氨基化硅胶载体固定化纤维素酶的研究

用硅胶作载体,戊二醛作交联剂,制备了固定化的纤维素酶。对制备固定化纤维素酶的偶联剂浓度、pH、给酶量3个影响因素进行了研究,通过正交试验优化得出最佳的固定化条件:交联剂戊二醛浓度为1%,固定化pH值为5,固载量为每克载体100mg纤维素酶。     

葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。     

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。     

过氧化氢酶对固定化葡萄糖氧化酶稳定性的影响

本文通过重氮盐共价键合法把葡萄糖氧化酶接到交联琼脂糖上制备固定化酶。为了提高固定化酶的使用稳定性,把过氧化氢酶和葡萄糖氧化酶同时接到载体上,并研究了这两种酶在不同比例时对固定化葡萄糖氧化酶活力和稳定性的影响,随着加入过氧化氢酶量的增加,固定化葡萄糖氧化酶稳定性显著增加。所制得的固定化葡萄精氧化酶-过氧化氢酶在25℃下连续使用36h,活力几乎不变,失活半衰期可达1155h。     

蔗糖酶和葡萄糖氧化酶的共固化

＃ 本文研究了用吸附交联技术共固定化蔗糖酶和葡萄糖氧化酶(GOD)的方法,考查了共固定化酶的动力学性质。试验结果表明:与溶液酶相比较,固定化蔗糖酶和GOD的响应滞迟期分别为3分钟和2分钟,稳态响应时间增加了6分钟和4分钟,Km值增大,pH—活力曲线变宽,最适pH值分别增大0.7和0.64,最适温度则降低7.3℃和16℃。 以活性氧化铝作载体,戊二醛作交联剂制备的共固定化蔗糖酶和GOD,其蛋白质固定化率为62.9％,分解葡萄糖的总速度为441.6IU,当蔗糖浓度为0.2％,以内时其反应速度与蔗糖的浓度呈正相关(r=0.996),使用半衰期1623次,在4℃下保存120天活力残存为83.7％。     

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。     

固定化原生质体生产葡萄糖氧化酶*

用溶壁酶处理对数生长前期的黑曲霉GP 024细胞,分离得到原生质体,再用光交联树脂包埋制备固定化原生质体,用于生产葡萄糖氧化酶。其胞外葡萄糖氧化酶的比产率几乎达到相应的游离细胞生产的胞内和胞外全部葡萄糖氧化酶的比产率。用光交联树脂制备的固定化黑曲霉细胞的产酶特性与游离细胞相似,而用溶壁酶处理过的固定化细胞的产酶特性与固定化原生质体相似。     

青霉素酰化酶在新型复合载体上的固定化研究

通过γ-氯丙基三甲氧基硅烷的媒介,将聚乙烯亚胺(PEI)化学偶联在硅胶微粒表面,制备了新型复合载体PEI/silica gel,然后通过双官能团试剂戊二醛的作用,将青霉素酰化酶固定在复合载体上;考察了戊二醛用量、pH值、固定化温度、固定化时间及给酶量等条件对固定化青霉素酰化酶表观活力、活性回收率等性能的影响;并通过测定复合载体在固定化前的ζ电位,探索了复合载体PEI/silica gel固定化酶的作用机理。研究结果表明,由于PEI分子链中含有大量胺基,共价键联与物理吸附相结合,使青霉素酰化酶被快速稳定地固定化,并具有高的催化活性与活力回收率。复合载体PEI/silica gel(0.5 g)固定青霉素酰化酶的适宜固定化条件为:固定化温度为30℃;固定化时间为14~15 h;戊二醛用量为1.2 mmol/g;pH=7.92;给酶量为0.1 mL/g。     

接枝淀粉载体固定化糖化酶的研究

合成了淀粉接枝丙烯腈及烯酰胺的两亲性高分子化合物,并以此为载体,用物理吸附方法固定化了糖化酶,最适偶联条件研究表明：缓冲液的浓度,ＰＨ值及吸附时间和加酶量都对固定化酶活力,比活有一定的影响,有最适固定化条件下,固定化酶的活力为１５００Ｕ／ｇ干胶,蛋白载量为２５ｍｇ／ｇ干胶,比活为６０Ｕ／ｍｇ蛋白,比天然酶的比活提高６倍,最适反应温度比天然酶提高１０℃。无底物存在下,固定化酶在５５℃的半衰期为２４ｈ     

Preparative production of hydroquinone from benzoquinone catalysed by immobilized d-glucose oxidase

Benzoquinone can replace O₂ as an electron acceptor in the oxidation of d-glucose catalysed by A. niger d-glucose oxidase. As a result, a useful chemical, hydroquinone, is formed in nearly 100% yield. A column packed with d-glucose oxidase immobilized onto alumina was operated for two weeks with no measurable decline in its catalytic efficiency and produced more than one hundred grams of hydroquinone.     

Two-step immobilized enzyme conversion of cephalosporin C to 7-aminocephalosporanic acid

The first large-scale production of 7-aminocephalosporanic acid (7ACA) from cephalosporin C (CPC) using a wholly enzymatic synthesis method is reported here. We produced 7ACA from CPC in as high a molar yield as 85% using the immobilized enzymes D-amino acid oxidase (D-AOD) and glutaryl-7-ACA acylase (GL-acylase). In the first reactor, CPC is converted to keto-adipyl-7-aminocephalosporanic acid (keto-7ACA) using an immobilized D-AOD isolated from a yeast, Trigonopsis variabilis. The keto-7ACA is then spontaneously converted to glutaryl-7-aminocephalosporanic acid (GL-7ACA) via a chemical reaction with hydrogen peroxide. The hydrogen peroxide is also a product of the D-AOD reaction. Near quantitative conversion of the keto-7ACA to GL-7ACA was observed. The second reactor converts GL-7ACA to 7ACA using an immobilized GL-acylase, which was isolated from a reconbinant Escherichia coli. The final 7ACA crystalline product is a high quality product. The reactions are conducted under very mild aqueous conditions: pH 8.0 and 20 degrees to 25 degrees C. The production of desacetyl side products is minimal. This process is currently being implemented on an industrial scale to produce 7ACA. (c) 1995 John Wiley & Sons, Inc.     

Reaction and diffusion in a gel membrane reactor containing immobilized cells

To investigate the effect of diffusional limitations and heterogeneous cell distribution in a gel-immobilized cell system, a gel membrane reactor has been constructed. The reactor consists essentially of a gel layer with immobilized cells, flanked by two well-mixed chambers. Through one chamber substrate is pumped, and this chamber is the equivalent of the outside of a spherical gel bead. The second closed measuring chamber contains a small quantity of liquid that can equilibrate with the inside surface of the membrane, eventually after a long transient. Analysis of the liquid in this chamber can give direct information on substrate and product concentrations at the gel surface, and is and indication of the situation in the center of a gel bead. The gel membrane reactor appears to be an excellent tool to study diffusion and reaction in a gel-containing immobilized cells. A mathematical model with time- and position-dependent cell concentration and diffusion coefficient is described. Experimental data show the effective diffusion coefficient of glucose in an alginate gel to decrease with yeast cell concentration. Moreover, kinetic parameters could be determined, using the mathematical model. Microscopic analysis confirmed the proliferation of the gel-entrapped microorganisms in the outer layer of the matrix, as predicted by the model. Potentially, this type of reactor has a clear potential to study the physiology of gel-immobilized cells. (c) 1992 John Wiley & Sons, Inc.     

A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing

A limited number of support matrices have so far been developed for use in magnetically stabilized fluidized bed (MSFB) applications. We have developed a versatile magnetic silica support which can be derivatized readily for both adsorption chromatography and enzyme immobilization by well-known techniques. A magnetic pellicular bead is prepared by electrostatically depositing alternating layers of colloidal silica and cationic polymer onto macroscopic nickel core particles. The polymer is then burned out and the silica partially sintered to yield a porous shell with 5-80 m(2)/g of surface area. This magnetic composite was tested as a support for immobilizing invertase. Sucrose was continuously converted to its component monosaccharides with nearly constant activity over the first 8 days and retention of 50% of initial activity after 25 days.     

Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^-3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^?3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis

A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, alpha-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound alpha-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized alpha-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass-enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons     

Biosilicification of dual-fusion enzyme immobilized on magnetic nanoparticle

Rapid recovery, immobilization, and silica encapsulation of a dual-fusion enzyme was achieved by using iminodiacetic acid (IDA) modified magnetic nanoparticle as a carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme in which a silica-precipitating peptide R5 and a metal ion complexing peptide (His)(6) were fused to its N- and C-terminal, respectively. After charging the magnetic particle with Cu(2+), the dual-fusion DAAO of 0.43 g could be directly recovered from the recombinant E. coli crude extract and immobilized on 1 g of the magnetic particle. Once in contact with hydrolyzed tetramethoxysilane (TMOS), the homogeneously dispersed immobilized dual-fusion DAAO was biosilicificated to form aggregates with size about 50 microm. The silica-encapsulated immobilized DAAO demonstrated a pyruvic acid production rate comparable with that of the naked immobilized DAAO in five repeated batch reactions when D-alanine was used as substrate. Furthermore, 85% of its activity remained after incubation at 60 degrees C for 1 h while the naked immobilized DAAO lost all its activity. This process provides the advantages that recombinant fusion enzyme can be directly recovered from crude extract, silica encapsulation protects the enzyme from leakage and denaturation, and the enzyme activity can be easily retrieved by applying a magnetic field.     

葡萄糖测定方法的比较研究

比较了传统斐林定糖,葡萄糖氧化酶－过氧化物酶比色法,葡萄糖氧化酶电极自动分析仪法测定葡萄糖。比较测定了的结果显示,三法的平均标准误差（ＳＤ）,变异系数（ＣＶ）均十分接近。通过对此三种方法的回归相关性分析显示：斐林法－酶终点比色法的回归方程为ｙ＝０．９８４３ｘ＋６．３２３９,相关系数Ｒ＾２＝０．９９８９,斐林法－自动分析仪法的回归方程为ｙ＝１．００８８ｘ＋２．０４８３,相关系数Ｒ＾２＝０．９９９１,