1株耐热脂肪酶产生菌的筛选及产酶条件研究

通过固体平板法,从食堂下水道污泥中筛选到1株耐热脂肪酶产生菌LJM-1,采用摇床培养法对该菌株的产酶条件进行了研究,结果表明,该菌株产脂肪酶的适宜培养基组成是:玉米粉淀粉6.0%﹑酵母粉3.5%﹑KH2PO40.3%﹑MgSO4.7H2O0.05%、(NH4)2SO40.5%﹑NaCl0.1%;适宜培养条件:培养温度40℃,初始pH7.7,摇床转速150r/min,装液量100mL/250mL三角瓶,接种量2.5%(体积分数),种龄72h,发酵时间48h。在此条件下,脂肪酶最高酶活为112.6U/mL。在单因素试验的基础上,采用正交试验对棉粕发酵脱毒条件进行了优化,得到最佳发酵条件是:初始pH7.7,培养温度40℃,接种量1.5%,发酵周期2d,酶活力达131.6U/mL。     

自筛菌株PM-1脂肪酶的提取及对脱脂乳风味改进的研究

本文将筛选到的、对乳脂肪有高度专一性的脂肪酶高产菌株PM-1发酵,上清液经硫酸铵沉淀、透析脱盐、蒸发浓缩后获得的粗酶提取液是原上清液酶活的4.5倍。将粗酶液发酵稀奶油产物用于90%脱脂乳中,感官评定效果良好,并通过测定增香90%脱脂乳中的游离脂肪酸和挥发性风味物质可知,增香后的90%脱脂乳中中链及部分长链脂肪酸增加,对奶香有突出贡献的特征性风味物质显著增长。从而肯定了该菌株所产脂肪酶的应用潜质。     

Purification and biochemical characterization of an intracellular lipase by Rhizopus chinensis under solid‐state fermentation and its potential application in the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)

BACKGROUND: Purification and characterization of an intracellular lipase produced by Rhizopus chinenesis cultured in solid‐state fermentation was investigated. The potential application in concentrating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil by the pure enzyme was also studied. RESULTS: Through four successive purification steps, the enzyme was purified to homogeneity with an apparent molecular mass of 36 kDa. The lipase was active for pH between 7.0 and 9.0 and temperatures 20–45 °C. Lipase activity was slightly increased in the presence of Ca²⁺ and Mg²⁺, but strongly inhibited by Hg²⁺ and SDS. The pure enzyme was most active on medium chain p‐nitrophenol esters, with the highest activity towards pNP‐caprylate (C8). The enzyme is a non‐specific lipase, because it cleaved not only the 1,3‐positioned ester bonds but also the 2‐positioned bond in triolein. High EPA (17.6%) and DHA (32.9%) contents were achieved using the pure lipase (100 U) within 10 h. CONCLUSION: The enzymatic activity of the lipase on a wide variety of substrates and its stability in the presence of some organic solvents suggest that the lipase should be investigated for a range of commercial applications. The pure lipase was proved to possess potential ability for the production and concentration of EPA and DHA from fish oil. Copyright © 2008 Society of Chemical Industry     

Enzymatic interesterification of lard and high-oleic sunflower oil with Candida antarctica lipase to produce plastic fats

Lard and high-oleic sunflower oil (Trisun® Extra) were interesterified at 55°C for 24 h with SP435 lipase from Candida antarctica to produce plastic fats. As the amount of trisun increased, percentage free fatty acid, unsaturated fatty acid/saturated fatty acid value, oxidizability, and the amount of 18:1 found at the sn-2 position of triglyceride products increased. Differential scanning calorimetry showed that the low-melting components in the product contained more 18:1 than the high-melting components. A 60:40 (w/w) ratio of lard to trisun had the widest plastic range (3–26°C). The scaled-up reaction to produce this blend resulted in a product that had 60.1% 18:1 at the sn-2 position compared to 44.9% for the physical blend. The solid fat content of the 60:40 interesterified mixture resembled soft-type margarine oil.     

Porcine pancreatic lipase immobilized on polysiloxane–polyvinyl alcohol hybrid matrix: catalytic properties and feasibility to mediate synthesis of surfactants and biodiesel

Polysiloxane–polyvinyl alcohol hybrid matrix was prepared by a sol–gel technique and its capacity to bind porcine pancreatic lipase investigated. The loading of 250 units g^?1 support was shown to be effective, resulting in an immobilized lipase with high catalytic activity. Both free and immobilized lipases were characterized by determining the activity profile as a function of pH, temperature, substrate concentration and thermal stability. Application of the immobilized lipase in non‐conventional biocatalysis for the synthesis of surfactants and biodiesel was also analyzed. Production of sugar fatty acid esters was found to be dependent on the carbohydrate and the highest molar conversion (50% in 3–4 h of reaction) was achieved for substrates containing fructose and lauric or oleic acids. Biodiesel synthesis from babassu oil and ethanol, propanol or butanol was feasible and regardless of the kind of alcohols, results revealed that the immobilized PPL could efficiently convert triglycerides to fatty acid alkyl esters attaining yields varying from 75 to 95%. Copyright © 2007 Society of Chemical Industry     

Screening catalytic lipase activities with an analytical supercritical fluid extractor

Two different screenings of several commercial lipases were performed to find a lipase with superior performance for the conversion of lipid moieties to their fatty acid methyl ester (FAME) derivatives under supercritical conditions. The first screening was done under hydrolytic conditions in a buffer. The second screening was done under supercritical conditions with CO₂, utilizing some of the same lipases for the methanolysis of different lipids. For the substrates studied, there was a significant difference in lipase activity under the two above conditions. Significant hydrolytic activity was demonstrated for three different lipid types (triglycerides, sterols, and phospholipids) with Lipase PS30, but when the same lipase was immobilized on an Accurel carrier (polypropylene), the activity decreased considerably. The opposite was found for Lipase G, which showed strong activity when immobilized and under supercritical conditions. Furthermore, Chirozyme L-1 was superior under supercritical conditions. The altered substrate specificity that some of these lipases show in supercritical CO₂ suggests several interesting synthetic options and applications under these conditions.     

Enantioselective esterification of ibuprofen with ethanol as reactant and solvent catalyzed by immobilized lipase: experimental and molecular modeling aspects

BACKGROUND: In recent years enantioselective esterification of racemic ibuprofen performed in organic co‐solvent media such as isooctane and cyclohexane and catalyzed by lipases, has been proposed as an effective way to increase the concentration of S‐ibuprofen in the racemic mixture. In this contribution, the enantioselective enzymatic esterification of (R,S)‐ibuprofen with ethanol catalyzed by commercial Novozym 435 without the addition of a co‐solvent is thoroughly investigated. Experimental data are further analyzed considering the results of extensive molecular modeling calculations. RESULTS: The conversion of ibuprofen towards the ethyl esters and the enantiomeric excess towards S‐ibuprofen are greatly affected by the ethanol and water contents of the reaction media. The optimum conditions for the esterification of racemic ibuprofen in a batch‐type reactor were as follows: molar ratio of ethanol to ibuprofen = 7, 4.8% v/v of water, 160 mg of Novozym 435, 45 °C and 200 rpm. Under these conditions an enantiomeric excess of 54% and 63% of ibuprofen conversion were reached. CONCLUSIONS: Results showed that the reaction in excess of the esterifying alcohol in a system free of additional organic solvents is possible if the proper conditions are set. Molecular modeling calculations demonstrated that the formation of dead‐end compounds between the enzyme and ethanol/water may account for lipase inhibition at high concentrations of those compounds. Copyright © 2009 Society of Chemical Industry     

Enhancement of activity and selectivity in lipase‐catalyzed transesterification in ionic liquids by the use of additives

BACKGROUND: Seven ionic liquids (ILs) based on 1-alkyl-3-methylimidazolium cation in combination with hexafluorophosphate and bis{(trifluoromethyl)sulfonyl}imide anions were tested as reaction media for lipase-catalyzed transesterification in low water conditions. With the aim of improving the activity and/or selectivity of the lipase, various treatments were applied to ionic liquid media such as equilibration with aqueous solutions of salts, NaHCO₃ or Na₂CO₃, or the addition of a catalytic amount of a non-reactive organic base to the reaction mixture, triethylamine. RESULTS: The treated ionic liquids were shown to be excellent media for lipase-catalyzed ester synthesis by transesterification compared with conventional organic solvents, such as n-hexane. All treatments were found to enhance the synthetic activity of the enzyme, the best results being achieved with the addition of triethylamine. The addition of a catalytic amount of this base to the ILs resulted in a significant increase in both the synthetic activity and selectivity values. For instance, the synthetic activity in [emim⁺][TfN₂⁻] was enhanced more than 12 times and the selectivity increased from 86% to 95% when triethylamine was used. CONCLUSION: These treatments could be easy-to-use approaches to improve the efficiency of enzymatic reactions in ionic liquids when the reaction does not proceed smoothly. Copyright © 2007 Society of Chemical Industry     

Fluorometric detection of interaction between lipase and glyceride

Previously, we devised the efficient modification of lipase, which can be dissolved and still maintain its activity in organic solvents. In this work, the fluorescence of the modified lipase could be detected in chloroform. When glycerides were added to the modified lipase solution, the intrinsic tryptophan fluorescence of the modified lipase decreased, which suggests that the environment of the tryptophan residue was affected by the substrate. The interaction between the modified lipase and glyceride was studied kinetically in terms of fluorescence intensity of the tryptophan residue. Because glyceride is not subject to hydrolysis in nonaqueous solution, the dissociation constant of the enzyme-substrate complex could be determined. Thus, insight into the direct interaction between enzyme and substrate provided some structural information regarding the active site of lipase.