Utilization of various sterols by lecithin-cholesterol acyltransferase as acyl acceptors

Highly purified lecithin-cholesterol acyltransferase of human plasma was used to study the utilization of various sterols as the acyl acceptor. The esterification of sterols was facilitated by the presence of a 3β-hydroxyl group and thetrans configuration of the A/B rings, as was evident from the lack of acceptor activity of all 3α-hydroxy sterols tested and coprostanol. Cholesterol analogs in which the side chain is modified, such as campesterol, β-sitosterol, desmosterol and stigmasterol, were less effective than cholesterol as acyl acceptors. However, androstan-3β-o1, which completely lacks the side chain, was found to be more active than cholesterol. The transfer of the acyl group to all effective sterols required the presence of the cofactor peptide apolipoprotein A-I.     

Lipase-catalyzed synthesis of unsaturated acyl l-ascorbates and their ability to suppress the autoxidation of polyunsaturated fatty acids

l-Ascorbic acid and various polyunsaturated fatty acids (PUFA) were condensed at 55°C by the immobilized lipase Chirazyme l-2 in dry acetone to produce the unsaturated acyl ascorbates. The PUFA moieties of the products were much more resistant to autoxidation at 65°C and nearly 0% relative humidity than the corresponding unmodified PUFA. The effects of the molar ratio of ascorbic acid or linoleoyl ascorbate to linoleic acid on the autoxidation of linoleic acid were examined. The autoxidation of linoleic acid was effectively suppressed at molar ratios greater than or equal to 0.2 when either ascorbic acid or linoleoyl ascorbate was mixed with linoleic acid. The addition of lauroyl ascorbate, synthesized through the enzyme-catalyzed condensation of ascorbic acid and lauric acid in acetone, to docosahexaenoic acid also significantly suppressed the autoxidation of docosahexaenoic acid at molar ratios of ≥0.2.     

Lipase-catalyzed esterification of lactic acid with straight-chain alcohols

Enzymatic synthesis of esters of lactic acid and straight-chain alcohols with different chain lengths (C₆–C₁₈) were investigated in batch reactions with hexadecanol (C₁₆) as the model alcohol. Cyclohexane was the best solvent for higher ester yields, and the best biocatalyst was the immobilized Candida antarctica lipase B (Novozym 435) as well as the textile-immobilized Candida sp. lipase. A method was established to obtain ester yields in the range of 71 to 82% for the different alcohols, and the most favorable conditions for the esterification reaction using Novozym 435 were an equimolar ratio of lactic acid to alcohol, each at a concentration of 120 mM each; a 50°C reaction temperature; 190 rpm shaking speed; and the addition of 100 mg molecular sieves (4 Å) for drying. The ester yield increased with increasing lipase load, and a yield of 79.2% could be obtained after 24 h of reaction at 20 wt% of Novozym 435. The immobilized Candida sp. lipase prepared in the laboratory also could be used to produce esters of lactic acid and straight-chain alcohols, but it had a much lower activity than Novozym 435 with a temperature optimum of 40°C.     

Lipase-catalyzed acidolysis of menhaden oil with pinolenic acid

Lipase-catalyzed acidolysis of menhaden oil with a pinolenic acid (PLA) concentrate, prepared from pine nut oil, was studied in a solvent-free system. The PLA concentrate was prepared by urea complexation of the FA obtained by saponification of pine nut oil. Eight commercial lipases from different sources were screened for their ability to catalyze the acidolysis reaction. Two different types of structured lipids (SL) were synthesized. The first type, which has PLA residues as a primary FA residue at the sn-1,3 positions of the TAG, was synthesized using a 1,3-regiospecific lipase, namely, Lipozyme RM IM from Rhizomucor miehei. The second type of SL, which has PLA residues as a primary FA residue at both the sn-1,3 and sn-2 positions of the TAG, was synthesized using a nonspecific lipase, namely, Novozym 435 from Candida antarctica. The effects of variations in enzyme loading, temperature, and reaction time on PLA incorporation into the oil were monitored by GC analyses. The optimal temperature and enzyme loading for synthesis of the two types of SL were 50°C and 10% of the total weight of substrates for both enzymes. The optimal reaction time for the synthesis with Lipozyme RM IM was 16h, whereas the optimal reaction time for the synthesis mediated by Novozym 435 was 36 h. Pancreatic lipase-catalyzed sn-2 positional analyses were also carried out on the TAG samples.     

脂肪酶催化合成单脂肪酸甘油酯

对脂肪本矣合成单甘酯中的催化作用作了综述。介绍了有机溶剂、反相胶束和无溶剂固相等反应体系中用不同脂肪酶对油脂选择水解、脂肪酸的酯化或脂肪酸酯与甘油的转酯反应、油脂甘油解以及甘油基团保护反应等合成方法。２     

Lipase-catalyzed production of biodiesel

Lipases were screened for their ability to transesterify triglycerides with short-chain alcohols to alkyl esters. The lipase fromMucor miehei was most efficient for converting triglycerides to their alkyl esters with primary alcohols, whereas the lipase fromCandida antarctica was most efficient for transesterifying triglycerides with secondary alcohols to give branched alkyl esters. Conditions were established for converting tallow to short-chain alkyl esters at more than 90% conversion. These same conditions also proved effective for transesterfying vegetable oils and high fatty acid-containing feedstocks to their respective alkyl ester derivatives. Presented in part at the 86th Annual Meeting of American Oil Chemists’ Society, San Antonio, Texas, May 1995.     

脂肪酶催化合成生物表面活性剂

脂肪酸单甘油酯、脂肪酸糖酯、聚甘油脂肪酸酯和长链脂肪酸蜡酯是重要的生物表面活性剂。传统化学法以碱为催化剂在高温下进行,不仅能耗高且产品纯度低。脂肪酶作为一种天然生物催化剂,可以温和条件下催化合成上述生物表面活性剂,能耗低且产品纯度高。综述对脂肪酶催化合成生物表面活笥剂工艺路线、反应体系及操作参数。     

Natural and unnatural silks

Natural silk is an important biopolymer with huge potential as it combines superb mechanical properties with environmentally sensitive production methods. Native silk dope taken straight from the gland can easily and without chemical assistance be drawn into strong fibres. Artificial silk fibres, on the other hand, rely on spinning dopes typically ‘reconstituted’ from natural silk fibres by strong chaotropic agents. Such fibres do not form readily, and often require chemical post-spin treatment for stabilisation. In addition these fibres tend to be brittle, and so far have been unable to match native fibres. Here we present novel rheometric data to argue that native and reconstituted silkworm silk dope differ in kind, not just in degree. While native silks behave like typical molten polymers, reconstituted silks do not. We conclude that rheology provides a powerful tool in the quest to learn from the Nature's polymer fibre technology.     

Lipase-catalyzed production of wax esters

The lipase (triacylglycerol acylhydrolase, E.C. 3.1.1.3) catalyzed synthesis of wax esters has been investigated via two different approaches. All studies were performed using an immobilized 1,3-specific lipase [Lipozyme from Novo Industries (Montréal, Québec, Canada)]. The first approach involves reacting stoichiometric amounts of a fatty acid and stearyl alcohol in the presence of lipase. The medium is solvent-free, which allows for high substrate concentrations (1.55 M) and use of 5% (w/w) Lipozyme. In this reaction, maximum wax ester synthesis was found to be dependent upon the efficient removal of the water produced by the reaction. Under optimal conditions, yields of 100% were routinely reached after only 2 hr. The medium was then exclusively composed of the wax and the enzyme, no purification was required. The second method involves alcoholysis of a triglyceride, in this case triolein, with stearyl alcohol to produce 1,2-diolein, 2-monoolein and the wax ester of oleic acid. Again, no organic solvent was used. The wax ester yield was found to be directly dependent upon the alcohol concentration that was used to modulate the outcome of the reaction towards either the wax or the partial glycerides. The process was applied to the synthesis of waxes from high erucic acid rapeseed oil.     

Lipase-catalyzed hydrolysis of palm oil

The hydrolysis of palm oil, palm olein and palm stearin, soybean oil, corn oil and peanut oil by the commercial lipase fromCandida rugosa (formerly known asC. cylindracea) was studied. The optimal conditions for the hydrolysis of palm oil by the lipase were established. The lipase fromC. rugosa exhibits an optimal activity at 37 C and at pH 7.5. The optimal oil to hexane ratio is 1 g of oil to 0.5 ml hexane. The rate of hydrolysis of palm oil by the lipase is linear on a logarithmic scale. Under the same conditions, palm oil and palm olein were hydrolyzed at the same rate, whereas palm stearin was hydrolyzed much more slowly.     

Lipase-catalyzed synthesis of ferulate esters

Four commercially available esterases were screened for their ability to esterify ferulic acid (4-hydroxy-3-methoxy cinnamate). Novozym^® 435 was found to be the only one of those screened to convert ferulic acid to ethyl and octyl ferulate at 20 and 14% yields, respectively. The highest percentage conversion was obtained using a 1∶1 mole ratio of alcohol to ferulic acid in stirred batch reactions in anhydrous 2-methyl-2-propanol at 60°C using one equivalent (wt/wt based on ferulic acid) of Novozym 435. Increased water content and a higher alcohol/ethyl ferulate ratio had adverse effects on the lipase-catalyzed esterification. The Novozym 435 activity was tested in less polar solvents (anhydrous toluene and hexane) by monitoring the alcoholysis of ethyl ferulate with 1-octanol, which resulted in a 50% yield of octyl ferulate. The alcoholysis was improved to 83% by applying a 16 mm Hg vacuum for 5 min every 24 h to remove the ethanol co-product. The optimal alcoholysis parameters were applied to the alcoholysis of ethyl ferulate with monoolein and the transesterification with triolein. The transesterification of ethyl ferulate with triolein in anhydrous toluene produced a combined 44% yield of ferulyl monoolein and ferulyl diolein, a 20% greater yield than that obtained for alcoholysis using monoolein. The highest yield, 77%, of ferulyl monoolein and ferulyl diolein was achieved using a threefold excess of neat triolein. The lipase-catalyzed transesterification of ethyl ferulate with triolein appears to be a technically feasible route to ferulyl-substituted acylglycerols, which are potentially useful sunscreen ingredients.     

Lipase-catalyzed transesterification of trilinolein or trilinolenin with selected phenolic acids

The enzymatic transesterification of selected phenolic acids with TAG, including trilinolein (TLA) and trillinolenin (TLNA), was investigated in an organic solvent medium. Maximal bioconversion of 66% was obtained with a dihydrocaffeic acid (DHCA) to TLA ratio of 1∶2 after 5 d of reaction. Similarly, the highest bioconversion of 62% was obtained with a DHCA to TLNA ratio of 1∶2, but after 12 d of reaction. However, a ratio of 1∶4 DHCA/TLA decreased the bioconversion to 53%. Transesterification reactions of ferulic acid with both TAG, using a ratio of 1∶2, resulted in low bioconversion of 16 and 14% with TLA and TLNA, respectively. The overall results indicated that bioconversion of phenolic MAG was higher than that of phenolic DAG. The structures of mono- and dilinoleyl dihydrocaffeate as well as those of mono- and dilinolenyl dihydrocaffeate were confirmed by LC-MS analyses. The phenolic lipids demonstrated moderate radical-scavenging activity.     

脂肪酶催化合成D-异抗坏血酸月桂酸酯

用固定化脂肪酶Lipozyme 435作催化剂,由D-异抗坏血酸和月桂酸合成D-异抗坏血酸月桂酸酯。考察了反应温度、反应溶剂、反应物配比、酶用量、反应时间、月桂酸浓度、分子筛用量、摇床转速对产物收率的影响。结果表明,以20mL叔戊醇为反应溶剂、0.10g脂肪酶为催化剂、2.0g 4A分子筛为脱水剂,在55℃、170r/min转速的恒温摇床中,0.88g D-异抗坏血酸和2.50g月桂酸反应36h,产物收率68%。产物结构经IR、1H NMR、13C NMR和MS表征。     

Lipase-catalyzed transesterification of ethyl ferulate with triolein in solvent-free medium

The lipase-catalyzed transesterification of ethyl ferulate with triolein in a solvent-free medium was investigated. Transesterification was catalyzed by immobilized lipase from Candida antarctica (Novozym 435), to form ferulyl oleins, a mixture of ferulyl diolein and ferulyl monoolein. These ferulated esters can be widely used as natural antioxidant in both lipid containing food and cosmetic applications. External mass transfer limitations were lowest, when the agitation speed was higher than 180 rpm. A linear relationship between the initial reaction rate and enzyme load up to 10% demonstrated that the internal diffusion limitations could be minimized. The effects of various parameters on yields and rates of reaction were studied in the absence of mass transfer limitations. The initial reaction rate increased when the reaction temperature was raised in the range of 45-65 °C, further increase to 70 °C decreased the final yield to 48.9%. The value of activation energy was calculated as 65.04 kJ/mol based on the Arrhenius law. Under the most favorable conditions, a kinetic model based on the ping-pong bi-bi mechanism with triolein inhibition was found to fit the initial reaction rate data very well and the kinetic parameters were evaluated by non-linear regression analysis.     

Lipase-catalyzed synthesis of chiral triglycerides

Under certain reaction conditions, the acidolysis of tripalmitin with oleic acid using immobilized lipase from Rhizomucor miehei resulted in a higher level of monosubstituted oleoyldipalmitoyl (OPP) triglycerides than had been predicted according to kinetic modeling. The reaction products were subjected to chiral analysis by high-performance liquid chromatography (HPLC), which indicated that the enzyme was more active at the sn-1 position of the triglyceride than at the sn-3 position, resulting in synthesis of the chiral triglyceride 1-oleoyl-2,3-dipalmitoyl-sn-glycerol. A kinetic model was developed and was correlated with the HPLC method to provide a simple means to predict the stereoselectivity of lipase-catalyzed reactions. By using the model, the stereoselectivity of immobilized Rhizomucor miehei lipase was found to depend strongly on the initial water activity (a _w) of the reaction mixture, with greater selectivity occurring at lower a _w. The sn-1 selectivity was essentially maintained using various solvents, or without solvent, when a _w was kept constantly low. Variation in the fatty acid composition of the triglyceride indicated that shorter-chain fatty acids result in greater stereoselectivity, while variation of the chainlength of the free fatty acid indicated an enhancement by the longest chainlength. The stereoselectivity of this lipase was confirmed using a new ¹³C nuclear magnetic resonance method. By using immobilized R. miehei lipase at low a _w approximately 80% of the chiral triglyceride found in the reaction mixture was the sn-1 enantiomer, at high reaction conversion.     

Lipase-catalyzed triglyceride hydrolysis in organic solvent

An investigation of lipases fromRhizomucor miehei,Candida rug osa and porcine pancreas revealed that these enzymes hydrolyzed triglycerides in an organic solvent system. The presence of secondary amines,i.e., diethylamine,N-methylbutylamine, or the tertiary amine, Methylamine, greatly increased the extent of hydrolysis. The lipolysis of tallow took place under mild conditions,e.g., room temperature, moderate shaking and within 20 hr. At 45°C, complete hydrolysis of tallow was obtained in 6 hr. Vegetable oils and a fish oil (cod liver oil) were also hydrolyzed at 20°C byR. miehei lipase in the presence of iV-methylbutylamine for 20 hr. The lipases were recovered for reuse with some loss of activity. Optimum yields of free fatty acids were obtained by usingR. miehei lipase as catalyst. Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Lipase-catalyzed synthesis of structured low-calorie triacylglycerols

Because of their unique fatty acid specificities and regioselectivities, lipases have been found to be effective catalysts for the synthesis of structured lipids that have a predetermined composition and distribution of fatty acyl groups on the glycerol backbone. The prospective plant-derived lipase found in the exudate of Carica papaya is known for its shortchain acyl group specificity, 1,3-glycerol regioselectivity, and sn-3 stereoselectivity. Carica papaya latex (CPL) was therefore examined for its potential ability to synthesize structured lowcalorie short- and long-chain triacylglycerols (SLCT). In this paper, we describe the utility of CPL in the lipase-catalyzed interesterification reaction of triacetin and hydrogenated soybean oil. Normal-phase high-performance liquid chromatography, combined with mass spectrometry, was used to distinguish the structured SLCT synthesized using the lipase from the corresponding SLCT produced by chemical synthesis.     

Lipase-catalyzed interesterification of oils and fats

Extracellular microbial lipases can be used as catalysts for the interesterification of oils and fats. Use of specific lipases gives products which are unobtainable by chemical interesterification methods. Some of these products have properties of value to the oils and fats industry. The catalysts for enzymatic interesterification are prepared by coating inorganic support materials with the lipases. For batch interesterification reactions, the catalyst particles are activated by addition of a small amount of water and then stirred with a reactant mixture dissolved in petroleum ether. At the end of the reaction period, the catalyst particles are removed by filtration, and the interesterified triglycerides isolated by conventional fat fractionation techniques. The catalyst can be used in subsequent batch reactions. As an alternative to the batch reaction system, continuous enzymatic interesterification processes can be operated by pumping water containg feedstock through a packed bed of activated catalyst.     

Lipase-catalyzed alcoholysis with supercritical carbon dioxide extraction 2: Phase behavior

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.