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。     

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。     

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。     

固定化假丝酵母1619脂肪酶催化油酸油醇酯的合成

比较了14种不同来源的脂肪酶催化油酸油醇酯的合成。其中,假丝酵母(Candidasp．)1619脂肪酶酯化能力最强,以硅藻土为载体,分别按0．1％添加椰子油、吐温80．按l％添加MgSO43种共固定物,醇化反应初速度提高了1．5倍。此固定化酶催化油酸油醇酯合成的最适温度为30℃,0～60℃下反应24h的酯化率均在90％以上,100℃下还有10．25％的酯化率。最适酯化pH6．0。反应中去水,可使终酯化率提高到99％。在添加的23种有机溶剂中,以异辛烷促进酯化的效果最好．正壬烷和正己烷次之。此固定化酶在28℃下批式重复反应的半衰期为990h,柱式固定床反应器中28℃连续运转1000h后酯化率为78％。     

乙二醇缩水甘油醚交联海藻酸钠-羧甲基纤维素钠固定化脂肪酶

以海藻酸钠、羧甲基纤维素钠(CMC)为载体,分别以乙二醇缩水甘油醚(EGDE)和戊二醛为交联剂,采用包埋交联法对脂肪酶进行固定化,结果显示EGDE的交联效果要优于戊二醛,添加EGDE的固定化酶酶活最好。得到制备固定化酶的最优方案为海藻酸钠2.5%,CMC浓度1.5%,给酶量800U/ml复配载体,氯化钙5%,以0.02%的EGDE交联固定30min,由此制备得到酶活约为380 U/g的固定化酶,酶活收率约为50.09%。固定化酶的最适反应p H为8.5,比游离酶增大0.5个单位;最适反应温度是45℃,比游离酶提高5℃;耐热性能变好,且重复使用7次后仍能保持60%左右的相对酶酶活。     

微水——有机溶剂两相体系中消旋萘普生的酶法拆分

研究了微水－有机溶剂两相体系中固定化脂肪酶催化的萘甲酯的立体选择性水解反应,固定化酶活性受载体极性、水含量、有机溶剂的ｌｏｇＰ值,产物抑制的影响,据此构建了一种可以连续拆分产生（Ｓ）－（＋）－萘普生的微水－有机溶剂两相体系。反应在一个具有回路的连续流搅拌反应器中进行,反应器中添加有采用吸附法固定化的脂肪酶,截体为一种弱极性的合成载体,水相连同固定化酶颗粒一起永久保持在反应器中,有机流动相带入底物,     

具有磁响应性的聚乙二醇载体固定化糖化酶的研究

以两亲性的聚乙二醇胶体粒子,通过吸附－交联法固定化糖化酶。研究了戊二醛浓度,缓冲液ｐＨ值及加酶量对酶固定化的影响,固定化酶活力可达１７０９５Ｕｐｅｒｇｒａｍ干胶,活力回收为６３％。磁性固定化酶最适温度比天然酶提高５℃,最适ｐＨ比天然酶提高０５个ｐＨ单位。并且对酸、碱、热的稳定性大大增强,贮存及操作稳定性也大大提高。该磁性载体合成简单,固定化方法简便,具有磁性,易于分离,因而为工业上的应用提供了一种新的可能。     

聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 *

聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0.75%交联剂浓度、交联温度35℃、交联时间3h、载体量1.25g、pH9.0、固定化温度55℃、固定化时间1h。对LX-1000EA-PEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA- PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7.0提高为8.0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78.84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。     

化学修饰的和固定化的脂肪酶在有机溶剂中的催化作用研究

本文对聚乙二醇修饰脂肪酶、多孔玻璃载体吸附酶、多孔玻璃载体丙酮沉积酶、硅藻土吸附酶、氧化铝吸附酶和琼脂珠疏水载体吸附酶在有机相中酯合成和酯交换反应的催化作用进行了研究。实验表明,不同形式的酶需要不同的最适加水量。而且,在各自最适条件下,对各种形式酶进行了比较,得出硅藻土和琼脂珠疏水载体是很好的固定化载体,疏水性琼脂珠固定化酶在有机相中的活力比酶粉高46.5％。     

聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶

聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0. 75%交联剂浓度、交联温度35℃、交联时间3h、载体量1. 25g、pH9. 0、固定化温度55℃、固定化时间1h。对LX-1000EAPEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA-PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7. 0提高为8. 0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78. 84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。     

Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres

A novel magnetic poly(vinyl acetate (VAc)–divinyl benzene (DVB)) material (8–34 μm) was synthesized by copolymerization of vinyl acetate and divinyl benzene using oleic acid-stabilized magnetic colloids as magnetic cores. The magnetic colloids and the copolymer microspheres were characterized with transmission and scanning electron microscopes, respectively. Magnetization of the microspheres could be described by the Langevin function. All the observations indicated that the microspheres were superparamagnetic. Magnetic sedimentation of the microspheres was achieved within 3 min, over 300 times faster than the gravitational sedimentation. Candida cylindracea lipase (CCL) was immobilized to the porous carrier at up to 6750 IU/g carrier, remarkably higher than the previous studies. The pH and temperature dependencies of the immobilized CCL were investigated and the optimum temperature and pH for the immobilized CCL were determined. Activity amelioration of the immobilized CCL for the hydrolysis of olive oil was observed, indicating an interfacial activation of the enzyme after immobilization. Moreover, the immobilized CCL showed enhanced thermal stability and good durability in the repeated use after recovered by magnetic separations.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system

Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 mug/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.     

Immobilization of penicillin acylase on acrylic carriers

Penicillin acylase obtained from E. Coli (E. C. 3.5.1.11) was covalently bound via glutaric aldehyde to acrylic carriers crosslinked with divinylbenzene or ethylene glycol dimethacrylate. The best enzymatic preparation was obtained by using ethyl acrylate/ ethylene glycol dimethacrylate copolymer. 1 cm³ of the carrier bound 6.4 mg of protein, having 72% activity in relation to the native enzyme. The preparation lost only 10% of its initial activity after 100 d of storage at 4°C. A negligible effect of immobilization on the enzyme activity at different temperatures or pH as well as significant increase of the stability of the immobilized enzyme at elevated temperatures were observed.Abbreviations BA butyl acrylate - AE ethyl acrylate - PA penicillin acylase - 6-APA 6-aminopenicillanic acid - EGDMA ethylene glycol dimethacrylate - DVB divinylbenzene     

Effect of solvents on hydrolysis of olive oil by immobilized lipase in reverse phase system

Summary Lipase fromCandida rugosa was immobilized by adsorption on three supports which could contain water available for the hydrolysis of olive oil in a reverse phase system. To select the most suitable solvent for this system, the effect of organic solvents on the stability and catalytic activity of immobilized lipase for the hydrolysis reaction has been examined. The results revealed that isooctane was superior to any other solvents tested in this study for enzymatic fat splitting in a reverse phase system. Also the effect of the solvent polarity on the hydrolysis of olive oil has been examined in detail using various organic solvents mixed with an equivolume of isooctane. It was found that the hydrolysis of olive oil by immobilized lipase was markedly affected by the polarity of reaction solvents.     

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。     

Synthesis of a mesoporous functional copolymer bead carrier and its properties for glucoamylase immobilization

A series of mesoporous and hydrophilic novel bead carriers containing epoxy groups were synthesized by modified inverse suspension polymerization. Glycidyl methacrylate and acryloyloxyethyl trimethyl ammonium chloride were used as the monomers, and divinyl benzene, allyl methacrylate, and ethylene glycol dimethacrylate as crosslinking agents, respectively. The resulting carriers were employed in the immobilization of glucoamylase (Glu) with covalent bond between epoxy groups and enzymes. The activity recovery of the three series of immobilized Glus could reach 76%, 79%, and 86%, respectively. The immobilized Glus exhibit excellent stability and reusability than that of the free ones.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

Covalent-bonded immobilization of lipase on poly(phenylene sulfide) dendrimers and their hydrolysis ability

Covalent-bonded immobilization of lipase from burkholderia cepacia onto two poly(phenylene sulfide) (PPS) dendrimers with different generations (two and three) was achieved using carbodiimide as a coupling reagent. The hydrolysis activity of olive oil to fatty acid was studied on enzyme-immobilized PPS dendrimers. Enzyme activity was proportional to the enzyme loading, and highest recovered activity was obtained at the medium enzyme loading for both G2 and G3 dendrimers. The immobilization improved the optimum pH and caused the temperature range to widen. Immobilization of enzyme has enhanced the thermal stability of enzyme activity in comparison with free enzyme. The immobilized enzyme as a biocatalyst for batch hydrolysis of olive oil retained 80 approximately 90% activity even after 20 times of recycling. This retention of activity after recycle is very valuable and powerful in enzyme technology. The present noteworthy and vital availability on enzyme reaction of the covalently bonded immobilized lipase on dendrimer came from the structure of dendrimer with a large number of functional terminal groups, which are easily available for immobilization of many lipases at the situation keeping reactive enzymes on the surface of dendrimer.     

The Effect of Substrate Hydrophobicity on the Kinetic Behaviour of Immobilized Candida rugosa Lipase

Candida rugosa lipase (EC 3.1.1.3.) was immobilized in a hydrophilic polyurethane foam and used in the hydrolysis of olive oil, in H-hexane. The results obtained were compared with those from a previous study, in which the same lipase preparation was used in the esterification of ethanol with butyric acid.

The initial rate of hydrolysis increased exponentially with increasing olive oil concentration. In contrast, for the esterification reaction, Michaelis-Menten kinetics with inhibition by both substrates, had been observed.

The effect of medium viscosity, stirring conditions and size of immobilization particles could not explain the observed kinetics of the hydrolytic reaction. However, a direct relationship was observed between the log P values of the reaction medium and the initial rate of hydrolysis, i.e., activation of the immobilized Candida rugosa lipase appears to be promoted by a high hydrophobicity of the reaction medium.

In the case of the esterification reaction, no similar correlation was found.