Lipase-catalyzed acidolysis of fish liver oil with dihydroxyphenylacetic acid in organic solvent media

Lipase-catalyzed acidolysis reaction of fish liver oil with dihydroxyphenylacetic acid (DHPA) was investigated in terms of enzyme specificity as well as the effects of enzyme concentration, molar substrate ratio and organic solvent mixture on the bioconversion yield. The highest bioconversion yield of 83% was obtained when Novozym 435 was used as biocatalyst in a hexane:2-butanone mixture of 75:25 (v/v) at a fish liver oil to DHPA substrate molar ratio of 4:1; however, lower bioconversion yield (15%) was obtained when Lipozyme IM 20 was used. The bioconversion yield of phenolic monoacylglycerols (MAGs) increased from 11 to 70% when the ratio of the hexane/2-butanone reaction medium was changed from 85:15 to 75:25 (v/v), whereas that of phenolic diacylglycerols (DAGs) remained relatively unchanged (13–16%). The results also showed that the acidolysis reaction resulted in an increase of C_20:5 ω-3 and C_22:6 ω-3 proportions from 11.5 and 20.2% in the original fish liver oil to 22.6–27.1 and 22.8–23.1% in the phenolic lipids, respectively. The radical scavenging ability of phenolic lipids was determined to be about half-time lower than that of α-tocopherol.     

Enzymatic synthesis of structured phenolic lipids by incorporation of selected phenolic acids into triolein

The synthesis of structured phenolic lipids by lipase-catalyzed transesterification of selected phenolic acids, including p-hydroxyphenyl acetic, p-coumaric, sinapic, ferulic and 3,4-dihydroxybenzoic acids, with triolein was investigated. The highest enzymatic activity (248 nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (62%) was obtained for the transesterification of p-hydroxyphenyl acetic acid with triolein. In addition, the transesterification of p-coumaric with triolein resulted in a higher enzymatic activity (87 nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (46%) than those obtained for the transesterfication of ferulic and sinapic acids. The results also showed that using p-hydroxyphenyl acetic, p-coumaric and ferulic acids as substrate, the maximum bioconversion of phenolic monoacylglycerols was close to that of phenolic diacylglycerols. Although p-coumaric acid had very low radical scavenging activity (2%) compared to that of ferulic acid (62%), the p-coumaroylated lipids demonstrated a higher scavenging potency (16%) than that of the feruloylated one (10%).     

Lipase-catalyzed acylation of quercetin with cinnamic acid

Acylation of quercetin with cinnamic acid catalyzed by Candida antarctica lipase B (CAL-B) or Pseudomonas cepacia lipase C (PCL-C) was investigated. Specifically, the effects of reaction duration, incubation temperature, and molar ratio of substrates on bioconversion yield, initial rate of reaction, and regioselectivity were investigated. Three new acylated quercetin analogues were produced: quercetin 4′-cinnamate (C₂₄H₁₆O₈), quercetin 3′,4′-dicinnamate (C₃₃H₂₂O₉), and quercetin 7,3′,4′-tricinnamate (C₄₂H₂₈O₁₀). The effects of the lipase-catalyzed acylation conditions on the bioconversion yields varied across the conditions. The initial rate of reaction of acylation of quercetin with cinnamic acid catalyzed by CAL-B and PCL-C was similar. In the presence of CAL-B, acylation mainly took place at the C-4′-OH, generating mostly quercetin 4′-cinnamate; whereas with PCL-C, acylation mainly took place at both the 4′- and 3′-hydroxyls, generating quercetin 3′,4′-dicinnamate. Thin-layer-chromatography analysis showed that the three acylated quercetin analogues had higher lipophilicity when compared with quercetin. In silico investigation revealed that quercetin 4’-cinnamate and quercetin 3′,4′-dicinnamate are likely to be orally active pharmacological drugs.     

Enzymatic synthesis of structured phenolic lipids by incorporation of selected phenolic acids into triolein

The synthesis of structured phenolic lipids by lipase-catalyzed transesterification of selected phenolic acids, including p-hydroxyphenyl acetic, p-coumaric, sinapic, ferulic and 3,4-dihydroxybenzoic acids, with triolein was investigated. The highest enzymatic activity (248?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (62%) was obtained for the transesterification of p-hydroxyphenyl acetic acid with triolein. In addition, the transesterification of p-coumaric with triolein resulted in a higher enzymatic activity (87?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (46%) than those obtained for the transesterfication of ferulic and sinapic acids. The results also showed that using p-hydroxyphenyl acetic, p-coumaric and ferulic acids as substrate, the maximum bioconversion of phenolic monoacylglycerols was close to that of phenolic diacylglycerols. Although p-coumaric acid had very low radical scavenging activity (2%) compared to that of ferulic acid (62%), the p-coumaroylated lipids demonstrated a higher scavenging potency (16%) than that of the feruloylated one (10%).     

Enzymatic synthesis of phenolic lipids in solvent-free medium using flaxseed oil and 3,4-dihydroxyphenyl acetic acid

The enzymatic synthesis of phenolic lipids (PLs) by transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA) was investigated in solvent-free medium (SFM), using Novozym 435 from Candida antarctica as the biocatalyst. The effects of selected reaction parameters, water activity (a_w), enzyme concentration and agitation speed, were studied and optimized. Increasing the a_w of the reaction mixture from 0.18 to 0.38 resulted in a significant increase in the bioconversion yield from 62 to 77%. APCI–MS analysis confirmed the formation of six 3,4-dihydroxyphenyl acetoylated lipids, which were monolinolenyl, dioleyl, dilinolenyl, linoleyl linolenyl, oleyl linolenyl and oleyl linoleyl dihydroxyphenyl acetates. The highest enzymatic activity (178 nmol of PLs/g solid enzyme/min) was obtained using 40 mg of solid enzyme (400 PLU)/mL at agitation speed 150 rpm. Using the optimized conditions, the phenolic lipids showed a high relative proportion of linolenic acid (C_18:3 n?3) that increased from 57% in the flaxseed oil to 75 and 64% in the produced phenolic mono- and diacylglycerols, respectively. In addition, the synthesized phenolic lipids demonstrated a 7.2-fold lower radical scavenging activity than that of DHPA but half that of α-tocopherol.     

Acidolysis between triolein and short-chain fatty acid by lipase in organic solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 degrees C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Optimization of reaction conditions and stabilization of phenylalanine ammonia lyase-containing Rhodotorula glutinis cells during bioconversion of trans-cinnamic acid to L-phenylalanine

Studies were performed to elucidate the optimal reaction conditions (pH, temperature, ammonia concentration and biocatalyst loading) for bioconversion of trans-cinnamic acid (t-CA) to L-phenylalanine (L-Phe) by L-phenylalanine ammonia lyase (PAL) containing Rhodotorula glutinis cells. All treatments with permeabilizing agents stimulated L-Phe production and also enhanced instability of the catalyst, except Triton X-100 which gave a superior (56%) increase in conversion as compared to the control and a significant stabilization of PAL enzyme. Inclusion of several activity modifiers and stabilizer additives in reaction mixtures were shown to enhance the yield of L-Phe and maintained PAL stability over several successive incubations during the bioconversion process. Maximum stabilization of PAL and enhancement of L-Phe production was achieved with addition of 20% polyhydric alcohol (glycerol). The production of L-Phe continued to the fifth cycle and the total yield increased 2.3 times compared to the yield produced by the control (without glycerol addition) during the repeated batch process. Reducing agents such as 2-mercaptoethanol and thioglycolic acid were added to the bioconversion mixture in order to reduce the effects of oxygen on PAL catalyst life. Production of L-Phe by addition of 400 mgL(-1) of thioglycolic acid was maximized over the control by 55%. When both 20% glycerol and 400 mgL(-1) thioglycolic acid were simultaneously present in the reaction mixture, reuseability and stability of biocatalyst (PAL) were extended to eight consecutive cycles and conversion rate and overall productivity of L-Phe were higher than that of the control. These results may lead to improvements in the production of the essential amino acid L-Phe.     

Lipase-catalyzed synthesis of structured phenolic lipids in solvent-free system using flaxseed oil and selected phenolic acids as substrates

Structured phenolic lipids (PLs) were obtained by lipase-catalyzed transesterification of flaxseed oil, in a solvent-free system (SFS), with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. A bioconversion yield of 65% was obtained for the transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA). However, the effect of the chemical structure of phenolic acids on the transesterification of flaxseed oil in SFS was of less magnitude as compared to that in organic solvent system (OSS). Using DHPA, the APCI-MS analysis confirmed the synthesis of monolinolenyl, dilinolenyl, linoleyl linolenyl and oleyl linolenyl dihydroxyphenyl acetates as phenolic lipids. A significant increase in the enzymatic activity from 200 to 270 nmol of PLs/g solid enzyme/min was obtained upon the addition of the non-ionic surfactant Span 65. However, upon the addition of the anionic surfactant, sodium bis-2-ethylhexyl sulfosuccinate (AOT), and the cationic one, hexadecyltrimethylammonium bromide (CTAB), the enzymatic activity was decreased slightly from 200 to 192 and 190 nmol of PLs/g solid enzyme/min, respectively. The results also showed that the increase in DHPA concentration from 20 to 60 mM resulted in a significant increase in the volumetric productivity (P(V)) from 1.61 to 4.74 mg PLs per mL reaction mixture per day.     

Enzymatic hydrolysis of hydrophobic triolein by lipase in a mono-phase reaction system containing cyclodextrin; Reaction characteristics

A hydrophobic substrate triolein was hydrolyzed by lipase in a mono-phase reaction system containing cyclodextrin(CD) as emulsifier. The triolein was transformed to an emulsion-like state in the CD containing reaction system in contrast to the oil-droplet like state without CD due to the formation of an inclusion complex between the lipids and CDs. The hydrolysis reaction increased substantially in the CD containing reaction system, and the optimum reaction conditions including the amount of lipase, β-CD concentration, and mixing ratio of triolein and β-CD, were determined. The performance of the enzyme reaction in a mono-phase reaction system was compared with that of a two-phase reaction system which used water immiscible hexane as the organic solvent. The role of a CD in the mono-phase reaction system was elucidated by comparing the degree of the inclusion complex formation with triolein and oleic acid, K_m and V_max values, and product inhibition by oleic acid in aqueous and CD containing reaction systems. The resulting enhanced reaction seems to be caused by two phenomena; the increased accessibility of lipase to triolein and reduced product inhibition by oleic acid through the formation of an inclusion complex.     

Acidolysis between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 °C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Lipase-catalyzed synthesis of phenolic lipids from fish liver oil and dihydrocaffeic acid

The enzymatic synthesis of phenolic lipids by lipase-catalyzed transesterification of dihydrocaffeic acid (DHCA) with fish liver oil was investigated in a selected organic solvent medium. These synthesized phenolic lipids have potential use as nutraceutical products. Using a molar ratio of 1:8 DHCA to fish liver oil in hexane:2-butanone mixtures of 75:25 and 85:15 (v/v), the lipase-catalyzed reaction resulted in maximum conversion of 55.8 and 65.4%, respectively. The maximum conversion of phenolic monoacylglycerols in hexane:2-butanone mixture of 75:25 and 85:15 (v/v) was 40.3 and 37.7%, respectively; using the same solvent mixtures, the conversions of the phenolic diacylglycerol were 15.8 and 36.8%, respectively. Hexane:2-butanone mixture of 75:25 (v/v) was, therefore, the best organic solvent mixture for the production of phenolic monoacylglycerols, while that of 85:15 (v/v) was best for the production of phenolic diacylglycerols. The phenolic lipids produced from the fish liver oil and DHCA demonstrated antioxidant property as indicated by its free radical scavenging capacity.     

High-yield enzymatic bioconversion of hydroquinone to α-arbutin, a powerful skin lightening agent, by amylosucrase

α-Arbutin (α-Ab) is a powerful skin whitening agent that blocks epidermal melanin biosynthesis by inhibiting the enzymatic oxidation of tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA). α-Ab was effectively synthesized from hydroquinone (HQ) by enzymatic biotransformation using amylosucrase (ASase). The ASase gene from Deinococcus geothermalis (DGAS) was expressed and efficiently purified from Escherichia coli using a constitutive expression system. The expressed DGAS was functional and performed a glycosyltransferase reaction using sucrose as a donor and HQ as an acceptor. The presence of a single HQ bioconversion product was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The HQ bioconversion product was isolated by silica gel open column chromatography and its chemical structure determined by 1H and 13C nuclear magnetic resonance (NMR). The product was determined to be hydroquinone-O-α-D-glucopyranoside with a glucose molecule linked to HQ through an α-glycosidic bond. However, the production yield of the transfer reaction was significantly low (1.3%) due to the instability of HQ in the reaction mixture. The instability of HQ was considerably improved by antioxidant agents, particularly ascorbic acid, implying that HQ is labile to oxidation. A maximum yield of HQ transfer product of 90% was obtained at a 10:1 molar ratio of donor (sucrose) and acceptor (HQ) molecules in the presence of 0.2 mM ascorbic acid.     

Lipid transfer between human plasma low-density lipoprotein and a triolein/phospholipid microemulsion catalyzed by insect hemolymph lipid transfer particle

Lipid transfer between human plasma low-density lipoprotein (LDL) and an LDL-size microemulsion of triolein and phosphatidylcholine stabilized with human apolipoprotein A-I was catalyzed by the lipid transfer particle from hemolymph of the tobacco hornworm (Manduca sexta). Net transfer of phospholipid and triacylglycerol from the emulsion to LDL was observed and the apparent initial rates of transfer were dependent on the amount of catalyst. Net transfer of phospholipid mass was twice as much as that of triacylglycerol with respect to both the initial rate and the final equilibrium state. The final amount of net transfer of both lipids was dependent upon the initial ratio of LDL: microemulsion present in the incubation mixture up to 1:1 on the basis of phospholipid. The microemulsion lipid composition was maximally altered from an initial weight ratio of 1.09 +/- 0.08 (phospholipid/triolein) to 0.90 +/- 0.03 by this reaction. Further increase of LDL in the incubation caused neither further net transfer nor further change in the lipid composition of the microemulsion. The catalyst neither affected spontaneous transfer of free cholesterol between the emulsion and LDL nor enhanced cholesteryl ester transfer in this reaction system. As a result of the facilitated reaction, LDL gained a significant amount of phospholipid and triacylglycerol causing up to an 8% increase in core lipids and 14% in phospholipid. Some free cholesterol is recovered in the emulsions via spontaneous exchange. Transfer or exchange of apolipoproteins during the course of facilitated lipid transfer did not occur.     

Lipase-catalyzed synthesis of phenolic lipids from fish liver oil and dihydrocaffeic acid

The enzymatic synthesis of phenolic lipids by lipase-catalyzed transesterification of dihydrocaffeic acid (DHCA) with fish liver oil was investigated in a selected organic solvent medium. These synthesized phenolic lipids have potential use as nutraceutical products. Using a molar ratio of 1:8 DHCA to fish liver oil in hexane:2-butanone mixtures of 75:25 and 85:15 (v/v), the lipase-catalyzed reaction resulted in maximum conversion of 55.8 and 65.4%, respectively. The maximum conversion of phenolic monoacylglycerols in hexane:2-butanone mixture of 75:25 and 85:15 (v/v) was 40.3 and 37.7%, respectively; using the same solvent mixtures, the conversions of the phenolic diacylglycerol were 15.8 and 36.8%, respectively. Hexane:2-butanone mixture of 75:25 (v/v) was, therefore, the best organic solvent mixture for the production of phenolic monoacylglycerols, while that of 85:15 (v/v) was best for the production of phenolic diacylglycerols. The phenolic lipids produced from the fish liver oil and DHCA demonstrated antioxidant property as indicated by its free radical scavenging capacity.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

Spores of Aspergillus niger as reservoir of glucose oxidase synthesized during solid-state fermentation and their use as catalyst in gluconic acid production

AIMS: To exploit conidiospores of Aspergillus niger as a vector for glucose oxidase extraction from solid media, and their direct use as biocatalyst in the bioconversion of glucose to gluconic acid. METHODS AND RESULTS: Spores of A. niger (200 h old) were shown to fully retain all the glucose oxidase synthesized by the mycelium during solid-state fermentation (SSF). They acted as catalyst and carried out the bioconversion reaction effectively, provided they were permeabilized by freezing and thawing. Glucose oxidase activity was found retained in the spores even after repeated washings. Average rate of reaction was 1.5 g l(-1) h(-1) with 102 g l(-1) of gluconic acid produced out of 100 g l(-1) glucose consumed after approx. 100 h reaction, which corresponded to a molar yield close to 93%. These results were obtained with permeabilized spores in the presence of a germination inhibitor, sodium azide. CONCLUSIONS: Spores of A. niger served as efficient catalyst in the model bioconversion reaction after permeabilization. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first detailed study on the ability of A. niger spores to act as reservoir of enzyme synthesized during SSF without its release into solid media. Use of this material served as an innovative concept for enzyme extraction and purification from a solid medium. Moreover, this approach could compete efficiently with the conventional use of mycelial form of the fungus in gluconic acid production.     

Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase

An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.     

Enzymatic esterification of dihydrocaffeic acid with linoleyl alcohol in organic solvent media

The enzymatic esterification of dihydrocaffeic acid with linoleyl alcohol, using immobilized lipases (Lipozyme IM 20 and Novozym 435), was investigated in selected organic solvent media. Novozym 435 was found to be more efficient for catalyzing the esterification reaction. The highest enzymatic activity of 0.89 μmol esterified linoleyl alcohol/g solid enzyme/min was obtained in a hexane/2-butanone mixture of 75:25 (v/v), with an esterification yield of 75%; however, an increase in the 2-butanone proportion in the mixture up to 50% (v/v) resulted in a decrease in enzymatic activity and esterification yield to 0.38 μmol esterified linoleyl alcohol/g solid enzyme/min and 40%, respectively. The maximum esterification yield of 99.3% was obtained with a dihydrocaffeic acid to linoleyl alcohol ratio of 1:8. The electrospray ionization-mass spectroscopic structural analysis of the end products confirmed the biosynthesis of dihydrocaffeic acid ester of linoleyl alcohol, which demonstrated an anti-radical activity using 2,2-diphenyl-1-picrylhydrazyl as a radical model.     

Directed Evolution of Toluene Dioxygenase from Pseudomonas putida for Improved Selectivity Toward cis-Indandiol during Indene Bioconversion

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S, 2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S, 2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.     

Triglyceride Interesterification by Lipases: 2. Reaction parameters for the reduction of trisaturated impurities and diglycerides in batch reactions

A model system consisting of pure triolein and palmitic acid and LipozymeTM, an immobilized lipase (E.C. 3.1.1.3.). has been used to determine the effects of various reaction parameters on the reaction rate and the formation of by-products in the interesterification reaction. The goal was to minimize the level of diglycerides and eliminate trisaturated triglycerides at an endpoint chosen so that the results could be applied to the production of cocoa butter substitutes. The levels of diglycerides, which are essential reaction intermediates, and trisaturated glycerides, which are believed to be formed as a result of spontaneous acyl migration of mono- and diglyceride intermediates, were determined at a defined endpoint. A lag period was observed in which no tripalmitate was formed. The content of Lipozyme used was the most powerful factor in eliminating tripalmitate formation and reducing diglycerides; by using large quantities of Lipozyme, the reaction reached the endpoint before the tripalmitate formation became measurable and low levels of diglycerides were formed. The effects of varying the ratio of palmitic acid to triolein were investigated. A complex relationship between the ratio of substrate components emerged in which the diglyceride content increased with increasing triolein concentration and the tripalmitate content was lowest at a molar ratio of palmitic acid to triolein of 3.5. The reaction was run at 70, 80, and 90°C; best results were obtained at 70°. The water activity of the reaction was adjusted prior to catalysis and maintained during the reaction by equilibrating the reaction mixture and enzyme and running the reaction in an atmosphere of controlled water activity. A direct relationship between diglycerides and water activity was observed, and the level of tripalmitate formed corresponded to the time required to reach the endpoint. The reaction system was tested using ethyl palmitate instead of palmitic acid as acyl donor; the diglyceride content again increased with increasing water activity, but larger amounts of diglycerides were formed. Much shorter reaction times were required, with small quantities of tripalmitate formed.