响应面法优化丁酸缩水甘油酯的酶法拆分工艺

在已有的酶法拆分丁酸缩水甘油酯单因素试验最适条件的基础上, 采用Plackett-Burrman设计在影响酶法拆分的6个因素中, 有效筛选出主效因素: 底物浓度、酶用量和温度。在此基础上, 再利用响应面分析法(RSM)对以上三个显著因子的最佳水平范围进行研究, 通过对二次多项回归方程求解得知, 酶法拆分最适条件为: 底物浓度0.499 mol/L、酶用量30.23 mg/(g底物)和温度29.68oC; 并结合单因素最适条件: pH 7.6; 时间4 h; 转速150 r/min进行酶促拆分实验, 得到R-酯的对映体过量值为93.28%。对比优化之前的单因素试验最适条件的结果, 最大对映体过量值84.65%, 有了显著的提高, 证明RSM法优化酶法拆分丁酸缩水甘油酯工艺是可行的。     

虎杖中白藜芦醇的酶法制备

以白藜芦醇得率为指标,通过对酶制剂的筛选和酶解条件的考察,分别选出酶法提取和酶法转化制备虎杖中白藜芦醇的最佳条件,并对两种酶法进行比较.结果显示:(1)酶法提取最佳酶解条件为:酶解温度45℃,酶解时间60 min,最适pH 5.0,底物浓度17%;酶法转化最佳酶解条件为:酶解温度40℃,酶解时间48 h,最适pH 5.0,底物浓度15%.(2)与空白样品(不加酶)相比,酶法提取白藜芦醇得率增加了3.12 mg/g;酶法转化白藜芦醇得率增加了12.72 mg/g.研究表明,两种方法与不加酶提取相比均提高了白藜芦醇的得率,但酶法转化效果更显著,可用于白藜芦醇的制备.     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

米糠蛋白抗氧化活性肽的制备

以水解度(DH％)和对DPPH自由基清除率为指标,筛选出制备米糠蛋白抗氧化活性肽的最适蛋白酶.研究最适蛋白酶的酶解条件,探讨底物浓度、蛋白酶的加入量、pH值、酶解时间等因素对水解度(DH％)和DPPH自由基清除率的影响;在单因素基础上采用Box-Behnken响应曲面中心组合设计法,对酶解米糠蛋白的工艺进行优化.试验结果表明,在加酶量13970.82 U/g,时间3.05h,底物浓度4.97％的水解条件下,米糠蛋白的水解度能够达到23.67％,活性肽对DPPH自由基清除率达到64.26％.     

双酶法制备螺旋藻多肽的工艺研究

选用碱性蛋白酶和木瓜蛋白酶结合的双酶法对螺旋藻蛋白进行水解。其中,对木瓜蛋白酶水解螺旋藻蛋白的工艺进行优化。以水解度为指标,研究了酶解时间、酶与底物比、pH和酶解温度4种因素对酶解反应的影响。在此基础上设计了3因素(加酶量、酶解温度和pH)3水平的响应面试验。结果表明碱性蛋白酶水解螺旋藻蛋白的最佳酶解条件为:加酶量4300 U/g,pH 7.0,酶解温度55℃,酶解时间160 min;木瓜蛋白酶的最佳酶解条件为:酶底比为4.5%,酶解温度60℃,pH 6.5,酶解时间210 min。利用碱性蛋白酶和木瓜蛋白酶结合的双酶法制得的多肽水解度可达32.90%,与单酶法相比,水解度明显提高。     

脂肪酶YCJ01拆分对位取代α-苯乙醇

利用脂肪酶YCJ01催化拆分对位取代α-苯乙醇衍生物。以异丙醚为反应介质,采用乙酸乙烯酯作为酰基供体,对180 mmol/L的1-(4-甲基苯基)乙醇进行选择性酯化,脂肪酶粗酶粉添加量为5 g/L,50℃反应21 h后,底物转化率可达49.96%,对映体过量值e.e.s、e.e.p值分别为97.1%和97.2%,对映体选择性E200;同样,对1-(4-甲氧基苯基)乙醇进行选择性酯化,酰基供体为丁酸乙烯酯,底物浓度150 mmol/L,脂肪酶粗酶粉添加量为2.5g/L,30℃反应12 h后,底物转化率为49.8%,e.e.s、e.e.p值分别为97.7%和98.4%,对映体选择性E200,显示了很好的手性拆分效果。     

微水相脂肪酶催化乙酸甲基苯甲酯立体选择性氨解反应*

从 4种不同来源的脂肪酶 ,2种不同来源的蛋白酶中筛选出了具有较高活性和对映体选择性的能催化乙酸甲基苯甲酯氨解反应的脂肪酶Novozym 43 5。进一步探讨了氨源、酶浓度、底物浓度等因素对该酶反应的影响。结果表明 ,在优化条件下 ,氨解反应 6h ,转化率为51.6 %,残留底物中 ( ) 乙酸甲基苯甲酯对映体过剩值可达 99%以上。     

酶法协同超声波提取赤芝菌丝体多糖条件的优化

目的:本研究旨在优化赤芝菌丝体多糖提取条件。方法:采用酶法协同超声波法提取赤芝菌丝体多糖,以赤芝菌丝体粗多糖提取率为考察目标,通过单因素试验和正交试验,确定赤芝菌丝体多糖提取的最佳条件。结果酶法最佳提取条件为:果胶酶用量2.0%,酶解温度45℃,p H值为6.0,酶解时间60 min,多糖提取率为2.38%。在酶法处理基础上,进行超声波处理,超声处理最佳提取条件是:超声功率550 W,超声时间30 min(占空比5s/5s),料液比为1:35,多糖提取率为3.12%。     

α-淀粉酶水解魔芋飞粉最佳条件优化的研究

本研究采用单因素和正交试验的方法对α-淀粉酶水解魔芋飞粉的酶用量、pH值、温度、[Ca2 ]和底物浓度等条件进行优化,结果表明当α-淀粉酶用量为4u/g淀粉、pH为6.0、温度为60℃、[Ca2 ]为0.01mol/L和底物浓度为8%时,水解度达20%,为最佳反应条件,研究为进一步利用α-淀粉酶水解魔芋飞粉生产燃料乙醇奠定了基础.     

固定化脂肪酶催化制备L-薄荷醇

用大孔树脂NKA固定高选择性的脂肪酶,催化有机相中转酯化反应,从而拆分八异构体消旋薄荷醇来制备L-薄荷醇。研究pH、载体与酶比例对固定化酶制备的影响及固定化酶的反应稳定性;考察温度、转酯化过程醇酯比例、及底物醇异构组成变化对拆分效果的影响。结果表明:固定化酶的最适pH为8,载体与酶的比例为5∶1时,所得固定化酶的反应稳定性比游离酶的反应稳定性提高了约50%;转酯化反应的最优温度为40℃,醇酯比例为1.5∶1时最佳,改进八异构体消旋薄荷醇组分比例后,非对映体选择率dep达到了95.1%。     

环氧丙醇酯立体专-性水解酶产生菌的筛选

从土壤中筛选出一株能拆分（Ｒ,Ｓ）－环氧丙醇丁酸酯的根霉（Ｒｈｉｚｏｐｕｓｓｐ．Ｂｃ０－０９）,该菌株所产胞外脂肪酶在水解环氧丙醇丁酸酯的反应中具有良好的立体专一性。在ｐＨ恒定７．０的条件下,以其发酵液水解底物,当转化率为５８％时,残留的（Ｒ）－环氧丙醇丁酸酯的光学纯度（对映体过量值）达９６．１％。     

Optimization of Lipase-catalysed Synthesis of Butyl Butyrate Using a Factorial Design

Summary A lipase from Candida rugosa immobilized on styrene-divinylbenzene copolymer was used to catalyse the direct esterification of butanol and butyric acid. A factorial design was employed to evaluate the effects of temperature (37–50 °C), substrate molar ratio of butyric acid to butanol (0.6 to 2.0) and enzyme amount (0.2–0.4 g) on the ester yield. The main effects were fitted by multiple regression analysis to a linear model and maximum ester yield could be obtained working at 41 °C with 0.4 g of lipase. The mathematical model obtained, representing the ester yield has been found to describe adequately the experimental results. Under optimal conditions, concentration of 32.4 g butyl butyrate/l that corresponds to a yield of 75% was obtained.     

Response surface method to optimize the production and characterization of lipase from Penicillium verrucosum in solid-state fermentation

Current studies about lipase production by solid-state fermentation involve the use of agro-industrial residues towards developing cost-effective systems directed to large-scale commercialization of enzyme-catalyzed processes. In this work, lipase production and partial characterization of the crude enzymatic extracts obtained by Penicillium verrucosum using soybean bran as substrate was investigated. Different inductors were evaluated and the results showed that there is no influence of this variable on the lipase production, while temperature and initial moisture were the main factors that affected enzyme production. The optimized cultivation temperature (27.5 °C) and initial moisture of substrate (55%) were determined using the response surface methodology. Kinetics of lipase production was followed at the optimized growth conditions. Optimum lipase yield was 40 U/g of dry bran. The crude enzymatic extract showed optimal activity in the range from 30 to 45 °C and in pH 7.0.     

Dynamic kinetic resolution: alternative approach in optimizing S-ibuprofen production

A lipase catalysed enantioselective hydrolysis process under in situ racemization of the remaining (R)-ibuprofen ester substrate with sodium hydroxide as the catalyst was developed for the production of S-ibuprofen from (R,S)-ibuprofen ester in isooctane. Detailed investigations on parameters study indicated that 0.5 M NaOH, addition of 20% (v/v) co-solvent (dimethyl sulphoxide), operating temperature of 45 °C, and 40 mmol/L substrate gave 86% conversion and 99.4% optical purity of S-ibuprofen in dynamic kinetic resolution. Meanwhile, in common enzymatic kinetic resolution process, only 42% conversion of the racemate and 93% enantiomeric excess of the product was obtained which are of lower values as compared to dynamic kinetic resolution. The S-ibuprofen produced during each process was evaluated and approximately 50% increment in concentration of S-acid product was produced when dynamic kinetic resolution was applied into the process.     

Optimization and kinetic study of immobilized lipase-catalyzed synthesis of ethyl lactate

Abstract

Enzymatic synthesis of ethyl lactate catalyzed by immobilized lipase has been investigated. The reaction variables (including the molar ratio of ethanol to acid, total substrate amount, temperature, reaction time and rotation speed) were selected in accordance with the Plackett–Burman design and were further optimized via response surface methodology. The molar ratio of ethanol to acid, total substrate amount and reaction time were screened out as significant variables for the optimization study. A 20-run, full-factorial, central composite design was used to construct the statistical model and the optimal conditions obtained were as follows: molar ratio of ethanol to acid of 8.3:1, total substrate amount of 0.4 g, reaction time of 26.87 h with temperature of 55°C and rotation speed of 150 rpm. Under the optimal conditions, the yield of ethyl lactate was up to 24.32%; close to the 25.13% obtained using the commercial lipase, Novozym 435. Due to the low cost and simple immobilization process, the lipase prepared in the present work could have great potential in enzymatic applications. Additionally, a kinetic model with inhibition by both ethanol and lactic acid following a ping-pong bi-bi mechanism was proposed.     

Enantioselective enzymatic hydrolysis of racemic glycidyl butyrate by lipase from Bacillus subtilis with improved catalytic properties

The lipase from Bacillus subtillus (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of glycidyl butyrate. A high enantiomeric ratio (E = 108) was obtained by using 1,4-dioxane as co-solvent (18%, v/v) and decreasing the reaction temperature to 5 °C. The ratio is about 16-fold more than that (E = 6.52) obtained in pure buffer solutions (25 °C, pH 7.8). Under the optimum conditions, the remained (R)-glycidyl butyrate with high enantiopure (ee > 98%) was obtained when the conversion was above 52%.     

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.     

Kinetic resolution of racemic glycidyl butyrate using a multiphase membrane enzyme reactor: experiments and model verification

A laboratory-scale multiphase hollow fiber membrane reactor was employed to investigate the lipase-catalyzed enzymatic resolution of racemic glycidyl butyrate. A mathematical formulation was feveloped to simulate the performance of this system. Model parameters were determined independently (except the effective rate constant, k(s)) and incorporated in the model simulations. In this study, two modes of operation are considered: subtractive resolution, in which the unreacted substrate is recovered in the organic stream; and product recovery, where the optically pure product of the enzymatic reaction is recovered in the aqueous stream. Good agreement was obtained between theoretical predictions and experimental results under a variety of conditions. The effect of mass transport limitations on the performance of this system was investigated. An increase in enzyme loading resulted in a higher Thiele modulus due to an elevated rate constant as well as a concomitant decrease in the effective diffusivity. Optical purity decreased in both subtractive resolution and product recovery at higher Thiele modulus with the effect being more pronounced in the product recovery mode. Finally, normalized plots were established to describe the effect of enzyme immobilization on both the effective enzymes activity and effective diffusivity. (c) 1993 Wiley & Sons, Inc.     

诱变选育脂肪酶高产菌株及其酶学性质

面包干酵母(Saccharomyces cerevisiae)为出发菌株,对其进行紫外和微波复合诱变,得高产突变菌株DX213,高产突变菌株酶活力为635 U/mL,为出发菌株的1.69倍。菌株富集培养5代,遗传性状稳定。DX213菌株的最优产脂肪酶条件为:培养温度30℃和培养液pH 7.5。酶学性质研究表明:脂肪酶的最适温度40℃、最适pH为7.5、脂肪酶在40℃以下稳定。Fe3+离子对脂肪酶有激活效应,当Fe3+离子浓度为0.03 g/mL时,脂肪酶酶活力高达720 U/mL。