磁性壳聚糖纳米复合材料固定化褶皱假丝酵母脂肪酶催化合成木质甾醇酯

以磁性壳聚糖纳米复合材料共价固定的褶皱假丝酵母脂肪酶为催化剂,以木质甾醇和油酸为原料,对木质甾醇油酸酯的酶法合成工艺条件进行了优化。得到的最佳工艺条件为：催化剂用量12.7%（以底物总质量计）,油酸与木质甾醇物质的量比为2∶1,木质甾醇质量浓度为122.9 g/L,反应温度50℃,反应时间24 h。在该条件下,木质甾醇转化率为96.42%。对月桂酸、肉豆蔻酸、棕榈酸不同碳链长度的脂肪酸或混合脂肪酸进行酯化反应,木质甾醇的转化率可达96.67%～98.74%,催化剂使用5次时,转化率仍可达82.45%。     

乙醛对假丝酵母脂肪酶催化活性的影响

采用紫外可见分光光度法和荧光发射光谱法研究了乙醛溶液对假丝酵母脂肪酶水解三油酸甘油酯的催化活性和构象的影响。结果表明,低浓度乙醛能提高酶的催化水解活力,当乙醛浓度为0.221 5 mmol/L时,酶活力提高了18.84%;高浓度乙醛对酶活力有抑制作用,当乙醛浓度为3.322 5 mmol/L时,酶活力降低了25.85%。低浓度乙醛使酶催化反应的最适p H向碱性方向偏移,紫外吸收光谱和荧光发射光谱均有显著增强和光谱峰偏移现象。动力学分析表明,加入乙醛后,酶的V_(max)增大,K_m减小。     

南极假丝酵母脂肪酶发酵条件优化及酶学性质

分别用摇瓶和15L发酵罐,对南极假丝酵母产胞外脂肪酶的培养基成分和操作条件进行了实验研究。得到最优的培养基组成为:豆粉40g／L,淀粉15g／L,豆油5mL／L,K2HPO4g／L,MgsO4·7H2O1g／L,Tween-800.1％,酵母膏5g／L;操作条件为:温度24℃,初始pH值为6.0,通气量为10.0L／min。在此培养条件下,发酵周期缩短至54h。由15L发酵罐生产的酶液酶活达到19.2U／mL。酶液在pH值为4.0～6.0和7.5～9.0范围内较稳定,其最适宜pH值范围为6～8.5;70℃时酶的催化活性最大.在40～70℃的温度范围内保持1h后残留酶活为60％。     

假丝酵母99-125脂肪酶促酯化合成生物柴油的研究

1 INTRODUCTION Biodiesel, that is long-chain fatty acid short-chain alcohol esters (methyl, ethyl, propyl and butyl ester), is produced by esterification of fatty acids or inter- esterification of oils and fats. These fatty acid alcohol esters are not only used as important industrial addi- tives and surfactants, but also used for biofuel. The biodiesel is a biodegradable, environmental friendly, renewable substitute of diesel fuel[1]. The traditional production of biodiesel is by chem- i…     

用蛋白质工程技术提取和修饰南极假丝酵母脂肪酶B

从南极假丝酵母中提取的脂肪酶B,具有较高的对应异构体选择性,广泛应用在培养基中,作为不对称有机化学的生物催化剂。近年来,一系列组合蛋白质工程研究和扩展了南极假丝酵母脂肪酶的催化和物理特性。这些工程不但生产出了指定的催化剂,还有助于阐明酶的结构一作用关系,并且有助于说明这些酶的对应异构体选择性。进一步的研究将在工程与酶的关系方面展开讨论。     

固定化假丝酵母脂肪酶合成棕榈酸异辛酯

开发了固定化假丝酵母脂肪酶99-125合成棕榈酸异辛酯的工艺.对反应温度、酶用量、底物摩尔比等酯化反应条件进行了研究.脂肪酶以吸附的形式固定在织物膜上,以1g棕榈酸、0.67g异辛醇、0.12g固定化脂肪酶和5ml石油醚组成的反应系统在40℃条件下反应24h,酯化率可达96.6%.固定化酶连续反应9批后酯化率仍维持在90%以上.     

响应面优化褶皱假丝酵母脂肪酶催化合成木质甾醇油酸酯

以木质甾醇转化率为指标,考察了10种常见商业化脂肪酶催化合成木质甾醇油酸酯的效果,确定褶皱假丝酵母脂肪酶(CRL)为优选生物催化剂,进一步筛选出正己烷为优选反应介质.在脂肪酶用量、油酸和木质甾醇的物质的量比、反应温度和反应时间这4个单因素考察基础上,通过响应面分析法对酶催化木质甾醇油酸酯合成工艺条件进行优化,并对优化条件进行验证和放大实验.CRL催化合成木质甾醇油酸酯的优化工艺参数为:CRL添加量为木质甾醇质量的10％,油酸与木质甾醇的物质的量比为3.8:1,反应温度为46℃,反应时间为28 h,木质甾醇的转化率为91.56％±0.25％.     

硫酸铵-丙酮协同沉淀法纯化南极假丝酵母产脂肪酶

采用硫酸铵-丙酮协同沉淀法,对南极假丝酵母产脂肪酶(CALB)进行了分离纯化研究.经正交实验确定纯化工艺为:向酶液中加入硫酸铵至饱和度45%的同时加入0.3倍酶液体积的丙酮;离心后再向上清液中加入硫酸铵至饱和度75%,分相后,相界面处沉淀即为脂肪酶粗品.经分离纯化后,酶活回收率为54%、纯化倍数为2.72.     

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     

以壳聚糖-戊二醛为载体柔性固定假丝酵母脂肪酶candida rugosa lipase

以壳聚糖-戊二醛(Chitosan-GA)为载体固定假丝酵母脂肪酶candida rugosa lipase(CRL)最高酶活可达240μ·g~(-1)。Chitosan-GA固定化酶循环使用4次后保留了20%左右的初始酶活力。固定化酶在水相中保存185d后保留了40%以上初始酶活力。固定化酶在有机溶剂(正庚醇)中浸泡120h后固定化酶酶活保留32.2%,而游离酶只保留原始酶活的8.5%。Chitosan-GA载体固定化脂肪酶在水相和有机相中的保存稳定性都好于游离酶。     

Synthesis of medium-chain glycerides using lipase from Candida rugosa

Enzymatic synthesis of medium-chain glycerides (MCG) from capric acid and glycerol was studied using lipase from Candida rugosa. The effects of various reaction parameters such as time, molar ratio of substrates (mmol capric acid/mmol glycerol), amount of lipase, type of organic solvents, and initial water activity (a _w ) were studied. The best conditions tested for MCG synthesis at 37°C were, respectively, time, 24 h; molar ratio of substrates, 2.5; and amount of lipase, 100.0 mg. The use of organic solvents greatly influenced the activity of lipase in the synthesis of MCG. Generally, activity of lipase was high in nonpolar solvents with log P values from 3.50 to 4.50, where P is the partition coefficient between water and 1-octanol. The enzymatic synthesis of MCG was preferably carried out at an initial a _w of 0.328, which resulted in maximal yield. Analysis of the products of reaction using gas chromatography showed that lipase from Candida rugosa seemed to produce more dicaprin and tricaprin than monocaprin.     

Synthesis of triacylglycerol from polyunsaturated fatty acid by immobilized lipase

More than 95% of polyunsaturated acid (PUFA) was converted to triacylglycerol by immobilized lipase fromCandida antarctica orRhizomucor miehei. The esterification was carried out at 50–60°C with shaking and dehydration for 24 h. The substrates consisted of glycerol and free fatty acid or ethyl esters of the fatty acid at a 1∶3 molar ratio. The docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) in the substrate polymerized during the reaction, and they required 5–10% more than the stoichiometric amount to compensate for the PUFA loss. On the contrary, ethyl esters of DHA and EPA were not polymerized. Pure tridocosahexaenoyl, trieicosapentaenoly and triarachidonoyl glycerol were isolated after passing the product through a basic aluminum oxide column. Industrial feasibility of this process was discussed for the ethyl ester as substrate. Portions of this article were presented at the annual meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry and at the annual meeting of The Japan Oil Chemists' Society held in Kyoto, Japan, March 31, 1991, and in Hamamatsu, Japan, October 4, 1991, respectively.     

Production of medium-chain glycerides by immobilized lipase in a solvent-free system

Enzymatic synthesis of medium- chain glycerides (MCGs) was studied by using capric acid (decanoic acid) and glycerol as substrates for immobilized lipase (Lipozyme^TM) without any solvents or surfactants. Quantitative analysis of the reaction mixture was conducted by using highperformance liquid chromatography (HPLC), which enabled the exact tracing of the capric glyceride synthesis. Oleic acid was also used for comparison. The esterification activity of Lipozyme was determined at 40°C in an open batch reactor; the activities were 400 and 200 units/g for the capric glyceride and oleic glyceride synthesis, respectively. Maximum initial reaction rate was obtained at 50°C for capric and 60°C for oleic glyceride synthesis. The time course of the capric glyceride synthesis was compared in terms of different molar ratios, from which we infer that this enzyme is 1,3- specific, but not absolute, in this esterification reaction. The final conversion was greatly influenced by the methods used to remove water, among which the cold trap method resulted in a noticeable improvement. *To whom correspondence should be addressed at Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, P.O. Box 150, Chongyang, Seoul 130-650, Korea.     

固定酶法生产生物柴油

探讨了利用本实验室自制的Candidasp.99-125脂肪酶转酯化合成生物柴油的过程。深入研究了甲醇对反应的抑制作用,酶用量、溶剂、底物浓度、反应温度、时间、水含量、pH值对生物柴油合成的影响,以及反应中固定化酶的寿命等问题。试验结果表明,采用最佳转酯化反应条件和分批加入甲醇的工艺条件下,最高单批转化率可以达到96%,固定化酶的使用半衰期可达到200h以上。     

Synthesis of n‐butyl acetamide over immobilized lipase

BACKGROUND: The conversion of carboxylic esters to amides can be accomplished efficiently by enzymatic catalysis. Amidation of benzyl acetate with n‐butyl amine was studied in non‐aqueous media using immobilized lipases. RESULTS: The activities of immobilized lipases, Novozym 435, Lipozyme RM IM and Lipozyme TL IM were evaluated in the synthesis of n‐butyl acetamide, among which Novozym 435 was the best. The process was optimized by studying various process parameters. Benzyl acetate conversion of 46% was achieved in 8 h for a mole ratio of 3:1 of n‐butyl amine to benzyl acetate with 3.67 g L^?1 Novozym 435 in toluene at 55 °C. A model based on an ordered bi–bi mechanism fitted the initial rate data very well and the rate constant and inhibition constants were calculated by non‐linear regression analysis. The initial rate studies showed that the Michaelis constant for benzyl acetate was low indicating high affinity between the enzyme and the reactant. CONCLUSION: A novel, efficient and environmentally benign enzymatic process is reported for the synthesis of n‐butyl acetamide. This method is general and can be used to synthesize analogous compounds in optically enriched form, since it is difficult to make such amides directly from carboxylic acids and amines by purely chemical means. The theoretical predictions and experimental data matched very well. Copyright © 2008 Society of Chemical Industry     

利用醋酸纤维/聚四氟乙烯复合膜中的微结构固定化脂肪酶

脂肪酶的固定化是降低其使用成本的有效途径之一.提出了利用亲水/疏水复合膜中的微结构固定化脂肪酶的新思路.首先制备由致密的亲水层和多孔的疏水层组成的醋酸纤维素/聚四氟乙烯（CA/PTFE）复合膜,然后利用复合膜的特殊微结构,用超滤的方法实现了脂肪酶的固定化.扫描电镜照片结果表明,大部分被截留的酶位于复合膜的界面处.制备得到的固定化酶膜应用于水解橄榄油的反应,其最高催化活力达到1.24 μmol FFA·min^-1·cm^-2,大大高于文献报道值.同时研究了固定化脂肪酶膜的催化动力学,考察了亲水层厚度和脂肪酶负载量对固定化效果的影响,优化了固定化条件.在经过10次（50 h以上）的重复使用后,固定化酶膜的活力仅降低了20%.     

Synthesis of monoglycerides by glycerolysis of rapeseed oil using immobilized lipase

Reaction conditions for the synthesis of monoglycerides (MG) by enzyme-catalyzed glycerolysis of rapeseed oil using Lipozyme® IM have been studied. Silica gel was used to adsorb the glycerol to overcome the problems of low glycerol solubility in the organic phase. An experimental design was used where temperature, time, the ratio of silica gel to glycerol (w/w), the water activity (a) _w , the isooctane concentration, and the ratio of glycerol to triglycerides (mol/mol) were varied. Response surface methodology was used to evaluate initial reaction rate and yield for the different products. The best yield of MG achieved under the studied conditions was 17.4% (mol fatty acid in substance/total mol fatty acid in mixture) (75°C, 20 h, silica gel/glycerol 2:1, a _w =0.17, 48% isooctane, glycerol/triglycerides 6:1). The same conditions yielded 36.8% diglycerides, 13.6% free fatty acids (FFA), and 36.9% triglycerides. This is at the same level as the equilibrium yield. The yield of MG is low compared to the final yield achieved with solid-phase glycerolysis. However, in solid-phase glycerolysis the reaction mixture becomes solid, and therefore the solid-phase process is not suitable for industrial application. The formation of FFA was very fast compared to the synthesis of MG. Equilibrium for FFA was reached within 2 h, and the yield was strongly affected by the a _w . Increasing a _w greatly increased the formation of FFA. In the a _w ratio 0.06–0.3, the yield of FFA increased from 4 to 19% while the yield of MG was nearly unaffected. As FFA is an undesired product, it is important to keep the a _w as low as possible.     

固定化脂肪酶催化制备香叶树籽生物柴油研究

研究了Novozym 435和Lipozyme TLIM混合脂肪酶催化香叶树籽油制备生物柴油,2种酶按1:3质量比混合使用时,既可提高反应转化率,又可降低酶的使用成本.应用响应面优化法确定了固定化酶催化香叶树籽生物柴油的最优工艺参数,采用叔丁醇作为反应体系的溶剂,最优反应条件为反应温度38.5℃、甲醇与油摩尔比4:1、叔丁醇与油体积比1:1.5、酶用量为油质量的4%,此时反应转化率达90.09%.分析表明香叶树籽油的甘油三酯主要由短链脂肪酸甘油酯组成,生物柴油中原油的甘油三酯已完全转变成脂肪酸甲酯.