脂肪酶的固定化及其性质研究

采用吸附与交联相结合的方法国定化脂肪酶,研究了脂肪酶固定化的工艺条件,并考察了固定化脂肪酶的催化性能和稳定性。试验结果表明,WA20树脂固定化脂肪酶的最适条件是：酶液pH7．0、给酶量300IU／g树脂、固定时间8h,所得固定化脂肪酶的活力约为165IU／g树脂;固定化酶稳定性较高,在冰箱内贮存6个月活力没有下降,操作半衰期约为750h,而未用戌二醛文联的固定化脂肪酶操作半衰期仅约290h;固定化脂肪酶催化橄榄油水解的最适条件是：PH8．0、温度55℃、底物浓度60％（V／V）、搅拌转速500r／m。     

固定化脂肪酶性质及其应用研究

利用以四甲氧基硅烷(TMOS)和甲基三甲氧基硅烷(MTMS)为前驱体的溶胶-凝胶法(sol-gel)固定洋葱假单胞菌属脂肪酶,考查了固定化酶和游离酶的酶学性质及催化不同油脂酯交换合成生物柴油的情况。结果表明,80℃以下固定化酶能保持80%以上的酶活,而游离酶在50℃以后活力急剧下降,到80℃残余酶活约为10%;固定化酶在体积分数50%的甲醇中处理48 h能保持85%的酶活,在体积分数90%的乙醇中处理48h能保持31%的酶活,而游离酶残余酶活只有69%和0;在酯交换反应中固定化酶的催化效率比游离酶高10%~20%,且固定化酶重复使用11次后仍能保持60%的酶活。结果显示,酶经过固定化后稳定性和催化活性显著提高。     

脂肪酶固定化及其稳定性研究

目的:研究脂肪酶的固定化工艺及其稳定性。方法:以四甲氧基硅烷(TMOS)和甲基三甲氧基硅烷(MTMS)为前驱体的溶胶-凝胶法(sol-gel)固定化黑曲霉属脂肪酶。结果:最优固定化条件是:TMOS 0.5mmol、MTMS 2.5mmol,水与硅烷前驱体摩尔比(R)12,PEG400 120μL,给酶量120mg。酶的固定化效率为93.7%,比活力为游离酶的2.2倍。固定化酶和游离酶在60℃处理2h,其残余酶活分别为91.8%和0;在pH 11的缓冲液中处理2h,其残余酶活分别为95.2%和82%。结论:酶经固定化后其活力、热稳定性和pH稳定性均有提高。     

海泡石吸附固定化猪胰脂肪酶

以海泡石作为猪胰脂肪酶(PPL)的固定化载体,考察采用物理吸附的方法制备固定化脂肪酶的条件。结果表明:在固载时间4 h、反应磷酸盐(PBS)溶液pH 6.0、反应温度25℃时,可达最大比酶活309 U/g,固定化酶的化学稳定性和热稳定性均较高。同时利用红外谱仪(FT-IR)和扫描电子显微镜(SEM)的分析手段对固定化猪胰脂肪酶试样进行分析,进一步确定了海泡石材料在固定化酶中的作用。     

改性壳聚糖固定化脂肪酶的研究

以化学改性后的壳聚糖为载体固定假丝酵母99-125脂肪酶,研究了不同的活化剂对壳聚糖表面羟基基团的活化程度,及以活化后壳聚糖为载体采用不同固定化方法对假丝酵母脂肪酶固定效果的影响。结果表明1-乙基-3-(3-甲基氨基)丙基碳二亚胺可有效的活化壳聚糖表面羟基,活化后的壳聚糖表面氨基与戊二醛偶联后形成的壳聚糖为良好的脂肪酶固定化载体,其固定脂肪酶的水解活力可高达86.8U/g。此外,还对影响固定化进程中的各种因素进行了研究,确定最优条件,比较了固定化前后酶的热稳定性、有机溶剂稳定性及最适反应温度。并考察了该固定化脂肪酶催化合成棕榈酸十六酯的操作稳定性,结果表明,连续反应16批之后棕榈酸十六酯的转化率仍能达到85%以上。     

固定化脂肪酶合成二元酸酯

The syntheses of dicarboxylic esters by immobilized lipase from Candida sp. -1619 were investigated. The reaction system was composed of 1 mmol dicarboxylic acid, 2 mmol alcohol, 3 mL hexane and 15 mg celite-adsorbed im mobilized lipase(300 u), in a closed 100 mL Erlenmeyer flask, shaken at 40°C for 5h. Sebacic acid was the best substrate among nine dicarboxylic acids selected. Among the 18 saturated fatty n-alcohols, the alcohols with carbon chain length rangin from C4～C18 had good reactivity. The primary alcohols had much better reactivities than corresponding secondary alcohols and multihydroxy-alcohols. Tertiary alcohols showed no reactivity. Hydrocarbons, benzene, toluene, xylene and te trachloride were favorite reactants among 15 organic solvents selected, in none-solvent stationary system, (5 mmol sebacic acid, 10 mmol dndecanol, 150 mg immobilized lipase(3000 u))reacted without plug for 3.5h, the optimum temperature was 60°C. The conversion degree was over 92% when reaction carried out at 50～90°C for 17h. The suitable reaction pH ranged from 6～8. The reactant was developed on GF254 plate(hexane ethyl ether acetic acid = 30201 ( V V V).There were three spots with different Rf value at 0.96, 0.55 and 0 corresponding to product, oleyl alcohol and sebacic acids, respectively.     

脂肪酶的固定化新技术

脂肪酶已被广泛应用于有机相催化体系中的水解、酯化和转酯反应,其固定化技术是得以工业化应用的关键因素。简要介绍了最新发展的几种固定化技术即交联酶晶体、硅基质包埋、脂质包被、共价固定、ＣＬＡｓ固定化等方法。     

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%。     

诱变选育脂肪酶高产菌株及其脂肪酶固定化

以紫外和微波复合诱变选育脂肪酶产生菌 Rhizopus sp. RXF12,获得高产突变株RZ13,其脂肪酶摇瓶发酵单位是出发株的2.62倍。菌株经多次传代,遗传性状稳定。对RZ13菌株的发酵条件进行了正交优化,在25 ℃、pH 8.0的条件下,接入5 %(v/v)的RZ13菌株单孢子悬液 (107个/ml) 振荡培养84 h,达到RZ13菌株最佳产酶状态,脂肪酶活可达95.08 U/ml。考察了脂肪酶性质,在低于40 ℃,pH 7.0~9.0范围内脂肪酶活稳定。经载体筛选及固定化过程优化,选用镁铝水滑石25℃吸附4 h,对RZ13脂肪酶进行了固定化。结果表明,固定化酶的最适作用温度为35~55℃,pH为7.5~9.0,较游离酶的均有较大扩展。     

Enzymic Interesterification of fats: Immobilization and Immunogold Localization of Lipase on Ion-Exchange Resins

A 1,3 specific lipase from Mucor miehei has been immobilized to two phenolformaldehyde resins with active tertiary amine groups and different porous structures (Duolite ES562 and ES568N). Duolite ES568N has a more uniform pore structure, allowing more rapid uptake of lipase. Immobilized lipase particles were treated by washing, dehydration with glycolmethacrylate and embedding in polymer. Following immunogold staining of thin sections, examined by electron or light microscopy showed that the location of the lipase was dependent on the pore structure of the support material and the immobilization time.     

溶胶凝胶法固定化黑曲霉脂肪酶的性质研究

近年来溶胶-凝胶法固定脂肪酶已成为研究热点。选用TMOS、MTMS、ETMS和PTMS 4种硅烷试剂对黑曲霉脂肪酶进行了固定化研究。固定化的最佳配方为ETMS/TMOS=5：1、水与硅烷试剂分子比为8;固定化脂肪酶的固定率为80.2%、相对活性为136.3%;以乳化橄榄油作为底物,在50℃和pH4.0的条件下,固定化脂肪酶与游离脂肪酶K_m分别为1.899×10^-4M和2.789×10^-4M;最适反应pH均为pH4.0,固定化脂肪酶在pH4.0~pH5.5之间其活性能保持95%以上;固定化脂肪酶最适反应温度为60℃,较游离脂肪酶提高了10℃;固定化脂肪酶的酸碱稳定性和热稳定性较非固定化酶有显著的提高。固定化脂肪酶的使用寿命和保存稳定性良好,使用12次后仍能够保留71.7%活性,在室温避光条件下保存180天后仍可保留79.2%活性。     

Enzymic Interesterification of fats: Immobilization and Immunogold Localization of Lipase on Ion-Exchange Resins

A 1,3 specific lipase from Mucor miehei has been immobilized to two phenolformaldehyde resins with active tertiary amine groups and different porous structures (Duolite ES562 and ES568N). Duolite ES568N has a more uniform pore structure, allowing more rapid uptake of lipase. Immobilized lipase particles were treated by washing, dehydration with glycolmethacrylate and embedding in polymer. Following immunogold staining of thin sections, examined by electron or light microscopy showed that the location of the lipase was dependent on the pore structure of the support material and the immobilization time.     

脂肪酶生物印迹研究进展

脂肪酶是一种广泛应用的水解酶。生物印迹技术是一项新兴技术,它使脂肪酶在有机溶剂中发挥更好的催化能力,如更高的活性和稳定性,从而扩大了脂肪酶作为生物催化剂在工业上的应用。本文介绍了脂肪酶蛋白质的结构特点,脂肪酶生物印迹的过程和原理,对生物印迹过程中印迹模板的种类和选择原则、保护剂的作用等主要影响因素进行了讨论,并对不同种类脂肪酶的生物印迹条件和适用的反应及生物印迹效果作了归纳总结,指出了目前脂肪酶生物印迹技术存在的问题和发展前景。     

高分子载体固定化酵母脂肪酶的研究

合成了一系列丙烯酸甲酯－双甲基丙烯酸乙二醇酯、甲基丙烯酸羟乙酯－双甲基丙烯酸乙二醇酯、苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物作为固定化酵母脂肪酶的载体,比较了这些载体固定化酵母脂肪酶催化橄榄油水解反应的效果,结果发现疏水性较强的苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物固定化酵母脂肪酶的效果最好．比较了酵母脂肪酶溶液和苯乙烯－双甲基丙烯酸乙二醇酯文联共聚物固定化酶催化橄榄油水解反应的最适ｐＨ值、最适离子强度、最适温度和Ｋ＿ｍ值．同时考察了苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物固定化酶在有机溶剂中催化十六酸戊酯合成反应的活力．     

Variables that Affect Immobilization of Mucor Miehei Lipase on Nylon Membrane

Nylon membrane was used to immobilize Mucor miehei lipase. Variables that affect this immobilization procedure were studied by experimental design. A 2³ full factorial design was employed for this purpose. The protein retention and hydrolytic activity of the immobilized lipase were used as response variables. The rapid loss of enzyme activity was the main problem during repetitive use. Two strategies were used to improve the low operational stability: nylon treated with HCl and nylon coated with polyvinyl alcohol (PVA). Lipase-nylon-PVA was the best enzyme derivative, allowing performance of five consecutive assays, with a retained activity of 0.5 U mg of protein⁻¹ g of support⁻¹.     

氨基载体共价结合固定化海洋假丝酵母脂肪酶

共价结合法是重要的工业酶固定化方法,利用稳定的共价键固定化工业酶,在载体和酶间形成多点共价连接,可以制备稳定性较好的固定化酶,更具有实际应用价值。利用氨基载体共价结合固定化海洋假丝酵母脂肪酶,采用较为廉价的戊二醛进行辅助交联,通过单因素和正交试验,确定最佳固定化条件为:25℃、pH5. 0、0. 1%戊二醛、0. 25g载体、交联0. 5h、固定化1h、加酶量为800U,最终得到的固定化酶酶活达到83. 01U/g。固定化脂肪酶的最适pH较游离酶向碱性方向偏移,最适反应温度提高10℃,固定化酶的热稳定性和酸碱稳定性比游离酶好且重复使用性和储存稳定性明显优于游离酶。同时发现交联剂是制备固定化脂肪酶的重要因素,因此探索新型交联剂对于固定化效果的提高具有重要意义,为海洋假丝酵母脂肪酶的固定化工艺技术和工业应用奠定了良好基础。     

Effect of the Immobilization Method of Lipase from Thermomyces lanuginosus on Sucrose Acylation

Lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized using granulation by incubating low-particle-size silica with the lipase. Granules with a particle diameter in the range 0.3-1 &#117 mm were obtained. The immobilized lipase was tested in the acylation of sucrose with vinyl laurate in mixtures of tert -amyl alcohol: dimethyl sulfoxide. Results were compared with immobilization of enzyme by adsorption on polypropylene (Accurel EP100), deposition on Celite by precipitation, and covalent attachment to Eupergit C. Granulated lipase converted >95% of sucrose into 6- O -lauroylsucrose in 6 &#117 h. Accurel-lipase was also very active, converting 70% of sucrose into monoester in 2 &#117 h. The residual activity of granules after five reaction cycles under the best reaction conditions was 72%; this value was considerably higher than the one observed for the same lipase adsorbed on Accurel (15% residual activity after five cycles).