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Journal of the American Oil Chemists' Society (v.74, #11)

Special issue: Biotechnology by Ching T. Hou (pp. 1343-1343).

Biocatalytic synthesis of some chiral drug intermediates by oxidoreductases by Ramesh N. Patel; Ronald L. Hanson; Amit Banerjee; Laszlo J. Szarka (pp. 1345-1360).
Chiral intermediates were prepared by biocatalytic processes with oxidoreductases for the chemical synthesis of some pharmaceutical drug candidates. These include: (i) the microbial reduction of 1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]-1-butanone (1) to R-(+)-1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]-1-butanol (2) [R-(+)-BMY 14802], an antipsychotic agent; (ii) the reduction of N-4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide (3) to the corresponding chiral alcohol (4), an intermediate for d-(+)-N-4-{1-hydroxy-2-[(-methylethyl)amino]ethyl}phenyl methanesulfonamide [d-(+) sotalol], a β-blocker with class III antiarrhythmic properties; (iii) biotransformation of Nɛ-carbobenzoxy (CBZ)-l-lysine (7) to Nɛ-CBZ-l-oxylysine (5), an intermediate needed for synthesis of (S)-1-[6-amino-2-{[hydroxy(4-phenylbutyl)phosphinyl]oxy}1-oxohexyl]-l-proline (ceronapril), a new angiotensin converting enzyme inhibitor (6) and (iv) enzymatic synthesis of l-β-hydroxyvaline (9) from α-keto-β-hydroxyisovalerate (16). l-β-Hydroxyvaline (9) is a key chiral intermediate needed for the synthesis of S-(Z)-{[1-(2-amino-4-thiazolyl)-2-{[2,2-dimethyl-4-oxo-1-(sulfooxy)-3-azetidinyl] amino}-2-oxoethylidene]amino}oxyacetic acid (tigemonam) (10), an orally active monobactam.

Keywords: Antihypertensive drug (ceronapril); antipsychotic agent [R-(+)-BMY 14802]; biocatalysis; β-blocker with class III antiarrhythmic properties (d-sotalol); chiral drug intermediates; monobactam (tigemonam); oxidoreductases

Chemoenzymatic approaches to SCH 56592, a new azole antifungal by Brian Morgan; Brent R. Stockwell; David R. Dodds; David R. Andrews; Anantha R. Sudhakar; Christopher M. Nielsen; Ingrid Mergelsberg; Arne Zumbach (pp. 1361-1370).
Chemoenzymatic approaches to the synthesis of two key chiral-precursors of a new azole antifungal agent, SCH 56592, are described. In particular, the enzymatic diastereoselective acylation of 2-benzyloxy-3-pentanol (7) was developed to produce (2S;3R)-7 in >97% diastereomeric excess (de) from otherwise unusable mixtures of (2S,3R)/(2S,3S)-7 (40–80% de). The selectivity and reactivity of commercially available Candida rugosa and Mucor miehei lipases are compared for the acylation of 7 and the hydrolysis of the corresponding butyrate 16a. Of the 17 C. rugosa enzyme preparations that were examined for acylation of 7, two purified enzyme preparations showed no reactivity, five enzymes showed high diastereoselectivity with preference for the (2S,3R)-isomer, and seven showed a slight preference for the (2S,3S)-isomer.

Keywords: Acylation; azole antifungal; biocatalysis; Candida rugosa ; diastereoselective; lipase; Mucor miehei ; vinyl esters

Facile purification of a C-terminal extended His-tagged Vibrio mimicus arylesterase and characterization of the purified enzyme by Ya-Lin Lee; Rey-Chang Chang; Jei-Fu Shaw (pp. 1371-1376).
Vibrio mimicus arylesterase, a 20 kDa protein, is a multifunctional enzyme with thioesterase and chymotrypsin-like activities. Because an affinity His-tag (six consecutive histidine affinity tag) directly to the protein caused the loss of enzyme activity, a hexadecapeptide with His-tag, ADPNSSSVDKLAAALEHHHHHH encoded from vector pET-20b(+) was constructed to extend from the carboxyl terminus of the arylesterase. This Histagged protein retained enzyme functions. Thermal unfolding behavior of both proteins was almost identical, and their T m values were near 54°C as monitored by circular dichroism. Tryptic cleavage of the functional His-tagged enzyme produced two smaller proteins, which still possessed enzyme activity and which suggested that the additional peptide extended on the protein surface. The spacing peptide between His-tag and arylesterase successfully prevented the interference of the His-tag to the enzyme functions. The kinetic studies showed that the esterase and thioesterase activities of the His-tagged enzyme were similar to those of the wild type. On the other hand, the catalytic efficiency of chymotrypsin-like activity of the His-tagged protein was two times higher than that of the wild type.

Keywords: Arylesterase; enzyme purification; His-tag; recombinant protein

Purification and characterization of a cold-active protease from psychrotrophic Serratia marcescens AP3801 by Yasutaka Morita; Kenji Kondoh; Quamrul Hasan; Toshifumi Sakaguchi; Yuji Murakami; Kenji Yokoyama; Eiichi Tamiya (pp. 1377-1383).
Protease activity was detected in the culture medium of Serratia marcescens AP3801 grown at 10°C, which was isolated from soil collected from the top of a mountain. The enzyme, designated as CP-58 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographies. The molecular mass of the protease was 58 kDa, and its isoelectric point was close to 6.0. Maximal activity toward azocasein was observed at 40°C and from pH 6.5 to 8.0. The activity was strongly inhibited by 1,10-phenanthroline, suggesting that the enzyme is a metalloprotease. The N-terminal amino acid sequence was Ser-Leu-Asn-Gly-Lys-Thr-Asn-Gly-Trp-Asp-Ser-Val-Asn-Asp-Leu-Leu-Asn-Tyr-His-Asn-Arg-Gly-Asn (or Asp)-Gly-Thr-Ile-Asn-Asn-Lys-Pro-Ser-Phe-Asp-Ile-Ala. A search through databases for sequence homology aligned CP-58 protease with metalloprotease. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-58 protease has a broader specificity than other proteases against the peptide substrate.

Keywords: Cold-active protease; extracellular protease; metalloprotease; psychrotrophic bacteria; Serratia marcescens AP3801

Enantioselective formation of an α, β-epoxy alcohol by reaction of methyl 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate with titanium isopropoxide by George J. Piazza; Thomas A. Foglia; Alberto Nuñez (pp. 1385-1390).
Methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate (threo isomer) was generated from linoleic acid by the sequential action of an enzyme and two chemical reagents. Linoleic acid was treated with lipoxygenase to yield its corresponding hydroperoxide [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid]. After methylation with CH2N2, the hydroperoxide was treated with titanium (IV) isopropoxide [Ti(O-i-Pr)4] at 5°C for 1 h. The products were separated by normal-phase high-performance liquid chromatography and characterized with gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Approximately 30% of the product was methyl 13(S)-hydroxy-9(Z), 11(E)-octadecadienoate. Over 60% of the isolated product was methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate. After quenching Ti(O-i-Pr)4 with water, the spent catalyst could be removed from the fatty products by partitioning between CH2Cl2 and water. These results demonstrate that Ti(O-i-Pr)4 selectively promotes the formation of an α-epoxide with the threo configuration. It was critically important to start with dry methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate because the presence of small amounts of water in the reaction medium resulted in the complete hydrolysis of epoxy alcohol to trihydroxy products.

Keywords: Epoxide; hydroperoxide; linoleic acid; lipoxygenase; titanium isopropoxide

Characterization of new yeast lipases by Ching T. Hou (pp. 1391-1394).
Previously, we used a simple, sensitive agar plate method to screen lipase activity from 1229 selected cultures, including 508 bacteria, 479 yeasts, 230 actinomycetes and 12 fungi, that covered many genera and species. About 25% of the cultures tested were lipase-positive. We also expanded our screening method to focus specifically on the pH dependence and thermostability of these lipase activities. In this report, we have characterized 25 yeast lipases, obtained from our screening program, on the basis of their positional specificity against triglycerides. Lipase was produced by growing cultures on nutrient medium in the presence of vegetable oil at 25°C for 4 d. Of the 25 new yeast lipases analyzed, 19 showed 1,3-positional specificity and 6 showed random specificity. No 2-positional specific lipases were found. Among those cultures with highest lipase activity are: Candida silvicola NRRL YB-2846 (random); Candida sp. 55 (random); Candida sp. 125 (random); Pichia americana NRRL Y-2156 (1,3-specific); P. muscicola NRRL Y-7005 (random); P. petersanii NRRL YB-3808 (1,3-specific); and Yarrowia lipolytica NRRL YB-423 (random). Characterization of Candida sp. strain 55 lipase on its substrate preference showed that this enzyme hydrolyzed soybean oil triglyceride species LLLn, LLL, LLO, and LLP more readily than LOO, LOP, OOO, LOS, and POO, where L=linoleic, Ln=linolenic, O=oleic, P=palmitic, and S=stearin.

Keywords: Lipases; positional specificity; yeasts

Changes in hydrolysis specificities of lipase from Rhizomucor miehei to polyunsaturated fatty acyl ethyl esters in different aggregation states by Yoshitsugu Kosugi; Qing-long Chang; Kenji Kanazawa; Hiroshi Nakanishi (pp. 1395-1399).
Hydrolysis specificities of lipase from Rhizomucor miehei were compared for various fatty acyl ethyl esters. Activity yields of immobilized lipases, measured with 1 mM substrate, were more than 100%. Differences in hydrolysis rate and affinity for the substrates between lipase preparations were also typically higher during hydrolysis of substrates at 100 mM than at 1 mM, indicating better mass transfer effects for 1-mM substrates. The native lipase showed higher affinity for polyunsaturated fatty acid substrates at 1 mM than at 100 mM. Hydrolysis rates for 1-mM substrates were observed with immobilized lipases, fixed on anion exchange resin with glutaraldehyde and on cation exchange carrier with carbodiimide, and suggested some modification of the basic amino acid related to the lid of R. miehei lipase. Activation with these bifunctional reagents was not observed for 100-mM substrates, indicating that interfacial activation always occurred because of aggregation of 100-mM substrates. These results show that lipase from R. miehei recognizes molecular aggregation of lipids, and that various changes occur in the hydrolysis specificities for fatty acids.

Keywords: Aggregation; fatty acid; immobilized lipase; interfacial activation; lipase; polyunsaturated fatty acid; specificity

Additive effects of acyl-binding site mutations on the fatty acid selectivity of Rhizopus delemar lipase by Robert R. Klein; Gregory King; Robert A. Moreau; Gerald P. McNeill; Pierre Villeneuve; Michael J. Haas (pp. 1401-1407).
The fatty acid specificity and pH dependence of triacylglycerol hydrolysis by the Rhizopus delemar lipase acylbinding site mutant Val206Thr+Phe95Asp (Val, valine; Thr, threonine; Phe, phenylalanine; Asp, aspartic acid) were characterized. The activity of the double mutant prolipase was reduced by as much as 10-fold, compared to the wild-type prolipase. However, the fatty acid specificity profile of the enzyme was markedly sharpened and was dependent on the pH of the substrate emulsion. At neutral pH, strong preference (10-fold or greater) for hydrolysis of triacylglycerols of medium-chainlength fatty acids (C8:0 to C14:0) was displayed by the variant prolipase, with no hydrolysis of triacylglycerols of short-chain fatty acids (C4:0 to C6:0) and little activity manifested toward fatty acids with 16 or more carbons. At acidic pH values, the fatty acid selectivity profile of the double mutant prolipase expanded to include short-chain triacylglycerols (C4:0, C6:0). When assayed against a triacylglycerol mixture of tributyrin, tricaprylin and triolein, the Val206Thr+Phe95Asp prolipase displayed a high selectivity for caprylic acid and released this fatty acid at least 25-fold more efficiently than the others present in the substrate mixture. When presented a mixture of nine fatty acid methyl esters, the wild-type prolipase showed a broad substrate specificity profile, hydrolyzing the various methyl esters to a similar extent. Contrastingly, the double mutant prolipase displayed a narrowed substrate specificity profile, hydrolyzing caprylic methyl ester at nearly wild-type levels, while its activity against the other methyl esters examined was 2.5- to 5-fold lower then that observed for the wild-type enzyme.

Keywords: Fatty acid specificity; lipase; methyl ester hydrolysis; molecular modeling; mutagenesis; protein structure; triacylglycerol hydrolysis

Enzymatic synthesis of position-specific low-calorie structured lipids by Casimir C. Akoh; Lisa N. Yee (pp. 1409-1413).
An immobilized sn-1,3-specific lipase from Rhizomucor miehei (IM 60) was used to catalyze the interesterification of tristearin (C18:0) and tricaprin (C10:0) to produce low-calorie structured lipids (SL). Acceptable product yields were obtained from a 1:1 mole ratio of both triacylglycerols with 10% (w/w of reactants) of IM 60 in 3 mL hexane. The SL molecular species, based on total carbon number, were 44.2% C41 and 40.5% C49, with 3.8 and 11.5% unreacted tristearin C57 and tricaprin C27, respectively, remaining in the product mixture. The best yield of C41 species (44.3%) was obtained with zero added water. Tricaprylin (C8:0) was also successfully interesterified with tristearin in good yields at 1:1 mole ratio. Products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters, such as substrate mole ratio, enzyme load, time course, added water, reaction media, and enzyme reuse, were also investigated. Hydrolysis by pancreatic lipase revealed the specific fatty acids present at the sn-1,3 positions of SL.

Keywords: Interesterification; lipase; low-calorie lipids; Rhizomucor miehei ; structured lipids; tricaprin; tricaprylin; tristearin

Water activity-adjusted enzymatic partial hydrolysis of phospholipids to concentrate polyunsaturated fatty acids by Masayo Ono; Masashi Hosokawa; Yoshikazu Inoue; Koretaro Takahashi (pp. 1415-1417).
Selective partial hydrolyses of egg yolk phospholipid and squid skin phospholipid were carried out. By keeping the water activity (a w) of Lipozyme IM at an intermediate level, it was easy to concentrate docosahexaenoic acid (DHA). It was also possible to concentrate both DHA and arachidonic acid (AA) simultaneously to a certain level under this a w range. However, it was impossible to concentrate AA alone when DHA was present. Though there is a limitation in concentrating AA exclusively, the proposed a w-adjusted hydrolytic reaction is a promising way for preparing phospholipids rich in DHA.

Keywords: Arachidonic acid; DHA; docosahexaenoic acid; formula; hydrolysis; lipase; Lipozyme; partial hydrolysis; phospholipid; water activity

The preparation of concentrates of eicosapentaenoic acid and docosahexaenoic acid by lipase-catalyzed transesterification of fish oil with ethanol by Gudmundur G. Haraldsson; Björn Kristinsson; Ragnheidur Sigurdardottir; Gudmundur G. Gudmundsson; Harald Breivik (pp. 1419-1424).
The objective of this study was to investigate the use of lipases as catalysts for producing concentrates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil as an alternative to conventional chemical procedures. Transesterification of fish oil with ethanol was conducted under anhydrous solvent-free conditions with a stoichiometric amount of ethanol. Among the 17 lipases tested, the results showed that Pseudomonas lipases had the highest activity toward the saturated and monounsaturated fatty acids in the fish oil, much lower activity toward EPA and DHA and, at the same time, good tolerance toward the anhydrous alcoholic conditions. With 10 wt% of lipase, based on weight of the fish oil triacylglycerol substrate (15% EPA and 9% DHA initial content), a 50% conversion into ethyl esters was obtained in 24 h at 20°C, in which time the bulk of the saturated and monounsaturated fatty acids reacted, leaving the long-chain n-3 polyunsaturated fatty acids unreacted in the residual mixture as mono-, di-, and triacylglycerols. This mixture comprised approximately 50% EPA+DHA. Total recovery of DHA and EPA was high, over 80% for DHA and more than 90% for EPA. The observed fatty acid selectivity, favoring DHA as a substrate, was most unusual because most lipases favor EPA.

Keywords: DHA; EPA; ethanolysis; n-3 fatty acids; fish oil; lipase; Pseudomonas fluorescens lipase (PFL); Pseudomonas species lipase (PSL); transesterification

Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid by Harald Breivik; Gudmundur G. Haraldsson; Björn Kristinsson (pp. 1425-1429).
Because of the complexity of marine lipids, polyunsaturated fatty acid (PUFA) derivatives in highly purified form are not easily prepared by any single fractionation technique. The products are usually prepared as the ethyl esters by esterification of the body oil of fat fish species and subsequent physicochemical purification processes, including short-path distillation, urea fractionation, and preparative chromatography. Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils. At room temperature in the presence of Pseudomonas sp. lipase and a stoichiometric amount of ethanol without any solvent, efficient transesterification of fish oil was obtained. At 52% conversion, a concentrate of 46% eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) was obtained in excellent recovery as a mixture of mono-, di-, and triacylglycerols. The latter can be easily separated from the saturated and monounsaturated ethyl esters and converted into ethyl esters either by conventional chemical means or enzymatically by immobilized Candida antarctica lipase. Urea-fractionation of such an intermediary product can give an EPA+DHA content of approximately 85%.

Keywords: Candida antarctica ; DHA; enrichment; EPA; esterification; fish oil; lipase; n-3 fatty acids; Pseudomonas sp

Production of high yields of docosahexaenoic acid by Schizochytrium sp. strain SR21 by T. Yaguchi; S. Tanaka; T. Yokochi; T. Nakahara; T. Higashihara (pp. 1431-1434).
The culture conditions for high-yield production of docosahexaenoic acid (DHA) by Schizochytrium sp. strain SR21 were investigated in a fermenter. With increasing carbon (glucose) and nitrogen (corn steep liquor and ammonium sulfate) sources (up to 12% glucose) in the medium, DHA productivity increased without a decrease in growth rate, i.e., 2.0, 2.7, and 3.3 g DHA/L/d with 6, 10, and 12% glucose, respectively. Eventually, 48.1 g dry cells/L and 13.3 g DHA/L were produced in 4 d with 12% glucose. DHA productivity was decreased with 15% glucose, i.e., 3.1 g/L/d. With 12% glucose, the lipid content was 77.5% of dry cells, and DHA content was 35.6% of total fatty acids. The lipid was composed of about 95% neutral lipid and 5% polar lipid. In polar lipids, the contents of phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylinositol were 74, 11, and 5%, respectively. The PC profile was simple, 70% of PC molecules were 1-palmitoyl-2-DHA-PC and 1.2-di-DHA-PC. These results indicate that Schizochytrium sp. strain 21 is an excellent source for microbial DHA production, including not only the acid form of DHA but also 2-DHA-PC.

Keywords: DHA production; docosahexaenoic acid; docosapentaenoic acid and n-6 DPA; DPA; Schizochytrium

Industrial high-performance liquid chromatography purification of docosahexaenoic acid ethyl ester and docosapentaenoic acid ethyl ester from single-cell oil by R. Yamamura; Y. Shimomura (pp. 1435-1440).
The use of polyunsaturated fatty acids (PUFA) as medicine or in functional diets requires high purity. An industrial purification method for PUFA from Schizochytrium sp. SR21 oil was investigated. This oil contains fewer unwanted components than fish oils. Docosahexaenoic acid and docosapentaenoic acid ethyl esters (DHA-E and DPA-E) were prepared by treatment of this oil with ethanol and 1 N potassium hydroxide in hexane. DHA-E and DPA-E were purified by an industrial high-performance liquid chromatography (HPLC) plant. The separation plant consists of two columns (400 mm i.d., 1,000 mml) with temperature-controlled water jackets and double-plunger (four heads) injection and eluent pumps. This plant was computer-controlled and equipped with an explosion-prevention system. The packed material was octadecylsilica (reverse-phase ODS), and the eluent was methyl alcohol/water (98:2). DHA-E and DPA-E from single-cell oil were highly purified by this industrial HPLC method in a one-step process. The DHA-E and DPA-E obtained were better than 99% purity.

Keywords: DHA; docosahexaenoic acid ester; n-6 docosapentaenoic acid ester; DPA; preparative high-performance liquid chromatography; single-cell oil

Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification by Yuji Shimada; Kazuaki Maruyama; Akio Sugihara; Shigeru Moriyama; Yoshio Tominaga (pp. 1441-1446).
Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield. To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but not with DHA. These results suggest that lauryl DHA was generated only by esterification.

Keywords: Docosahexaenoic acid; hydrolysis; lipase; Pseudomonas sp; Rhizopus delemar ; selective esterification; tuna oil

Carica papaya latex-catalyzed synthesis of structured triacylglycerols by T. A. Foglia; P. Villeneuve (pp. 1447-1450).
One impediment to the industrial use of enzymes in fat and oil transformations is the higher cost often associated with an enzymatic process compared with the corresponding chemical process. Processes that utilize plant enzymes, however, may have advantages because of their lower cost and ready availability. One example of such a plant-derived enzyme is Carica papaya latex (CPL), the principal source of the protease papain. Recently, it has been shown that this latex also catalyzes the lipolysis of triacylglycerols and that this latex lipase has a selectivity for short-chain acyl groups as well as a 1,3-glycerol selectivity. These selectivities can be used in the synthesis of structured triacylglycerols. In this paper we describe the utility of CPL in lipase-catalyzed reactions, specifically the synthesis of low-calorie triacylglycerol analogs.

Keywords: Hydrogenated soy oil; low-calorie fats; short-chain triglycerides; triacylglycerols; tributyrin

Improvement of palm oil through breeding and biotechnology by B. S. Jalani; S. C. Cheah; N. Rajanaidu; A. Darus (pp. 1451-1455).
The oil palm Elaeis guineensis is the highest oil-yielding crop and has the potential to become the major supplier of both edible oil and renewable industrial feedstock. The oil yield from wild groves is presently less than 0.5 t/ha/y. However, through breeding and selection, the oil yield of commercial plantations could reach as much as 8 t/ha/y. New planting materials also have the capability of better oil yields with high iodine value (IV), slow height increment, and larger kernels. The oil also contains considerable amounts of carotenoids (500–700 ppm), vitamin E (600–1000 ppm), and sterols (250–620 ppm). The oil yield of another oil palm species, E. oleifera, is approximately 0.5 t/ha/y with high contents of carotenoids (700–1500 ppm), vitamin E (700–1500 ppm), and sterols (3500–4000 ppm). The above traits could be improved through breeding and biotechnology. Biotechnological efforts at the Palm Oil Institute of Malaysia are directed toward the production of oil with high IV and high monounsaturated fatty acids for edible purposes and industrial uses. Isolation and manipulation of the genes involved in the biosynthesis of fatty acids are the main focus. The aim is to increase the efficiency of conversion of palmitate (C16:0) to oleate (C18:1). Levels of palmitate and oleate are controlled by the enzymes acyl-acyl carrier protein (ACP) thioesterase and β-keto acyl ACP synthase II. The chain termination reactions of C16:0 and C18:1 are independent, thus paving the way for the possibility of reducing palmitate levels by switching off the palmitoyl ACP thioesterase gene.

Keywords: Biotechnology; breeding; genetic engineering; oil palm; palm oil

Kinetics of transesterification of soybean oil by H. Noureddini; D. Zhu (pp. 1457-1463).
Transesterification of soybean oil with methanol was investigated. Three stepwise and reversible reactions are believed to occur. The effect of variations in mixing intensity (Reynolds number=3,100 to 12,400) and temperature (30 to 70°C) on the rate of reaction were studied while the molar ratio of alcohol to triglycerol (6:1) and the concentration of catalyst (0.20 wt% based on soybean oil) were held constant. The variations in mixing intensity appear to effect the reaction parallel to the variations in temperature. A reaction mechanism consisting of an initial mass transfer-controlled region followed by a kinetically controlled region is proposed. The experimental data for the latter region appear to be a good fit into a second-order kinetic mechanism. The reaction rate constants and the activation energies were determined for all the forward and reverse reactions.

Keywords: Biodiesel; kinetics; methyl esters; mixing; renewable fuels; soybean oil; transesterification; triglycerides

Purification of γ-linolenic acid from borage oil by a two-step enzymatic method by Yuji Shimada; Akio Sugihara; Masahiro Shibahiraki; Hiroyuki Fujita; Hirofumi Nakano; Toshihiro Nagao; Tadamasa Terai; Yoshio Tominaga (pp. 1465-1470).
γ-Linolenic acid (GLA) was purified from borage oil by a two-step enzymatic method. The first step involved hydrolysis of borage oil (GLA content, 22.2 wt%) with lipase, Pseudomonas sp. enzyme (LIPOSAM). A mixture of 3 g borage oil, 2 g water, and 5000 units (U) LIPOSAM was incubated at 35°C with stirring at 500 rpm. The reaction was 91.5% complete after 24 h. The resulting free fatty acids (FFA) were extracted from the reaction mixture with n-hexane (GLA content, 22.5 wt%; recovery of GLA, 92.7%). The second step involved selective esterification of borage-FFA with lauryl alcohol by using Rhizopus delemar lipase. A mixture containing 4 g borage-FFA/lauryl alcohol (1:2, mol/mol), 1 g water, and 1000 U lipase was incubated at 30°C for 20 h with stirring at 500 rpm. Under these conditions, 74.4% of borage-FFA was esterified, and the GLA content in the FFA fraction was enriched from 22.5 to 70.2 wt% with a recovery of 75.1% of the initial content. To further elevate the GLA content, unesterified fatty acids were extracted, and esterified again in the same manner. By this repeated esterification, GLA was purified to 93.7 wt% with a recovery of 67.5% of its initial content.

Keywords: Borage oil; hydrolysis; lauryl alcohol; γ-linolenic acid; Pseudomonas sp. lipase; purification; Rhizopus delemar lipase; selective esterification

Synthesis of (Z)-3-hexen-1-yl butyrate in hexane and solvent-free medium using Mucor miehei and Candida antarctica lipases by S. Bourg-Garros; N. Razafindramboa; A. A. Pavia (pp. 1471-1475).
(Z)-3-Hexen-1-yl butyrate is an important flavor and fragrance compound as it represents the model of a natural herbaceous (green) note. Two immobilized lipases from Mucor miehei (Lipozym IM) and from Candida antarctica (Novozym 435) were investigated for their use in the synthesis of (Z)-3-hexen-1-yl butyrate by direct esterification in n-hexane. To determine optimal conditions for esterification, we examined the following parameters: temperature, amount of lipase, acid/alcohol ratio, and absence of solvent. In n-hexane, bioconversion yields reached 95 (after 4 h) and 92% (after 6 h) for, respectively, Lipozym IM [17 (w/w reactants)] and Novozym 435 [2% (w/w reactants)]. In the absence of solvent, at 60°C, Novozym 435-catalyzed esterification afforded the title compound in 80% yield. Up to 250 g (in hexane) and 160 g (without solvent) of ester were easily prepared, in a single operation, at a laboratory scale, in few hours, using 2% (w/w reactants) lipase.

Keywords: Biosynthesis; Candida antarctica ; direct esterification; n-hexane; (Z)-3-hexen-1-yl butyrate; lipases; Mucor miehei ; solvent-free medium

Synthesis of cocoa butter equivalent by lipase-catalyzed interesterification in supercritical carbon dioxide by Kuan-Ju Liu; Hong-Ming Cheng; Rey-Chang Chang; Jei-Fu Shaw (pp. 1477-1482).
With supercritical carbon dixoide as a reaction medium, the syntheses of cocoa butter equivalent by interesterification with various lipases were investigated. The study showed that among those five lipases tested, lipase IM-20 from Mucor miehei was the most effective and specific in synthesizing this cocoa butter equivalent product by interesterification. The yields of cocoa butter equivalent are affected by pressure, substrate oil composition, solubility and co-solvent. The best reaction conditions were: reaction pressure at 1500 psi, triglyceride with high content of POP (P, palmitate; O, oleate) and POO, reaction medium with 5.0% water, and reaction temperature at 50°C. The major component of cocoa butter, POS (S, stearate), can be increased by 6.0% by adding a small amount of carbon dioxide. The yield and melting point of the purified cocoa butter equivalent are 53.0% and 34.3°C, respectively.

Keywords: Cocoa butter equivalent; interesterification; lipase; palm oil; supercritical carbon dioxide

Lipase-catalyzed alcoholysis with supercritical carbon dioxide extraction 1: Influence of flow rate by Helga Gunnlaugsdottir; Björn Sivik (pp. 1483-1490).
A combined process of lipase (E.C. catalysis and extraction of product with supercritical carbon dioxide was studied. The effect of different flow rates of the extraction fluid on the selective removal of the ethyl esters (EE) synthesized in a lipase-catalyzed alcoholysis of cod liver oil with ethanol was investigated. The faster the flow rate, the faster the extraction rate and the higher the recovery of EE. For example, after a 270-min extraction, the total recovery of EE was 1520 mg for a flow rate of 0.3 liter carbon dioxide at atmospheric pressure and room temperature/min (NL/min) as compared to 250 mg when 0.015 NL/min was used. The concentration of EE in the carbon dioxide was found to decrease with increasing flow rate, which indicates that the rate of diffusion of EE limits their extraction at fast flow rates. A high flow rate was found to result in a more selective extraction of EE, i.e., less amounts of other lipid components present in the reaction mixture were coextracted with the EE. Further, by increasing the flow rate, the equilibrium of the reaction was shifted slightly toward ester synthesis. An increase in the flow rate from 0.015 to 0.075 NL/min resulted in an approximately 10% increase in total conversion (from 73 to 82%), whereas only a negligible increase was obtained when the flow rate was increased further to 0.15 NL/min.

Keywords: Alcoholysis; enzyme; extraction; fish oil; flow rate; lipase; supercritical carbon dioxide

Lipase-catalyzed alcoholysis with supercritical carbon dioxide extraction 2: Phase behavior by Helga Gunnlaugsdottir; Anders Å. Karlsson; Björn Sivik (pp. 1491-1494).
The phase behavior of systems containing ethanol plus lipid samples with different lipid compositions plus carbon dioxide was studied visually at 40°C and pressures of 0.01, 9, 15, and 23 MPa by means of a high-pressure sapphire cell. The systems studied represent the main components present in a lipase-catalyzed alcoholysis reaction of cod liver oil in supercritical carbon dioxide. Two phases, a vapor and a liquid phase, were observed in all systems studied at supercritical conditions.

Keywords: Alcoholysis; enzyme; ethanol; lipase; lipids; phase behavior; supercritical carbon dioxide

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