Enantioselective esterification of glycidol by surfactant-lipase complexes in organic media

Enantioselective esterification of glycidol has been performed with lauric acid in organic media dosed with surfactant-lipase complexes as catalysts. Lipase derived from various biomaterial sources was complexed with nonionic surfactant, dioleyl-N-D-glucono-L-glutamate, prior to use. Surfactant-lipase D (from Rhizopus delemar) complex had a higher enantioselectivity (v R /v S = 7.6) than the other lipases and the corresponding initial reaction rate was averaging 100-fold better than that of native powder lipase D in cyclohexane at 35°C.     

Characterization and catalytic property of surfactant-laccase complex in organic media

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated. The addition of HBT at the preparation stage of the enzyme complex did not accelerate the catalytic reaction because HBT was converted to an inactive benzotriazole (BT) by laccase. However, the addition of HBT at the reaction stage enhanced the catalytic performance by a factor of five compared to that without HBT.     

Optimization of methyl propionate production catalysed by Mucor miehei lipase

Lipase from Mucor miehei was used to catalyse the esterification reaction between propionic acid and methyl alcohol in modified organic media. Small-scale model studies were performed in order to define the optimal conditions. The specific activity of immobilized lipase, adsorbed onto hydrophilic supports, compared to free lipase, showed that enzyme activity was altered by immobilisation. Non-polar solvents were shown to be less harmful for the biocatalyst than solvents with higher polarity. Diethyl ether was used as the cosolvent of hexane to improve the solubility of substrates in the organic phase thus increasing contact with enzyme. An optimal ratio of 90/10 (v/v) was determined for a hexane/diethyl ether mixture. The mass of enzyme preparation must be high enough to display optimal diffusion of the reagents and hydration of the catalytic sites. Increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilisation, in repeated runs, occurred. Water saturation of the organic medium greatly lowered the biosynthetic activity of the enzyme. It was possible to reach a 96% methyl propionate biosynthesis yield after 2.30 h reaction, underlining the free-enzyme operational capacity in a quasi-anhydrous modified organic medium.     

脂解麻疯树油脂肪酶产生菌的筛选鉴定及其催化特性

[目的]分离筛选具有脂解麻疯树油能力的脂肪酶产生菌株,为以麻疯树油为原料酶法生产生物柴油奠定基础.[方法]以麻疯树油为唯一碳源,从麻疯树种子粉末处理过的土壤中分离筛选出1株具有脂解疯树油能力的脂肪酶产生菌,考察该菌株及其脂肪酶对有机溶剂耐受性以及脂肪酶催化酯化和转酯反应的能力,并通过生理生化特征和16S rDNA序列分...     

Long-term continuous production of optically active 2-(4-chlorophenoxy)propanoic acid by yeast lipase in an organic solvent system

Summary Long-term continuous optical resolution of 2-(4-chlorophenoxy)propanoic acid was carried out by stereoselective esterification with Celite-adsorbed lipase OF 360 from Candida cylindracea using n-tetradecanol as the second substrate in organic solvent systems. The water content of the Celite-adsorbed lipase affected productivity, 1.0 l water·mg lipase^–1 being optimal for preparation of the adsorbed lipase. Water-saturated carbon tetrachloride-isooctane (8:2, v/v) was found to be an excellent organic solvent for the continuous operation. The particle size of Celite had no effect on productivity. Under optimized conditions, the (R)-enantiomer of the acid was continuously esterified with high stereoselectivity in a packed-bed column reactor for 34 days. Furthermore, it was found that treatment of the reactor with acetone made it possible to restore productivity and extend the period of continuous operation for further 29 days. Offprint requests to: A. Tanaka     

Catalytic and structural properties of surfactant-horseradish peroxidase complex in organic media

A surfactant-horseradish peroxidase (HRP) complex that is catalytically active in organic media has been successfully prepared by a method utilizing water-in-oil (W/O) emulsions. To optimize conditions for preparation of the HRP complex, the effects of some key parameters in the aqueous phase of W/O emulsions were investigated. The surfactant-HRP complex prepared with a nonionic surfactant exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. At the preparation step, the pH of the aqueous solution had a prominent effect on the enzymatic activity of the HRP complex in organic media. Several kinds of salts present in the HRP complex could be employed to enhance the catalytic performance in organic media. However, anionic ions present in the preparation process appeared to lower the catalytic activity owing to the complexation with heme iron. UV-visible absorption spectra of the HRP complex in benzene, which were prepared from a KCN solution (pH 7.0) or an alkaline solution (pH 12), were comparable with those of native HRP in aqueous solution under the same conditions. Resonance Raman spectroscopic studies also revealed that no significant change in the coordination state of the heme iron occurred even after coating the enzyme with surfactant molecules, lyophilization, and solubilization in nonaqueous media.     

华根霉脂肪酶有机相合成酶活的研究

通过比较7种微生物脂肪酶的有机相合成酶活、水相水解酶活及在正庚烷中催化己酸乙酯合成的能力,证明了合成酶活与水解酶活相关性不高,合成酶活比水解酶活更能反映脂肪酶的合成能力。通过比较两株华根霉(Rhizopus chinensis)脂肪酶酶活,发现合成酶活相差较大,表明相同种属微生物的脂肪酶合成酶活存在不同。对．Rhizopus chinensis-2液态发酵产脂肪酶进程研究发现,水解酶活高峰先于合成酶活高峰大约12h。将不同培养时间的Rhizopus chinensis-2全细胞脂肪酶用于催化己酸乙酯合成,具有高合成酶活的全细胞脂肪酶催化己酸乙酯合成反应较快。因此,全细胞脂肪酶用于催化有机相酯合成反应时,具有高脂肪酶合成酶活的菌体具有较好的催化酯合成能力。     

A surfactant-coated lipase immobilized in magnetic nanoparticles for multicycle ethyl isovalerate enzymatic production

Gum arabic coated magnetic Fe₃O₄ nanoparticles (GAMNP) were prepared by chemical co-precipitation method and their surface morphology, particle size and presence of polymer-coating was confirmed by various measurements, including transmission electron microscopy (TEM), X-ray diffraction (XRD), thermo gravimetric analysis (TGA), and Fourier transform infra red (FTIR) analysis. Magnetic particles were employed for their potential application as a support material for lipase immobilization. Glutaraldehyde was used as a coupling agent for efficient binding of lipase onto the magnetic carrier. For this purpose, the surface of a Candida rugosa lipase was initially coated with various surfactants, to stabilize enzyme in its open form, and then immobilized on to the support. This immobilized system was used as a biocatalyst for ethyl isovalerate, a flavor ester, production. The influence of various factors such as type of surfactant, optimum temperature and pH requirement, organic solvent used, amount of surfactant in coating lipase and effect of enzyme loadings on the esterification reaction were systematically studied. Different surfactants were used amongst which non-ionic surfactant performed better, showing about 80% esterification yield in 48 h as compared to cationic/anionic surfactants. Enhanced activity due to interfacial activation was observed for immobilized non-ionic surfactant–lipase complex. The immobilized surfactant coated lipase activity was retained after reusing seven times.     

Influence of the nature of modifier in the enzymatic activity of chemical modified semipurified lipase from Candida rugosa

Semipurified lipase of Candida rugosa (CRSL) was subjected to chemical modification, and the activities of the modified lipase, in hydrolysis and esterification reactions, were examined. The esterification reactions were carried out in the absence and presence of isooctane. When the enzyme was modified with polyethylene glycol (PEG), two methodologies were studied. The activation of PEG with p-NO(2)-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG. The chemical modification with PEG increases the stability of pure lipases in isooctane at 50 degrees C (extreme conditions). The chemically modified enzymes are useful for biotransformations in organic solvents. In addition the nitration of tyrosines with tetranitromethane was also studied. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 252-260, 1997.     

Lipase immobilized by adsorption

Summary Candida cylindraceae lipase was immobilized by adsorption to various hydrophilic and hydrophobic supports and studied with respect to the esterification rates of a primary and a secondary alcohol, respectively, in organic media. The reaction rates were compared with the rate of esterification with free lipase. When the secondary alcohol, (R, S)-1-phenylethanol, was used the highest reaction rates were measured for lipase adsorbed to the hydrophobic supports. When the primary alcohol, heptanol, was used free lipase exhibited the highest reaction rate. A kinetic explantation of these results is proposed.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Water activity as a key parameter of synthesis reactions: The example of lipase in biphasic (liquid/solid) media

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (a_w = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high a_w (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration. The mass of the enzyme preparation was shown to be a parameter affecting the capacity of the lipase to produce enough water in its immediate environment. The lack of activity observed for a small quantity of enzyme was eliminated by addition of heat-denaturated lipase.     

Enantioselective synthesis of ibuprofen esters in AOT/isooctane microemulsions by Candida cylindracea lipase

The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipase, was studied in a water-in-oil microemulsion (AOT/isooctane). By using n-propanol as the alcohol, an optimal W(0) ([H(2)O]/[AOT] ratio) of 12 was found for the synthesis of n-propyl-ibuprofenate at room temperature. The lipase showed high preference for the S(+)-enantiomer of ibuprofen, which was esterified to the corresponding S(+)-ibuprofen ester. The R(-)-ibuprofen remained unesterified in the microemulsion. The calculated enantioselectivity value (E) for S-ibuprofen ester was greater than 150 (conversion 0.32). The enzyme activities of n-alcohols with different chain lengths (3-12) were compared, and it appeared that short- (propanol and butanol) and long-chained (decanol and dodecanol) alcohols were better substrates than the intermediate ones (pentanol, hexanol, and octanol). However, unlike secondary and tertiary alcohols, all of the tested primary alcohols were substrates for the lipase. The reversible reaction (i.e., the hydrolysis of racemic ibuprofen ester in the microemulsion) was also carried out enantioselectively by the enzyme. Only the S form of the ester was hydrolyzed to the corresponding S-ibuprofen. The reaction yield was, however, only about 4% after 10 days of reaction. The corresponding yield for the esterification of ibuprofen was about 35% (10 days). The high enantioselectivity displayed by the lipase in the microemulsion system was seen neither in a similar esterification reaction in a pure organic solvent system (isooctane) nor in the hydrolysis reaction in an aqueous system (buffer). The E value for S-ibuprofen ester in the isooctane system was 3.0 (conversion 0.41), and only 1.3 for S-ibuprofen in the hydrolysis reaction (conversion 0.32). The differences in enantioselectivity for the lipase in various systems are likely due to interfacial phenomena. In the microemulsion system, the water in which the enzyme is dissolved is separated from the solvent by a layer of surfactant molecules, thus creating an interface with a relatively large area. Such interfaces are not present in the pure organic solvent systems (no surfactant) nor in aqueous systems. (c) 1993 John Wiley & Sons, Inc.     

Modification of hydrophilicity/hydrophobicity of the microenvironment of lipase of Candida rugosa by dextrans

Replacing the lactose used in the commercial preparation of lipase from Candida rugosa by dextrans with different molecular weight, several preparations with enhanced activities in esterification of (R,S)-ibuprofen in organic medium were obtained. The presence of carbohydrates modifies the microenvironment of the enzyme and maintains the hydration of the biocatalyst. We can modulate the hydrophilic/hydrophobic balance on the surface of the biocatalyst creating non covalent enzyme-dextran complexes.     

微水相超声波协同固定化脂肪酶催化酯交换过程优化

超声波协同固定化脂肪酶催化制备生物柴油的最佳工艺条件为：超声波功率70W、叔丁醇为反应介质、叔丁醇用量3％（v／v）、醇油比3：1且甲醇分三批加入、反应温度40℃、水含量为2％（v／v）。副产物甘油对固定化脂肪酶使用寿命影响最大,使用后的固定化脂肪酶用丙酮洗去表面的甘油,进行酯交换反应,酶的稳定性大为提高,可连续使用16批次。     

High-yield synthesis of wax esters catalysed by modified <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

Candida rugosa lipase (CRL) was applied in a non-solvent esterification reaction to yield twelve wax esters. All products were obtained in nearly 100% yield for 10 h at 50°C when immobilized PEG₂₀₀₀-activated C. rugosa lipase was added to the reaction mixture. The surfactant had also a beneficial effect on the stability of the biocatalytic preparation with 83% of its activity conserved after the seventh run of repeated batch reactions.     

Surfactant enhancement of (S)-naproxen ester productivity from racemic naproxen by lipase in isooctane

In the enantioselective esterification of racemic Naproxen with trimethylsilyl methanol in isooctane by Candida cylindracea lipase, improvements in (S)-naproxen ester productivity and enzyme selectivity were demonstrated by adding bis(2-ethylhexyl) sodium sulfosuccinate (AOT) as the best surfactant. The effect of water content on the enhancement of enzyme activity was elucidated from the reduced adsorption of surfactant molecules on the lipase. A competitive inhibition by the alcohol and a noncompetitive inhibition by the surfactant to the enzyme were found from the kinetic analysis. By using a two-phase extraction, a complete separation of the surfactant from the organic solution was obtained. (c) 1996 John Wiley & Sons, Inc.     

Lipase in biphasic alginate beads as a biocatalyst for esterification of butyric acid and butanol in aqueous media

Esterification of organic acids and alcohols in aqueous media is very inefficient due to thermodynamic constraints. However, fermentation processes used to produce organic acids and alcohols are often conducted in aqueous media. To produce esters in aqueous media, biphasic alginate beads with immobilized lipase are developed for in situ esterification of butanol and butyric acid. The biphasic beads contain a solid matrix of calcium alginate and hexadecane together with 5 mg/mL of lipase as the biocatalyst. Hexadecane in the biphasic beads serves as an organic phase to facilitate the esterification reaction. Under optimized conditions, the beads are able to catalyze the production of 0.16 mmol of butyl butyrate from 0.5 mmol of butyric acid and 1.5 mmol of butanol. In contrast, when monophasic beads (without hexadecane) are used, only trace amount of butyl butyrate is produced. One main application of biphasic beads is in simultaneous fermentation and esterification (SFE) because the organic phase inside the beads is very stable and does not leach out into the culture medium. SFE is successfully conducted with an esterification yield of 6.32% using biphasic beads containing iso-octane even though the solvent is proven toxic to the butanol-producing Clostridium spp.     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.