Regiospecific ethanolysis of homogenous TAG with immobilized Candida antarctica lipase (Novozym 435) was studied using trioleoylglycerol (TO) as a model substrate. Optimization of the reactant weight ratio revealed that the 2-MAG reaction yield increased when a larger amount of ethanol was used. These results suggested that Novozym 435 showed strict regiospecificity in an excess amount of ethanol. The process optimization (reaction temperature and reactant molar ratio) and a study of lipase specificity for various substrates were performed. Under the optimized conditions (ethanol/TO molar ratio = 77:1 and 25°C), 2-monooleoylglycerol (2-MO) was obtained in more than 98% content among glycerides of the reaction mixture and approximately 88% reaction yield in 4 h. The above reaction conditions were applied for ethanolysis of tridocosahexaenoylglycerol, trieicosapentaenoylglycerol, triarachidonoylglycerol, tri-α-linolenoylglycerol, and trilinoleoylglycerol. Reaction yields ranging from 71.9 to 93.7% were obtained in short reaction times (2.5 to 8 h). Purified (>98%) 2-MO and 2-monodocosahexaenoylglycerol (2-MD) were reesterified with caprylic acid by immobilized Rhizomucor miehei lipase (Lipozyme IM) to afford symmetrical structured TAG. At a stoichiometric ratio of 2-MAG/caprylic acid, 25°C and 2-5 mm Hg vacuum, the glyceride composition of the esterification mixture was approximately 95% 1,3-dicapryloyl-2-oleoylglycerol (COC) at 4 h, and 96% 1,3-dicapryloyl-2-docosahexaenoylglycerol (CDC) at 8 h. The regioisomeric purity of both COC and CDC was 100%.Although 2-monoacylglycerols (2-MAG) have very good emulsifying properties and are physiologically essential molecules involved in lipid adsorption, they have limited industrial applications owing to the difficulty of their synthesis. Chemical synthetic methods have many reaction steps that require tedious purification, and the final yields are often very low (1). Enzymatic hydrolysis of triacylglycerols (TAG) with 1,3-specific lipases has limited success as a result of high acyl migration rates, which affect the reaction yield and final product purity. This method was improved by engineering the reaction media, i.e., working in a microemulsion system (2) or in a mixture of organic solvents containing a low amount of water (3). Although the rates are higher, they involve the use of surfactants such as sodium bis(2-ethylhexyl)sulfosuccinate (AOT), which complicate the product recovery (2), and unusual solvents, such as trichlorotrifluoroethane (3).Another enzymatic method using 1,3-specific lipases is the alcoholysis (usually ethanolysis) of TAG (4-6). The reaction was performed in an organic solvent and required a strict control of the water activity in the system (5,6). All the preceding enzymatic methods gave low reaction yields for polyunsaturated 2-MAG because of the low activity of available 1,3-specific lipases on polyunsaturated TAG.2-MAG are convenient synthetic blocks for the synthesis of symmetrical structured TAG (SSTG) by enzymatic esterification with a desi...

Resting cell suspensions of seven Nocardia species catalyzed the production of 10-hydroxystearic acid from oleic acid. Nocardia cholesterolicum N R R L 5767 gave a good yield with optimum conditions at pH 6.5 and 40 ° C. Yields exceeding 90% can be obtained within 6 h with 0.1 g cells (dry weight) and 178 mg oleic acid in 10 ml of 0.05 M sodium phosphate buffer (pH 6.5). In addition, minor amounts of 10-ketostearic acid were formed as a by-product. The reaction proceeded via hydration of the double bond as shown by labeling experiments with deuterium oxide and 180-labeled water. The system was specific for fatty acids with cis unsaturation at the 9 positon.

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Sixty‐two cultures from the Agricultural Research Service (ARS) Culture Collection and 10 cultures isolated from soil and water samples in Illinois were screened for their ability to convert agricultural oils to value‐added industrial chemicals. A new compound, 7,10‐dihydroxy‐8(E)‐octadecenoic acid (DOD), was produced from oleic acid at a yield of greater than 60% by bacterial strain PR3 which was isolated from a water sample in Morton, IL. To our knowledge, DOD has not been previously reported. The optimum time, pH and temperature for the production of DOD were 2 days, 7.0, and 30°C, respectively. The production of DOD is unique in that it involves hydroxylation at two positions and rearrangement of the double bond of the substrate molecule.

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C 1 to C 8 ), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH 2 or NADPH 2 could act as an electron donor. Formate and NAD + (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD + and secondary alcohol dehydrogenase generated the NADH 2 required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.

The bioconversions of oleic acid, linoleic acid, and ricinoleic acid by Pseudomonas aeruginosa PR3 were investigated. The conversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was improved to better than 80% yields by modifying the culture medium and reaction parameters. The microbial cultures were stable and retained the same level of DOD production capacity for up to 6 mon as examined herewith. Strain PR3 did not bioconvert linoleic acid. However, strain PR3 converted ricinoleic acid to a novel compound, 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD), with a yield of about 35%. The product was further metabolized by strain PR3. TOD was purified by thin-layer chromatography, and its structure was determined by 1 H and 13 C nuclear magnetic resonance and mass spectrometry. This is the first report on the production of 7,10,12-trihydroxy unsaturated fatty acid by microbial transformation. JAOCS 75, 875-879 (1998).

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Among the three groups of natural products (starch, protein and fat), fat and oil are the most under-investigated. The US has a large amount of surplus soybean oil annually, and using vegetable oils or their component fatty acids as starting material provides a new opportunity for bioindustry. Vegetable oils are relatively inexpensive and can be used to manufacture value-added products such as oxygenated oils and fatty acids. Oxygenated fatty acids are common in nature and are important industrial materials. They exist both in mammals and plants. Microorganisms oxidize fatty acids either at the terminal carbon or inside of the acyl chain to produce hydroxyl or keto fatty acids. In our continuing effort to produce value-added products from vegetable oils, we discovered more than one dozen novel oxygenated fatty acids through biotransformation. Microbial hydratase is a carbon 10 positional specific enzyme. Many of these new oxygenated fatty acids possess physiological activities and can be used as biomedicals, in addition to their known applications such as specialty chemicals. The position of hydroxyl groups on the fatty acyl carbon chain plays an important role in the activity against certain specific plant pathogenic fungi. Bacillus megaterium ALA2 converted polyunsaturated fatty acids (PUFA) in different ways. It converted omega-6 PUFAs to a mixture of diepoxy bicyclic, tetrahydrofuranyl rings, and/or trihydroxy groups in their molecules while the products from omega-3 PUFAs produced only hydroxyl tetrahydrofuranyl ring products. The monooxygenase gene of strain ALA2 was sequenced and is a soluble, self-sufficient P450(BM-3) subclass that was highly homologous with the wild-type protein. This new enzyme also possessed a significant high homology in all of the expected reductase regions as well. Fat and oil represent an area with tremendous opportunity for new biotechnology to explore.