Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

Enzymatic asymmetrization of prochiral 2-benzyl-1,3-propanediol through esterification in solvent media

The enzymatic esterification of the prochiral substrate, 2-benzyl-1,3-propanediol, has been studied in solvent media. Among the five tested lipases, Lipozyme and Novozym 435 led to higher reaction rates. Novozym 435 catalyzed faster reactions at low water activity and in solvents having log P above 2. However, the two positions of the diol, pro-(R) and pro-(S), led to the same reaction rate trends and no prochiral selectivity was obtained. When using Lipozyme in toluene, the reaction rates for the formation of both (R) and (S) products presented an optimum at a water activity of 0.22. In this case, the prochiral selectivity increased with the water activity, from a value of 5 at a _w < 0.01, to a value of 8 at a _w = 0.22, at which point it remained constant.     

Enzymatic synthesis of sialic acid derivative by immobilized lipase from Candida antarctica

The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.     

复合脂肪酶催化生物柴油的初步研究

初步探讨了复合脂肪酶催化生物柴油的工艺。优化了复合酶配比条件和叔丁醇反应体系。在无溶剂体系中,Novozym435分别与Lipozyme TLIM和Lipozyme RMIM均以70／30质量比混合时,甲酯得率分别达到94．52％和96．25％,比Novozym435单独催化时的甲酯得率分别提高了9．52％和9．99％。在叔丁醇体系中,当Novozym435与Li-pozyme TLIM和Lipozyme RMIM分别以60／40和80／20的质量比混合时,其甲酯得率分别为85．06％和81．5％,比Novozym435单独催化的效率分别提高了9．89％和7．48％。优化叔丁醇体系中复合酶催化条件后,甲酯得率达92％。     

Practical synthesis of 1,3-oleoyl 2-docosahexaenoylglycerol by lipase-catalyzed reactions: An evaluation of different reaction routes

Three new synthetic routes were critically evaluated for the lipase-catalyzed production of 1,3-oleoyl-2 docosahexaenoylglycerol (ODO) in relatively large-scale (approximately 200 g). First, the production of 1,3-diolein by the reaction of glycerol and oleic acid followed by incorporation of docosahexaenoic (DHA) ethyl ester at the sn-2 position was studied. 1,3-Diolein was produced in 68.3% and 84.6% yield when stoichiometric amounts of the substrates were reacted at 25 °C for 8 h in the presence of 10% Novozym 435 and Lipozyme RM IM, respectively. Further increase in reaction temperature and time led to decrease in the 1,3-diolein yield. However, only a 9.4% yield of triacylglycerol was obtained in the subsequent reaction step when the 1,3-diolein was reacted with DHA ethyl ester in the presence of Novozym 435. Secondly, the feasibility of direct acidolysis was studied. Acidolysis of single cell oil (SCO) in excess oleic acid using Novozym 435 as the catalyst occurred twice as fast in solvent (tert-butanol) compared to a solvent-free system, and 63% oleic acid was incorporated into SCO. However, the regio-isomeric purity of the product was poor. Finally, the ethanolysis of SCO to produce DHA-enriched 2-monoacylglycerol followed by esterification with oleic acid or ethyl oleate was investigated. ODO was obtained in 50.9% regio-purity by Lipozyme RM IM-catalyzed esterification. The latter method was the most feasible for preparing ODO in large-scale. This synthetic route could be adapted for related triacylglycerols containing highly polyunsaturated when their productions in large-scale and high regio-purity are required.     

Enzymatic Synthesis of Lipophilic Caffeoyl Lipids Using Soybean Oil as the Novel Acceptor

Soybean oil-based caffeoyl lipids are the novel lipophilic derivatives of caffeic acid, which can be used as UV absorbers and antioxidants in the food and cosmetic industries. In the work, the novel lipophilic structured lipids were prepared using soybean oil as the novel caffeoyl acceptor by enzymatic transesterification. The effects of the reaction variables on the transesterification were investigated, and response surface methodology was used to optimize the reaction variables. Reactions were monitored by HPLC-UV. Different enzymes (Novozym 435, Lipozyme RMIM, and Lipozyme TLIM) were used as biocatalysts, and Novozym 435 showed the best performance for the reaction. The results showed that a high lipophilic soybean oil-based caffeoyl lipids yield (73.5 ± 1.2%) was achieved under the optimal conditions (reaction temperature 85°C, substrate molar ratio 1:6 (ethyl caffeate (EC)/soybean oil), enzyme load 25% (w/w), and 60 h at atmosphere pressure). The activation energies of EC conversion, hydrophilic glyceryl caffeates (GC) and lipophilic caffeoylated acylglycerol (CAG) formations were 32.92 kJ/mol, 17.21 kJ/mol and 57.36 kJ/mol, respectively. Km and Vm were 0.022 mol/L and 0.033 × 10^-3 mol/(Lmin), respectively.     

Novel and highly effective chemoenzymatic synthesis of (2<Emphasis Type="Italic">R</Emphasis>)-2-[4-(4-cyano-2-fluorophenoxy)phenoxy]butylpropanoate based on lipase mediated transesterification

(2R)-2-[4-(4-Cyano-2-fluorophenoxy)phenoxy]butylpropanoate (cyhalofop-butyl, CyB) was synthesized by a chemoenzymatic route involving enantioselective transesterification with Candida antarctica lipase B (Novozym 435). The optimum organic solvent, acyl donor, a _w , reaction temperature and shaking rate for the transesterification were acetonitrile, n-butanol, 0.11, 45°C and 200 rpm, respectively. Under the optimum conditions, the maximum substrate conversion and the enantiomeric purity of the product were 96.9 and >99%, respectively. The total yield and enantiomeric purity of CyB by this chemoenzymatic synthesis were 60.4 and >99%, respectively; 15.3 and 21% higher than that of the traditional way (45 and 78%).     

Penicillium expansum lipase-catalyzed production of biodiesel in ionic liquids

Penicillium expansum lipase (PEL) was used to catalyze biodiesel production from corn oil in [BMIm][PF₆]¹ (an ionic liquid, IL) and tert-butanol. Both systems were optimized in terms of MeOH/oil molar ratio, reaction temperature, enzyme loading, solvent volume, and water content. The high conversion obtained in the IL (86%) as compared to that in tert-butanol (52%) demonstrates that the IL is a superior solvent for PEL-catalyzed biodiesel production. Poor yields were obtained in a series of hydrophilic ILs. Addition of salt hydrates affected biodiesel production predominantly through the specific ion (Hofmeister) effect. The impact of methanol on both activity and stability of PEL in the IL and in hexane was investigated, in comparison to the results obtained by two commonly used lipases, Novozym 435 and Lipozyme TLIM. The results substantiate that while different lipases show different resistance to methanol in different reaction systems, PEL is tolerant to methanol in both systems.     

硬脂酸和月桂酸在Novozym 435催化下对芦丁酯化及分子筛对酯化率的影响

为了增加芦丁的脂溶性从而使其具有更优秀的抗氧化活性,以硬脂酸和月桂酸为酰基供体,在脂肪酶Novozym 435催化下对芦丁选择性酯化.经色谱柱提纯,得到两种带不同长度烃基的芦丁脂肪酸酯.用红外光谱和核磁共振波谱对芦丁硬脂酸进行了结构鉴定,表明该类酯化物的酯化反应位为鼠李糖的C4′″位羟基.以高效液相色谱监测酯化反应进程,分子筛添加时间对酯化率的研究结果显示,分子筛对酯化率和反应速率有提高的作用.分子筛添加时间对酯化率有影响.对于硬脂酸为酰基供体的情况,反应24h后添加分子筛的酯化反应可以得到最大的酯化转化率46%.以月桂酸为酰基供体的酯化反应,反应11 h后添加分子筛可以得到最大的酯化转化率64.5%.     

Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium

tert-Butanol, as a novel reaction medium, has been adopted for lipase-catalyzed transesterification of rapeseed oil for biodiesel production, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. Combined use of Lipozyme TL IM and Novozym 435 was proposed further to catalyze the methanolysis and the highest biodiesel yield of 95% could be achieved under the optimum conditions (tert-butanol/oil volume ratio 1:1; methanol/oil molar ratio 4:1; 3% Lipozyme TL IM and 1% Novozym 435 based on the oil weight; temperature 35 °C; 130 rpm, 12 h). There was no obvious loss in lipase activity even after being repeatedly used for 200 cycles with tert-butanol as the reaction medium. Furthermore, waste oil was also explored for biodiesel production and it has been found that lipase also showed good stability in this novel system.     

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.     

Long-chain acyl thioesters prepared by solvent-free thioesterification and transthioesterification catalysed by microbial lipases

Long-chain acyl thioesters (thio wax esters) have been prepared in high (80% to more than 90%) yields by solvent-free esterification of fatty acids (lauric, myristic, palmitic and stearic acids) with long-chain thiols, such as decane thiol, dodecane thiol, tetradecane thiol and hexadecane thiol, catalysed by lipases from Candida antarctica (Novozym) and Rhizomucor miehei (Lipozyme) in the presence of a 0.4-nm molecular sieve. In the thioesterification reaction Novozym was a more effective biocatalyst than Lipozyme. The extent of thioesterification increased with increasing molar ratio of fatty acid to alkane thiol (1:1 to 3:1) and with temperature (40 °C compared to 60 °C), as well as with the amount of the enzyme preparation and the amount of 0.4-nm molecular sieve. Decreasing the chain length of the alkane thiol from C₁₆ to C₁₀ also increased the extent of thioesterification. Lipase-catalysed solvent-free transthioesterification of fatty acid methyl esters with alkane thiols was less effective for the preparation of acyl thioesters than was thioesterification of fatty acids with alkane thiols. In transthioesterification, Lipozyme was slightly more effective as a biocatalyst than Novozym. Received: 3 September 1998 / Received revision: 18 November 1998 / Accepted: 21 November 1998     

Process intensification of immobilized lipase catalysis by microwave irradiation in the synthesis of 4-chloro-2-methylphenoxyacetic acid (MCPA) esters

4-Chloro-2-methylphenoxyacetic acid (MCPA) is a selective systemic herbicide which is absorbed by leaves and roots. MCPA esters are preferred due to their low water solubility and environmental friendliness. Esterification of MCPA with n-butanol was investigated as a model reaction using immobilized enzymes under the influence of microwave irradiation. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM and Lipase AYS Amano were studied under microwave irradiation amongst which Novozym 435 (immobilized Candida antarctica lipase B) was the best catalyst. Effects of various parameters were systematically studied on rates and conversion. Under microwave irradiation, the initial rates were observed to increase up to 2-fold. Under optimized conditions of 0.1 mmol MCPA and 0.3 mmol n-butanol in 15 mL 1,4-dioxane as solvent, Novozym 435 showed a conversion of 83% at 60 °C in 6 h. Based on initial rate and progress curve data, the reaction was shown to follow the Ping Pong bi–bi mechanism with inhibition by MCPA and n-butanol. Esterification of MCPA was also studied with different alcohols such as isopropyl alcohol, n-pentanol, n-hexanol, benzyl alcohol and 2-ethyl-1-hexanol.     

Immobilized lipase-catalysed synthesis of cinnamyl laurate in non-aqueous media

Esters of cinnamyl alcohol find many applications in food, cosmetic and pharmaceutical industries as flavor and fragrance compounds. The current work focuses on the synthesis of cinnamyl laurate from cinnamyl alcohol and lauric acid, including screening of various immobilized lipases and optimization of reaction conditions such as catalyst loading, speed of agitation, mole ratio and temperature. Among different lipases screened such as Novozym 435, Lipozyme RM IM and Lipozyme TL IM, Novozym 435 was found to be the best catalyst with 60% conversion in 2 h at 30 °C for equimolar quantities of the reactants using 0.33% (w/v) of catalyst and toluene as solvent. An ordered bi–bi mechanism with dead-end complex of lauric acid was found to represent the kinetic data.     

Different enzyme requirements for the synthesis of biodiesel: Novozym 435 and Lipozyme TL IM

Enzymatic syntheses of biodiesel via alcoholysis of different vegetable oils (sunflower, borage, olive and soybean) have been studied. Loss of lipase activity induced by the nucleophile is greater with methanol than with ethanol, and is greater for Lipozyme TL IM than for Novozym 435. The optimum volume of ethanol depends on the loading of solid biocatalyst and is higher for preparations of Novozym 435 than for Lipozyme TL IM. Maximum rates were obtained with Lipozyme TL IM, for a molar ratio of alcohol to FA residues of 0.33. By contrast, Novozym 435 requires at least a 2:1 ratio. Alcoholysis of the vegetable oils is faster with Lipozyme TL IM than with Novozym 435. Use of a high loading of Novozym 435 (50% w/w) and a large molar excess of ethanol are required to obtain an initial rate similar to that obtained with Lipozyme TL IM at a lower enzyme loading (10% w/w) and an equimolar ratio of ethanol and FA residues. Novozym 435 produces quantitative conversions in only 7h at 25 degrees C, but complete conversions are not obtained with Lipozyme TL IM. Three stage stepwise addition of ethanol yields 84% conversion to ethyl esters for Lipozyme TL IM. Hence use of Novozym 435 is preferred. After nine cycles in a batch reactor Novozym 435 retained 85% of its initial activity.     

Production of extremely pure diacylglycerol from soybean oil by lipase-catalyzed glycerolysis

Research work was objectively targeted to synthesize highly pure diacylglycerol (DAG) with glycerolysis of soybean oil in a solvent medium of t-butanol. Three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) were screened, and Novozym 435 was the best out of three candidates. Batch reaction conditions of the enzymatic glycerolysis, the substrate mass ratio, the reaction temperature and the substrate concentration, were studied. The optimal reaction conditions were achieved as 6.23:1 mass ratio of soybean oil to glycerol, 40% (w/v) of substrate concentration in t-butanol and reaction temperature of 50 °C. A two-stage molecular distillation was employed for purification of DAG from reaction products. Scale-up was attempted based on the optimized reaction conditions, 98.7% (24 h) for the conversion rate of soybean oil, 48.5% of DAG in the glycerolysis products and 96.1% for the content of DAG in the final products were taken in account as the results.     

Stability and activity of lipase in subcritical 1,1,1,2-tetrafluoroethane (R134a)

The stability and activity of commercial immobilized lipase from Candida antarctica (Novozym 435) in subcritical 1,1,1,2-tetrafluoroethane (R134a) was investigated. The esterification of oleic acid with glycerol was studied as a model reaction in subcritical R134a and in solvent-free conditions. The results indicated that subcritical R134a treatment led to significant increase of activity of Novozym 435, and a maximum residual activity of 300% was measured at 4 MPa, 30 °C after 7 h incubation. No deactivation of Novozym 435 treated with subcritical R134a under different operation factors (pressure 2–8 MPa, temperature 30–60 °C, incubation time 1–12 h, water content 1:1, 1:2, 1:5 enzyme/water, depressurization rate 4 MPa/1 min, 4 MPa/30 min, 4 MPa/90 min) was observed. While the initial reaction rate was high in subcritical R134a, higher conversion was obtained in solvent-free conditions. Though the apparent conversion of the reaction is lower in subcritical R134a, it is more practicable, especially at low enzyme concentrations desired at commercial scales.     

The influence of cosolvent concentration on enzymatic kinetic resolution of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives

A method to improve the enantioselectivity of lipase-catalyzed kinetic resolution (KR) of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives in water–acetone solution is presented. Two different approaches were compared: enzyme-catalyzed esterification and enzymatic hydrolysis of the target ester. A substantial influence of enzyme type, ethoxy group donor, and solvent on conversion and enantioselectivity of the enzymatic esterification was noted. While enzymatic esterification proceeds with poor enantioselectivity, the hydrolysis of target ester proceeds efficiently. Studies on the influence of cosolvent used for the enzymatic hydrolysis reaction showed that kinetic resolution can be performed in acetone and water buffer mixture predominantly containing organic solvent. Any change in organic solvent content resulted in a substantial decrease in enantioselectivity from almost E = 150 to less than 5.     

Supercritical CO2 as a reaction medium for synthesis of capsaicin analogues by lipase-catalyzed transacylation of capsaicin

Capsaicin analogues having different acyl moiety were synthesized by lipase-catalyzed transacylation of capsaicin with a corresponding acyl donor in supercritical CO₂ as a reaction medium. Transacylation with methyl tetradecanoate using Novozym 435 as a catalyst gave vanillyl tetradecanamide in a 54% yield at 80 °C and 19 MPa over 72 h. Vanillyl (Z)-9-octadecenamide, olvanil, was synthesized from triolein in a 21% yield over 7 d.     

Enzymatic Preparation of Mono- and Di-Stearin by Glycerolysis of Ethyl Stearate and Direct Esterification of Glycerol in the Presence of a Lipase from Candida Antarctica (Novozym 435)

Mixtures of 1(3)-monostearin and distearin were prepared by direct esterification of glycerol with stearic acid or transesterification using ethyl stearate as acyl donor in the presence of Candida antarctica lipase (Novozym 435) using a variety of solvents of differing polarity. In all cases, the transesterification resulted in higher product yields. In n-heptane as reaction medium the addition of water (3%) was essential for high product yields, with mono- and distearin being produced in almost equal amounts. Using more polar solvents as reaction media, such as acetonitrile or acetone, again the highest yields were obtained in the transesterification mode; employing these solvents the reactions were much more selective towards the formation of monostearin.