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。     

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。     

蚕丝固定化脂肪酶的研究

研究了蚕丝固定化脂肪酶的工艺条件,并考察了固定化脂肪酶的稳定性。试验结果表明：蚕丝与对-β-硫酸酯乙砜基苯胺(SESA)进行反应的最适条件是PH=10．8,SESA：2．0g／g蚕丝,反应生成的对氨基苯磺酰乙基蚕丝(ABSE-蚕丝)经重氮化后与脂肪酶偶联的最适条件是：pH=7．5,偶联时间>10h。加酶量为168～308u／g蚕丝时,所得固定化脂肪酶活力为106～160u√g蚕丝．此时固定化冀的活力回收率较高(>52％)。固定化脂肪酶稳定性较高．其操作半衰期约为250h。     

微生物脂肪酶稳定性研究进展

脂肪酶广泛应用于食品、药物、生物燃料、诊断、生物修复、化学品、化妆品、清洁剂、饲料、皮革和生物传感器等工业领域,微生物脂肪酶是商品化脂肪酶的重要来源。高温、酸性、碱性和有机溶剂等恶劣的工业生产环境使得脂肪酶的进一步工业应用受到限制,获取稳定性好的脂肪酶成为打破这一限制的关键环节。本文重点对提高微生物脂肪酶稳定性的策略进行了综述:挖掘极端微生物脂肪酶资源;利用定向进化、理性设计和半理性设计等蛋白质工程策略改造脂肪酶;利用物理吸附、封装、共价结合和交联等酶的固定化技术提高脂肪酶的稳定性;利用物理/化学修饰、表面展示以及多种改良策略相结合提高脂肪酶的稳定性。结合作者前期对酶工程的研究发现,新型酶催化剂的获得应该基于明确的设计思路,结合多种改造方法,基于定向进化-理性设计、定向进化-半理性设计、蛋白质工程-酶的固定化、蛋白质工程-物理/化学修饰、酶的固定化-物理/化学修饰等组合改造,比单一的改造方法具有更高的效率。     

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。     

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。     

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。     

海藻酸-SiO2杂化凝胶固定化洋葱伯克霍尔德茵脂肪酶的研究

利用四乙氧基硅烷（TEOS）原位水解法将SiO2掺杂于海藻酸（ALG）凝胶中,通过双交联制备出新型ALG—SiO2杂化凝胶以固定化洋葱伯克霍尔德菌脂肪酶。结果表明,固定化酶的最优条件：质量分数为2．0％的ALG、0．2mol／LCaCl2、V（ALG）／V（TEOS）为5、加酶量为1gALG加100mg酶粉、固定化60min、采用直径为0．8mm的针头滴定、真空冷冻干燥。在此条件下,酶蛋白的包埋率可达100％,酶活回收率可达91％。固定化酶的最适pH为8．0,最适作用温度为50℃,重复使用8次后,酶活性仍能保持80％以上。ALG—Si02杂化凝胶的场扫描电镜（FESEM）观察发现凝胶的整体构造仍然是海藻酸凝胶骨架;与ALG凝胶平滑的内部相比较,杂化凝胶仍具有完整的网络结构,但内部更为粗糙,结构更为致密。     

壳聚糖固定化德氏根霉脂肪酶的研究

研究了壳聚糖吸附和戊二醛交联对脂肪酶固定化条件,在室温条件下将0．4g酶粉溶于pH6．0缓冲液中,加入10g壳聚糖,摇匀,再加入浓度为0．6％戊二醛交联6h,得到固定化酶,酶活力回收率约为54．2％。固定化酶的半失活温度比游离酶的高,半失活温度由游离酶的47℃提高到100℃,最适反应温度由40℃上升至80℃,最适pH由6下降到5．5,固定化酶K’m值由游离酶的Km 50mg／mL增加到56mg／mL。该固定化脂肪酶用于酯的合成;在80℃条件下经过10批次连续水解植物油反应,固定化酶的活力仍保持在82．6％以上。     

Immobilization of Candida rugosa lipase on colloidal gas aphrons (CGAs)

A novel technique for immobilization of Candida rugosa lipase onto anionic colloidal gas aphrons (CGAs) is described. CGAs are spherical microbubbles (10-100 microm) composed of an inner gas core surrounded by a surfactant shell. In this initial study, greater than 80% lipase (w/w) was effectively retained on the CGAs. Leakage of protein from the CGAs and the activity of the adsorbed lipase decreased with increasing enzyme loading; this indicates that multilayers of lipase may be adsorbing onto the CGAs. The CGA-immobilised lipase displayed normal Michaelis-Menten dependence on substrate concentration and also exhibited greater activity than the free enzyme.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.     

帆布固定化脂肪酶及其性能的研究

研究了用高碘酸钠氧化帆布纤维,使其纤维衍生化成为醛基,与脂肪酶交联进行固定化的过程。通过醛基被交联程度来评价交联过程的优劣。首先对纤维的氧化过程进行了简单优化,进而通过反复交联法与酶蛋白交联。以大豆油和橄榄油水解作为固定化酶的性能评价指标。实验结果表明,通过采用反复交联的方法,可提高载体表面酶蛋白质量分数30％左右。酶活力平均达到4U／cm^2,其对温度、pH的耐受性相比游离酶均有不同程度提高。同时利用油脂在固定化酶过程对酶进行保护,使其对温度、pH等的耐受性进一步增强。在维持较高水解率条件下,可在温和条件下连续反应7批,反应半衰期达140h以上。     

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.     

D301树脂固定化假丝酵母脂肪酶

本研究选择7种吸附和离子交换树脂进行了假丝酵母脂肪酶(Candida sp.lipase)的固定化试验,通过测定固定化后各脂肪酶的酶活,筛选出固定化效果较好的弱碱性阴离子交换树脂D301;并通过扫描电镜将D301与脂肪酶Novozym 435的表面形貌做比较,进一步选定D301树脂作为载体,并对其采用戊二醛交联固定化,研究并优化了其固定化条件。结果表明,5%戊二醛溶液的加入量为8mL,处理时间为5h,酶液浓度为1.0g/L,磷酸缓冲盐溶液pH6.0,固定化处理10h效果最好,获得的固定化酶活力可达35U/mg,酶的固定化效率约为3.5U/(mg·h)。     

Stability analysis ofBacillus stearothermopilus L1 lipase fused with a cellulose-binding domain

This study was designed to investigate the stability of a lipase fused with a cellulose-binding domain (CBD) to cellulase. The fusion protein was derived from a gene cluster of a CBD fragment of a cellulase gene inTrichoderma hazianum and a lipase gene inBacillus stearothermophilus L1. Due to the CBD, this lipase can be immobilized to a cellulose material. Factors affecting the lipase stability were divided into the reaction-independent factors (RIF), and the reaction-dependent factors (RDF). RIF includes the reaction conditions such as pH and temperature, whereas substrate limitation and product inhibition are examples of RDF. As pH 10 and 50°C were found to be optimum reaction conditions for oil hydrolysis by this lipase, the stability of the free and the immobilized lipase was studied under these conditions. Avicel (microcrystal-line cellulose) was used as a support for lipase immobilization. The effects of both RIF and RDF on the enzyme activity were less for the immobilized lipase than for the free lipase. Due to the irreversible binding of CBD to Avicel and the high stability of the immobilized lipase, the enzyme activity after five times of use was over 70% of the initial activity.