can influence the performance of the immobilized enzyme reactor. To examine which basic features that need to be considered to obtain an industrially beneficial procedure continuous and easily operated glycerolysis was studied in different lipase packed columns. Immobilized Candida antarctica lipase B was used to catalyze the glycerolysis reaction between glycerol and sunflower oil dissolved in a binary tert-butanol:tert-pentanol medium. Practical design-related issues such as required reaction time, enzyme capacity, expansion of the enzyme during wetting, and the effect of different column length-to-diameter ratios, fluid velocities and particle sizes of the enzymes were evaluated.
Growth of Oleaginous Microorganisms Utilizing Glycerol as the Sole Carbon Source. J. Thomas, R. Hernandez, T. French, E. Alley, W. Holmes, Mississippi State University, Mississippi State, MS, USA
The growing popularity of biodiesel as an alternative to petroleum-derived diesel fuel has created a dramatic increase in the production of crude glycerol. Currently crude glycerol has a low market value and many biodiesel producers are treating the crude glycerol as a waste product. If biodiesel is to succeed as an alternative to petroleum-based fuels, it is essential that the crude glycerol produced be given value. Oleaginous microorganisms have the ability to use many different types of carbon sources and convert them into oils that can then be used for the production of fuels. The purpose of this study is to investigate the feasibility of oleaginous microorganisms to utilize glycerol as a carbon source for the production of oils. The study will focus on several different species of oleaginous yeasts and bacteria. Kinetics of glycerol removal and cell growth will be presented, as well as quality and yield of oil from the oleaginous microorganisms.
Enzymatic Production of Diacylglycerol Oil. T.K. Yang, Dalian University of Technology, Dalian, China
The aim of this paper was to investigate the approaches for production of diacylglycerols (DAG) from vegetable oil. With mild reaction conditions, enzymatic process produced no waste and was friendly to environments. Lipase-catalyzed glycerolysis of vegetable oil, interesterification of triacylglycerol (TAG) with monoacylglycerol (MAG)ï¼Œand interesterification of MAGs were conducted to produce diacylglycerols in a stirred tank and through a packed-column reactor. After screening, Novozym 435 was chosen as the catalyst in the lipase-catalyzed reactions, which was active and stable during the production of DAG oil. Glycerolysis of oil and interesterification of triacylglycerol with monoacylglycerol resulted in the DAG products containing 20-25% TAG. TAG was very difficult to separate from DAG in the down-processing (short path distillation). Intramolecular interesterification of MAG could produce DAG product with more than 90% purity.
Update on Hydrocarbon Partitioning into Soybean Oil. D. Hildebrand1, R. Li1, K. Yu1, T. Hatanaka2, 1University of Kentucky, Lexington, KY, USA, 2Kobe Univ., Kobe, Japan
With growing interest in seed oils as renewable sources of fuel and chemicals there is a need to better understand how to increase hydrocarbon flux into seed oil. Soybeans are among the lowest in oil (and highest in protein) among major renewable oil sources with ~ 20% oil. We have good evidence that much of the final synthesis of the triacylglycerol (TAG) of soybean oil is synthesized by two diacylglycerol acyltransferase 1s (DGAT1s). Collaborations with the soybean TILLING project led by Khalid Meksem at SIU are in progress to further confirm this. The TAG accumulates in oil bodies and the ontogeny of oil body formation from specialized ER structures in soybeans is being further examined. We have over expressed DGATs and the penultimate step for TAG synthesis, lysophosphatidate acyltransferase genes. These lines with enhanced “pull” of hydrocarbon into TAG are being combined with lines with reduced phytate and raffinosaccharides. Changes are also being made to enhance the flow of triose and hexose phosphates into plastids to “push” hydrocarbon into fatty acid synthesis. Methods and their limitations of rapid and accurate determinations of seed compositions coupled with this research will be discussed including transmission and reflectance NIR of bulk samples and single seeds.
Properties of Algal Oil to be Used as Feedstock for Biofuels. N. Abunasser2,1, D. Garcia1, K.Y.S. Ng1,2, S. Salley1,2, J. Wilson3, 1Wayne State University, Detroit, MI, USA, 2National Biofuels Energy Lab, Detroit, MI, USA, 3Next Energy, Detroit, MI USA
Interest in using algal oil as a source for biofuels has increased in recent years. However there are few comprehensive studies of the properties of the oil extracted from the different species being considered. Several of the algae strains under consideration, Dunaliella tertiolecta and Botryococcus braunii, have been cultivated for this purpose. The Dunaliella tertiolecta UTEX LB 999 was grown in Erdschreiber's Medium at room temperature with a 16-8 light/dark cycle, and the Botryococcus braunii UTEX LB 572 was grown in Waris+Soil Extract Medium at room temperature with a 12-12 light/dark cycle. The algal oil was extracted using a hexane extraction, and the amount of oil collected from each strain was noted and compared to literature values. Properties such as the fatty acid content and profiles were measured using GC-MS. The effects of the properties such as fatty acids, unsaturated bonds and viscosity on the quality of resulting biofuels such as biodiesel were considered.
Manipulating Seed Oil Composition for Feedstock Applications. M. Smith, National Research Council of Canada, Plant Biotechnology Institute, Canada
The suitability of seed oils to function as feedstocks for industrial applications could be improved by creating oils with more homogeneous fatty acid composition, and by introducing fatty acids with novel functionality. We are using a transgenic approach to explore the pathways of hydroxy fatty acid, and very long chain monounsaturated fatty acid accumulation in seed oils. Our goal is to produce oil with high levels of a single fatty acid.
Crop Biofactories Initiative – Investigation, Production and Uses of Novel Hydroxylated Fatty Acids. F. Graichen1, A. Warden2, S. Kyi1, V. Haritos2, S. Singh3, X. Zhou3, M. O′Shea1, 1CSIRO Molecular and Health Technologies, Melbourne, Victoria, Australia, 2CSIRO Entomology, Canberra, ACT, Australia, 3CSIRO Plant Industry, Canberra, ACT, Australia
The Crop Biofactories Initiative (CBI) is a jointly funded initiative between CSIRO and GRDC. CBI is a 12 year research initiative with the aim of adding value to the agriculture and the chemicals industries through the development of technologies to produce novel industrial compounds from genetically modified non-food grains crops. Focus areas are: Industrial oils; Complex monomers and Protein BiopolymersThe research in the areas of industrial oils and complex monomers is aimed at the production, evaluation and modification of novel oil and fatty acids for a range of industrial applications.There are significant opportunities in the production of novel hydroxylated oils and fatty acids with novel hydroxy-group positions. We have investigated the synthetic and biological production of hydroxylated fatty acids of a range of structures and their utility in the chemicals and polymers industries.For example alpha–hydroxy fatty acids have a range of interesting properties, some of which are strongly influenced by the self assembly properties of the 2-hydroxy fatty acids. Uses for these novel fatty acids include: monomer for use in commodity polymers, modifiers and adhesives.This paper will cover some synthesis methods for the saturated and unsaturated alpha hydroxy fatty acids as well as industrial applications.
Chair(s): R. Patel, Bristol-Myers Squibb, USA
Composition and Characterization of Solvent-Fractionated Mustard Oil.
M.A. Alim, C.R. Lee, H.J. Kim, K.T. Lee, Department of Food Science and Technology, Chungnam National University, Daejeon, South Korea
Mustard oil (MO) was fractionated at low temperature with acetone. Among all fractions obtained from MO, -24L, i.e., the liquid fraction obtained at -24°C, showed higher levels of oleic (24.7%) and linoleic (22.0%) acids, and lower level of erucic acid (24.5%) compared to other fractions. The results demonstrated that the lowest temperature (-24°C) was most effective for lower levels of erucic acid through crystallization of triacylglycerol (TAG) molecules. Obtained fractions were transesterified with capric acid for sytnehsis of structured lipid. From the economic point of view and reaction efficiency, the ideal mole ratio was 1:3 for both cases when observed the effect of mole ratio on the incorporation of capric acid (CA) into fractions. The content of erucic acid was found reasonable lower level for 24 h reaction time of both cases. The -16L contained 36.7 mg/100g of α-tocopherol which was the highest than those of others. All liquid fractions (-16L = 36.7 and -24L = 33.9 mg/100g, respectively) obtained higher amount of α-tocopherol than those of all solid fractions (-16S = 3.7 and -24S = 4.5 mg/100g, respectively). Within 12 h reaction periods, higher amount of α-tocopherol content was observed for all reaction time courses.
Monitoring of Trans Fatty Acids Content in Dietary Foods from Chungcheong Area of South Korea.
Y.M. Kim, C.R. Lee, H.J. Kim, K.T. Lee, Dept. of Food Science and Technology, Chungnam National University, Daejeon, Korea
This study was designed to determine the trans fatty acid (tFA) contents of 83 take-out and bakery food items in Daejeon and Chungcheong Province. After crude fat extraction by the Folch method, methylation was carried out to analyze the fatty acid compositions by GC, using a SP-2560 column and flame ionization detector. The total fat contents of the bakery foods were in the approximate range of 2~34%. The take-out food maximum fat content among the observed samples was obtained from theater popcorn 13.74~38.09%, while minimum content was observed from the glutinous rice doughnut 2.31~5.44%. The contents of tFA (g/100 g food) were as follows: whipping cream cake = 0.03~1.31 (g/100 g food), pastries = 0.21~2.64 (g/100 g food), cream puff = 0.09~0.43 (g/100 g food), croquette = 0.22~1.99 (g/100 g food), and glutinous rice doughnut = 0.03~0.38 (g/100 g food), toasts, glutinous rice doughnut and twisted doughnut sold in streets = 0.02 ~ 0.56 (g/100 g food), fried items = 0.05 ~ 3.08 (g/100 g food). As a result, most samples showed the tFA content as less than 1 g (g/100 g food) except for sample from theater popcorn and pastries which contained about 2~3 (g/100 g food) as tFA content.
The Enzymatic Extraction of Coconut (Cocos Nucifera Linn. Var. Laguna) Oil.
L.J. Pham1, C.B. Pham2, 1BIOTECH,U.P. Los Baños, Los Baños, Laguna, Philippines, 2BIOZYM Systems Ent., Bay,Laguna, Philippines
One of the major problems of the coconut industry is the low quality of the coconut oil. Oil is obtained from copra meat by pressing in an expeller. However, the copra-derived oil still has to undergo drastic refining which reduces the quality of the oil. Our work aimed to develop a process of coconut oil extraction using enzymes to increase yield and quality of the oil by establishing process steps of enzymatic extraction in a batch mode and optimization of process conditions . Enzyme used was Gamanase from NOVO Industries A/S (Bagsvaard, Denmark) and had an activity of 1,500,000 VHCU/g). Using a batch process at 300 rpm agitation speed in a stirred tank bioreactor,optimization of the effect of pH, temp. and enzyme conc. was performed. Oil yield was 85-92% under optimized process conditions of pH 4.5,temp. at 50C and 0.75 ml gamanase /100 g of coconut meat of enzyme concentration. Oil obtained is a clear colorless oil with a fresh coconut odor and low moisture content (0.6%). Fatty acids of the enzymatically extracted oil was comparable to the refined coconut oil. Quality of the oil produced by the enzymatic extraction minimized the need for refining the oil. This does not only mean a reduction in the processing/refining cost but would also render the oil competitive over other oils in terms of quality and even cost.
Preparation and Esterification Activity Evaluation of CLEAs of Lipases.
B. Lakshmi Anu Prabhavathi Devi, Z. Guo, X. Xu, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark
Commercially available microbial lipases such as Candida antarctica (CALB-L), Candida rugosa (TL100L), Lipase AK Amano 20, Lipase PS Amano 30, Lipase A Amino 12, Lipase M Amano 10, Lipase D Amano 350 and Newlase F were immobilized as their cross-linked enzyme aggregates (CLEAs) using different precipitants and glutaraldehyde as cross-linker. These CLEAs were assayed for esterification of lauric acid with n-propanol in solvent free system. CLEAs of CALB were found to be more active for esterification and others with less or no activity. Among different CLEAs of CALB, the ones obtained using PEG200 (CALB-CLEA-PEG200), acetone (CALB-CLEA-acetone), PEG600 (CALB-CLEA-PEG200) and ammonium sulfate (CALB-CLEA-(NH4)2SO4) as precipitants were found to be more active towards the synthesis of n-propyl laurate. A systematic kinetic study of most active CLEA-CALB-PEG200 was carried out in comparison with its free enzyme powder and commercial Novozym 435. The comparative study revealed that the CALB-CLEA-PEG200 is more stable, reusable and more active heterogeneous biocatalyst with favorable perspectives for its commercial application in esterification process.
Physicochemical and Volatiles Characterization of trans-Free Solid Fats Produced by Lipase-Catalyzed Interesterification.
J.H. Lee1, J.Y. Kim1, C.C. Akoh2, K.T. Lee1, 1Dept. Of Food Science and Technology, Chungnam National University, Daejeon, Korea, 2Dept. of Food Science and Technology, University of Georgia, Athens, GA, USA
Trans-free solid fats were synthesized from fully hydrogenated soybean oil (FHSBO), olive oil (OO) and palm stearin (PS) at different substrate weight ratios (10:20:70, 10:40:50 and 10:50:40) via lipase-catalyzed interesterification. The interesterified products contained mostly TAG (98.8% to 99.0%), and small amounts of MAG and DAG as by-products. The major fatty acids were oleic, palmitic and stearic acid in the interesterified products, and the melting points ranged from 39 to 45 °C . The amount of α-tocopherol was reduced by 75 to 92%. Volatile analysis by solid-phase microextraction indicated that OO and PS had distinct volatile profiles, in which 18 volatiles were retained in interesterified products. Furthermore, some volatiles disappeared or formed during processing. Electronic nose showed that the odor of substrates (OO and PS) were different from each other, and the odors of interesterified products were distinguishable from that of OO or PS. Among the interesterified products, the odor of blend FHSBO:OO:PS of 10:40:50 or 10:50:40 was different from that of blend FHSBO:OO:PS (10:20:70). However, no odor difference was observed between products blend FHSBO:OO:PS 10:40:50 and 10:50:40.
Enzymatic Synthesis and Physicochemical Properties of Fractionated Rice Bran Oil and Conjugated Linoleic Acid to Produce Zero-Trans Margarine Spreads.
J.A. Shin1, P. Adhikari1, C.C. Akoh2, K.T. Lee1, 1Dept. of Food Science and Technology, Chungnam National University, Daejeon, Korea, 2Dept. of Food Science and Technology, University of Georgia, Athens, GA, USA
Rice bran oil (RBO) was fractionated into solid (S-RBO) and liquid fractions (L-RBO) by means of an acetone fractionation at -18°C using a 2-L manufactured lab-scale vessel. The yields of each fraction part were 46.4% (S-RBO) and 53.6% (L-RBO). S-RBO contained just total tocopherols (T-toc) of 7.8 mg/100 g whereas L-RBO contained 33.7 mg/100 g (T-toc) and 0.46 mg/100 g (gamma-oryzanol). Functional-margarine fat stock (SL) was synthesized from S-RBO, palm stearin (PS), and conjugated linoleic acid (CLA) mixture (1:1 mole, CLA: TAG) using Lipozyme TL IM (10% of total substrates, by weight). The effect of various molar ratios (1:1, 1:1.5, 1:2, 1:2.5, and 1:3) with S-RBO and PS was investigated. As increasing PS amount in molar ratio, equivalent carbon number (ECN) 48-50 (POP, PPP, POS and PPS) were slightly increased. Their slip melting points were also increased with ranges from 32-42°C. Finally, large-scale synthesis of SL was performed in a 1-L batch type reactor with 1:2.5 molar ratio of S-RBO:PS. CLA was incorporated around 13-14% into triacylglycerol (TAG) backbone. The ratio of saturated to unsaturated fatty acid in SL was 47-53%. SL synthesized was much spreadable than blends or butterfat spreads when we prepared the spreads using FBF, blends and butterfat.
Development of High Oil Soybeans for Biodiesel Markets Using Nuclear Magnetic Resonance and Molecular Markers.
G. Samii Saket1, G.R. Ablett2, I. Rajcan1, R. Fletcher2, 1University of Guelph, Department of Plant Agriculture, Guelph, ON, Canada, 2Ridgetown Campus, University of Guelph, Ridgetown, ON, Canada
In the era of an increasing product demand and constant price pressure, finding a suitable substitute for petroleum-based fuels is both crucial and inevitable. One of the promising sources of renewable fuels is soybean oil for biodiesel production. Increasing the oil content in soybean seeds would increase the competitiveness of soybean as a source of biodiesel. Crosses between unrelated genetically diverse moderately high oil parents have been made to study the potential for developing high oil soybean cultivars designed specifically for the biodiesel market. Nuclear Magnetic Resonance (NMR) spectroscopy was used in combination with the Soxhlet wet chemistry method, to develop a method for measuring oil content on individual soybean seeds. The new method has been used to measure the seed oil content in different generations of breeding populations to calculate genetic parameters governing the oil content including heritability. Simple Sequence Repeat (SSR) markers have been used to determine the allelic composition at the quantitative trait loci responsible for oil content in the selected breeding materials. The results of this study will help to enhance our understanding of the genetic basis of high oil content and facilitate the development of high oil soybeans for the biodiesel market.
Production and Physicochemical Characterization of Butterfat Fortified α-Linolenic Acid through Enzymatic Interesterification in a Packed-Bed Continuous Reactor.
J.A. Shin1, C.C. Akoh2, K.T. Lee1, 1Dept. of Food Science and Technology, Chungnam National University, Daejeon, Korea, 2Dept. of Food Science and Technology, The University of Georgia, Athens, GA, USA
Functional-butterfat (FBF) was synthesized with four blends (8:6:6, 6:6:8, 6:6:9, and 4:6:10, by weight) of anhydrous butterfat (ABF), palm stearin (PS) and flaxseed oil (FSO) through lipase-catalyzed interesterification in a packed-bed reactor. Flow rate effect of 3, 5, 8 and 10 mL/min on enzymatic interesterification was investigated. As reducing the flow rate, not only melting and crystallization points but also triacylglycerol profiles were changed. All reactions were performed at 5 mL/min in a continuous reactor packed with Lipozyme RM IM. Iodine values (IV) of FBFs were 90 (8:6:6), 104 (6:6:8), 101 (6:6:9), and 117 (4:6:10), respectively. After interesterification, the areas (%) of ECN 36-38 and 48-50 were decreased. Newly produced ECN 42-46 were increased. Alpha-linolenic acid in individual 8:6:6, 6:6:8, 6:6:9 and 4:6:10 FBFs were 16, 21, 23, and 25%, respectively. Respective 8:6:6, 6:6:8, 6:6:9 and 4:6:10 FBFs were 69, 88, 80, and 92% softer than pure butterfat at refrigeration temperature. The polymorphic form of FBFs was changed from β form (the blends) to desirable β′ form (the FBFs). Fat crystal size in FBFs interesterified was smaller (within 1 µm) and agglomerated into spherical crystals.
Synthesis of 3-Ketosteroids Using Cholesterol Oxidase: Effects of Sterol Side Chain Structure.
E.J. Parish, H. Sun, A. Bell, Department of Chemistry and Biochemistry, Auburn University, Auburn, AL, USA
Cholesterol oxidase is an enzyme that has the abilityto selectively oxidize cholesterol to an unsaturated 3-ketoderivative. The usefulness of this transformation is limitedby its selective oxidation of steroids that only contain theisooctal side chain of cholesterol. In order to expand theusefulness of this oxidation technique we have developedthe use of chemical derivatives to construct temporaryside chains of certain steroids that would allow them to beoxidixed by cholesterol oxidase. The side chain of androst-5-en-3,17-diol (a poor substrate for cholesterol oxidase)has been modified to mimic the bulky hydrophobic(isooctal) side chain of cholesterol and various substrateshave been synthesized for investigating the methodologyof applying cholesterol oxidase on poor substrates.
Interruption of Ergosterol Biosynthesis and Growth Impairment of Cryptococcus neoformans by Rationally Designed High Energy Intermediates Analogs of Sterol Catalysis.
R. Vatsyayan, S. Chamala, K. Hernandez, K. Ganapathy, J. Liu, W. Zhou, W.D. Nes, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, USA
The sterol composition of Cryptococcus neoformans (CN) has been characterized and found to contain a lanosterol-ergosterol based pathway. Ergosterol isolated from cultures grown in the presence of deuterated methionine [methyl-2 H3 l contained two 2 H atoms showing that one 2 H atom is lost during the transmethylation reaction. A series of high energy intermediate analogs of the sterol C-24 methyltransferase (SMT) catalyzed reaction and various other enzyme analogs in the sterol pathway were synthesized and tested for their fungicidal activity in vitro against CN. Several of them displayed promising anti-cryptococcal activity with IC50 values ranging from 0.1 mM to 4 mM in vitro against CN. Sterol analysis of the inhibitor treated CN revealed that several inhibitors specifically target the rate-limiting enzyme, SMT, as evident by the cellular accumulation of lanosterol. Transcript profiling of the CN SMT at IC50 and IC90 after inhibitor treatment revealed that the level of SMT transcript increased, including itraconazole treated cells used as a CYP51 control. This previously unrecognized catalyst specific inhibition in CN indicates that the SMT, not synthesized in humans, can be a target for chemotherapy of ergosterol-dependent disease in these opportunistic pathogens.
Lipase-Catalyzed Synthesis of Poly (Hydroxy Fatty Acid) Co-Polymers.
V.K. Mannam, D.G. Hayes, University of Tennessee, Knoxville, TN, USA
Unimolecular polymeric micelles, or UPMs, have been formed biocatalytically in our laboratory in an environmentally-friendly manner for potential utilization as drug delivery vehicles. The UPMs consist of a polyhydric alcohol (pentaerythritol) core, esterified to oligo(ricinoleic acid), and exhibit excellent physical and transport-related properties desired for viscosity index modifier ingredients of biolubricants, namely, high viscosity index and low melting point temperature. To further improve the performance of UPMs for drug delivery and lubricants, the poly(hydroxyacyl) chain length has been increased via lipase-catalyzed co-polymerization of ricinoleic acid and long-chain omega-lactones. Although co-polymerization increased the average degree of polymerization 2-3-fold, to 9-10 monomeric units per oligomer, the melting point point temperature increased significantly. A second means of improvement is to increase the density of the grafted oligo(hydroxyacyl) chains onto the UPM's core is by the enzymatic synthesis of triglycerides that contain a polymerizable acyl group at the middle position and oligo(ricinoleic acid) and the 1 and 3-positions. These molecules can then be joined together via free radical polymerization.
Single Step Biodiesel Production from Soybean Oil Using a Pseudo 4 Stage CSTR System.
D.K. Kim, J.Y. Park, J.S. Lee, S.C. Park, J.P. Lee, K.R. Hwang, Korea Institute of Energy Research, Daejeon, South Korea
With growing concern about renewable fuel, various biodiesel production processes have been studied extensively. To achieve high yield and purity, most commercial biodiesel processes have adopted 2 step production processes with 2 set of reactor and separator. In this study, biodiesel production characteristics were investigated in a pseudo 4 stage continuous stirred tank reactor (CSTR) to develop single step biodiesel production process. The pseudo 4 stage CSTR was manufactured by dividing a cylindrical reactor into 4 parts with separating plates having a hole providing some area for agitator and reactant flow. The term-pseudo was used because the reactants in each stage of the CSTR were mixed with the reactants in the adjacent stage of the CSTR. The production capacity of the pseudo 4 stage CSTR were investigated with various reaction temperatures and total flow rates at a constant molar ratio of methanol to oil, 9:1 and KOH catalyst concentration, 0.8% based on oil mass and 250 rpm agitating speed. FAME content at the outlet of the CSTR was increased with reaction temperature increase. The best performance of the CSTR was accomplished at 80°C reaction temperature, 20 minutes of retention time, methanol to oil molar ratio of 9:1, agitating speed of 250 rpm and 0.8% KOH concentration.
Immobilized Enzymatic Production of Low trans Blend from Fractionated Mustard Oil and Palm Stearin with Linoleic Acid by Response Surface Methodology.
M.A. Alim, M.S. Jeon, J.Y. Kim, Y.J. Kim, K.T. Lee, Department of Food Science and Technology, Chungnam National University, Daejeon, South Korea
Low-trans fat stock(LT) was produced from the fractionated mustard oil (solid phase, S-MO) and palm stearin (PS) through lipase-catalyzed reaction, in which linoleic acid (LA) was incorporated intentionally. For optimizing the reaction condition, response surface methodology was employed with three reaction variables such as substrate mole ratio of S-MO to PS, reaction temperature and reaction time. The predictive model was adequate due to no significant lack of fit and satisfactory level of coefficient of determination (R2 = 0.93). The melting point of LT was affected by substrate mole ratio and reaction time, whereas reaction temperature had no significant effect. Based on ridge analysis, the combination of 1:2 (X1), 65.17 °C (X2) and 21.38 h (X3) was optimized for producing LT with target melting point of 34.57 °C. The LT contained mainly 37.8% palmitic, 24.8% linoleic, 21.3% oleic and 9.7% erucic acid. Solid fat contents were 38.7 and 17.8% at 20 and 30 °C, respectively. These results suggested that RSM can be used to optimize the lipase-catalyzed production of LT.
Physicochemical and Flavor Characteristics of Commercial Olive Oils.
H.Y. Nam, J.Y. Kim, Y.J. Kim, K.T. Lee, Dept. of Food Science and Technology. Chungnam National University, Daejeon, Korea
Physicochemical and volatile characterizations of commercial olive oils (Extra virgin, EVOOL; Pure and refined, PROOL) were investigated. Solid-phase micro extraction (SPME) GC-MS and electronic nose (EN) were used for flavor discrimination. The volatiles identified in EVOOLs include hexanal, 4-hexen-1-ol, (Z)-3-hexen-1-ol, acetic acid, and 2,4-dimethyl-heptane, etc. Proportion of the first principal component was 99.70%, suggesting that each aroma pattern of the 13 EVOOLs could be discriminated by EN. In color study, EVOOL showed L* value of 81.7~92.9, a* value of -28.3~13.5 and b* value of 52.2~139.0. While PROOL showed L* value of 92.2~99.0, a* value of -22.2~-3.2, and b* value of 18.5~55.0. Total phenol and α-tocopherol contents of EVOOL were 6.2~24.9 mg/100g and 5.5~12.8 mg/100g, respectively. Total phenol contents of PROOL ranged from 1.9 to 13.3 mg/100g while α-tocopherol contents showed 7.91~13.88 mg/100g. In Rancimat test, the induction period of 13 EVOOLs showed 31.76~54.04 h while their POV were ranging from 13.5 to 22.9 meq/kg oil. The induction period of PROOLs ranged from 17.37 to 34.72 h while their POV was from 6.83 to 20.31 meq/kg oil.
Enzymatic Conversion of Aromatic Compounds Obtained from Crop Residues.
R. Holser1, R. Harry O'kuru2, 1Richard Russell Research Center, USDA-ARS, Athens, GA, USA, 2NCAUR, USDA, ARS, Peoria, IL, USA
Biomass obtained from crop residues and the related processing wastes typically contain minor amounts of aromatic compounds such as ferulic and p-coumaric acids. These compounds occur as esters and ethers associated with plant cell wall structures and as components of lignin. These compounds exhibit bioactivity and could be used for antimicrobial or antioxidant applications. Additionally, it has been shown that the yield of ethanol increases when biomass is delignified. This suggests that the availability of these compounds will increase with the production of biofuels from biomass. This investigation examined the development of new products incorporating ferulic and p-coumaric acids to impart bioactivity. These compounds were esterified to a variety of substrates including cellulosics and a series of PEG's using commercial esterases. Potential applications include surgical gauzes and controlled release therapeutics.
Development and Characterization of Trans-Free Margarine from Lipase-Catalyzed Interesterification of Avocado and Palm Oil.
Y.J. Lee, Y.M. Kim, H.J. Kim, K.T. Lee, Dept. of Food Science and Technology, Chungnam National University, Daejeon, Korea
Trans-free margarine stock (TFMS) was produced by lipase-catalyzed synthesis of fully hydrogenated soybean oil (FHSO), avocado oil (AO) and palm oil (PO) at 1:5:4, 1:4:5, and 1:2:7 (by vol.). Each reaction was conducted at a shaking bath for various reaction times (1,2,3,6,12,24 and 48h) at 65°C using Lipozyme RM IM. Major fatty acid profiles of TFMS were oleic (44.9-39.4 area%), palmitic (28.3~36.2%), and stearic acid (10.9~12.6%). After differential scanning calorimetry (DSC) analysis, each thermogram was used for obtaining solid fat content (SFC). SFC of FHSO:AO:PO (1:5:4, 12h reaction) was 9.17% at 35°C with the melting point of 37.3°C. The selected reaction condition (1:5:4 ratio of FHSO:AO:PO) leads to appropriate SFC and melting profiles for margarine stock and then performed in a 1-L batch type reactor for scale-up production of TFMS. The trans fatty acids content in TFMS was below 0.1 area%. Besides, acid, saponification and iodine values of TFMS were 0.4, 173.9 and 58.6, respectively. The newly synthesized peaks of triacylglycerol molecules in TFMS were observed by reverse-phase HPLC equipped with ELSD. Total tocopherol contents of TFMS were 9.5 mg/100g. SFC (%) and hardness of scaled-up TFMS were analyzed, and its slip melting point (SMP) was 37.5°C.
Physical and Chemical Effects on Gum Phase from Enzymatic Treated Crude Oil.
K. Christensen, J. Borch Soe, Danisco A/S, Brabrand, Denmark
Alteration of the degumming process towards bio processes increasingly has come into sight, owing to the growing oil prices. The application of enzymes is valuable, however, often associated with production facility costs. In this paper, a highly advantages new enzyme will be introduced. The enzyme has been explored with regard to degumming and has demonstrated great performance both in respect to phospholipid degradation as well as to process optimization in forms of (oil yield), capacity and cost reduction. The focus particular is held on chemical modifications, accomplished by the enzyme as well as on favourable physical changes, observed in the enzymatic process compared to conventional degumming.
Physicochemical Properties of Medium- And Long-Chain Triacylglycerol (MLCT)-Enriched Shortening.
A. Norlelawati 1, L. Seong-Koon1, C. Ling-Zhi1, K. Soo-Peng1, L. Kamariah3, L. Oi-Ming1, T. Chin-Ping2, Y. Mohd Suria Affandi4, 1Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia, 2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia, 3Department of Biotechnology. Malaysian Agricultural Research and Development Institute (MARDI), Kuala Lumpur, Malaysia, 4Golden Hope Research Centre (GHRC), Selangor, Malaysia
Six binary blends of medium-and long-chain triacylglycerols (MLCT) and soft stearin (PS) and four ternary blends of MLCT: PS: palm olein IV60 (PO) containing 13-21% of medium chain fatty acid (MCFA) were produced. The liquefied MLCT and PS were mixed in proportions ranging from 40-90% with 10% increments (w/w) for the binary blends while for the ternary blends, 40% and 50% of MLCT were selected with substitution of 10% and 20% of palm olein (PO) to PS. The solid fat content (SFC) and heating and cooling profiles using differential scanning calorimeter (DSC) of these blends were determined. Results obtained from both SFC and melting profile showed that all the blends melted completely at 55°C. The high melting point is due to the stearic acid content in MLCT. Three ratios of both binary and ternary blends had % SFC in the range of 15-25% at 25°C which is suitable for producing cake shortenings. Shortenings formulated from 4:4:2, 5:4:1 and 5:3:2 (MLCT: PS: PO) blends were subsequently chosen, with production cost considered. The score given by the untrained panelists on the Madeira cakes produced showed that the samples with 5:3:1 and 5:3:2 had no significant difference (p<0.05) compared to commercial shortenings in terms of overall acceptability.
Lipoxygenase, a Key Enzyme in Bioconversion of Linoleic Acid into Trihydroxy-Octadecenoic Acid by Pseudomonas Aeruginosa PR3.
J.H. Bae1, M.J. Suh1, C.T. Hou2, H.-R. Kim1, 1Dept. of Animal Science & Biotechnology, Kyungpook National University, Daegu, Korea, 2Microbial Genomic and Bioprocessing Research Unit, NCAUR/ARS/USDA, Peoria, IL, USA
Lipoxygenases catalyze the oxidation of unsaturated fatty acids with (1Z,4Z)-pentadiene structure leading to the formation of conjugated (Z,E)-hydroperoxydienoic acids, which are in turn resulting into the production of hydroxy lipid. These enzymes are widely distributed in plants, animals, and microorganisms. Productions of hydroxy fatty acids from different fatty acid substrates by microorganisms were well studied. Among those microorganisms, Pseudomonas aeruginosa PR3 is well known to produce mono-, di- and trihydroxy fatty acids from their corresponding substrates. PR3 converted linoleic acid to trihydroxy fatty acid suggesting that lipoxygenase could possibly be involved in this bioconversion. Therefore, we tried to isolate a lipoxygenase from P. aeruginosa PR3. As a result, we partially purified a lipoxygenase with 20.6 of purification fold and 3.08 % of recovery. The Km and Vmax values of the purified enzyme were 4.923 mM and 0.815 Î¼Mâ€¢ min-1â€¢mg-1, respectively. The optimal pH and temperature for activity were pH 6.0 and 60â„ƒ, respectively. The purified enzyme was highly heat stable and showed substrate specificity only for polyunsaturated fatty acids carrying double bonds more than two.
Supercritical Fluid Purification of Phytosterol Esters from an Enzymatically Modified Soybean Oil Deodorizer Distillate.
C.F. Torres, T. Fornari, G. Torrelo, F.J. Señoráns , G. Reglero, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
Separation of phytosteryl esters, fatty acid ethyl esters and tocopherols via countercurrent supercritical carbon dioxide extraction was studied. The raw material consisted of a product obtained after a two-step enzymatic reaction carried out upon soybean oil deodorizer distillates. This material was comprised of fatty acid ethyl esters, tocopherols and phytosteryl esters, together with minor amounts of squalene, free fatty acids, free sterols and triacylglycerols. Different extraction conditions were studied in an isothermal countercurrent column, without reflux, in a range of pressure from 200 to 280 bar, temperatures of 45-55 ï‚°C and solvent-to-feed ratios from 15 to 35 kg/kg. Total extraction of fatty acid ethyl esters was observed and the fractionation of tocopherols and phytosteryl esters was also evaluated. Hence, a raffinate product containing up to 82.4 % w/w of phytosteryl esters with satisfactory yield (72 %) was obtained at 250 bar and 55ï‚°C (solvent-to-feed = 35).
A Modular Biosynthetic Approach for the Metabolic Engineering of Functionalized Terpenoids in E. Coli.
D. Morrone, R. Peters, Iowa State University Department of Biochemistry, Biophysics, and Molecular Biology, Ames, IA USA
Natural products derived from plants provide a rich source of chemical diversity. Plants produce 100,000 known chemically diverse low molecular weight compounds classified as secondary metabolites, which perform an array of biological functions including antimicrobial defense. Terpenoids, comprised of various numbers of five carbon isoprene units, form the largest family of known natural products (> 40,000 to date) and their extreme chemical complexity is often refractory to synthesis. Some of these intricate terpenes are elaborated, often via cytochrome P450 enzymes, into therapeutic agents such as artemisinin and Taxol™. Recently, metabolic engineering from central metabolism in E. coli has focused on production of the lipophilic terpene olefins by their according synthases. Oxygen incorporation into these molecules by plant cytochrome P450s provides a ready means for further semi-synthetic derivitizations of these hydrocarbons. In addition to sequential involvement in pathways, the inherent plasticity of terpene synthases and P450 oxygenases with respect to substrate selection and product outcome allows for their modular utilization in combinatorial biosynthetic pathways. Accordingly, we have developed a modular biosynthetic approach for the metabolic engineering of functionalized terpenoids in E. coli.
Effect of PH on Selective Esterification of Borage Oil Fatty Acid to Gamma-Linolenic Acid Concentration.
M.E. Zúñiga, C. Soto, Universidad Catolica de Valporaiso, Valparaiso, Chile
Borage oil is the richest source of gamma-linolenic fatty acid (GLA), which is the first intermediate formed during the conversion of LA to prostaglandins. The later has anti-inflammatory and anti-thrombotic properties. Due to this fact, the oil is applied in several diseases as arthritis, certain skin problems and cardiovascular diseases among others. To obtain better therapeutic results it is suggested to concentrate the GLA. One of the techniques employed to fatty acid concentration is enzymatic esterification with lipases. These enzymes are selectives and they operate under soft conditions preventing the fatty acid damage. The purpose of this work is to evaluate the pH effect on GLA concentration by means of selective enzymatic esterification of borage oil fatty acid. Borage oil fatty acids were obtained by an unspecific enzymatic oil hydrolysis, then the free fatty acids were recovered using hexane, which was retired in a vacuum rotary evaporator. The experiences were done with Lipolase 100 T lipase, at 37º C, 100 mg of fatty acid and 20 uL of 200 mM phosphate buffer and a pH of 6, 7 and 8. The esterification degree was determined by free fatty acid titration. Also the fatty acid profile was determined by gas chromatography.