酶膜反应器在生物工程中的应用

利用膜作为固定化酶载体的酶膜反应器,能有效地将酶固定,可实现产物的原位分离,消除位阻效应和抑制效应,且可连续操作,便于放大,已广泛应用于蛋白质、纤维素、淀粉等的酶解、污水处理等。本文介绍酶膜反应器的原理、三种常用的酶膜反应器类型(连续搅拌釜、中空纤维、含辅酶的)及其在生物工程中的应用。     

固定化青霉素酰化酶反应器研究进展

介绍固定化青霉素酰化酶反应器的研究现状，将这类反应器分为搅拌釜反应器、柱式填充床反应器、中空纤维膜反应器以及电渗析耦合反应器，从工程角度分析了各类反应器的优缺点，并展望其今后的发展趋势。     

青霉素酰化酶研究进展

青霉素酰化酶除了在医药工业上用于大规模生产β－内酰胺类抗生素中间体和半合成β－内酰胺类抗生素之外,还有其它的一些潜在应用;对青霉素酰化酶的研究一直吸引着各国科研人员的兴趣,近年来,用一些经典的和现代的技术手段,对青霉素酰化酶产生菌进行改良,通过理化诱变,使酶活力提高;通过定点突和化学修饰,将酶活性中心的丝氨酸变成半胱氨酸或含硒原子的半胱氨酸,使酶的合成活力相对于水解活力大幅提高,通过基因工程技术提高酶的表达水平,促进分泌或改进生产菌的缺陷,出现了介孔分子筛,二甘醇二甲基酯丙烯酸酯接枝尼龙共聚物等新的;固定化材料及酶膜反应器,多室固定化酶反应器,有望用于工业化生产。ββ     

葡萄糖氧化酶电极中酶固定化的研究

研究了以丝纤维为载体，三聚氯氰为交联剂固定化葡萄糖氧化酶膜的方法和条件。实验表明，用二氧六环－二甲苯（质量比１１）为三聚氯氰的溶剂，固定化酶膜效果较好。酶的偶联条件以温度２５℃、ｐＨ＝９、酶液浓度４ｍｇ／ｍｌ为最佳，此时固定化酶膜稳定性好，反复使用３０次酶膜活性保留９０％以上，贮存在离子强度较高的溶液中，酶膜的半衰期为４０～５０周。     

治疗用固定化酶和固定化细胞——人工细胞和人工器官以及酶的截留问题

<正> 一、治疗用固定化酶的体外支路系统 1.固定化脲酶酶板——人工肾体外应用固定化酶的主要方面为固定化酶组成的固定床反应器,例如可处理全血的板状反应器(人工肾)即为一例。过去处理尿毒症所用的人工肾脏主要是用半透膜透析的原理把血     

一种酶反应器筛选胰蛋白酶抑制剂方法的建立

目的建立从黄酮化合物库中筛选胰蛋白酶抑制剂的新方法。方法利用毛细管固定化酶反应器技术,通过环氧基的开环反应,将胰蛋白酶与poly(GMA-co-PEGDA)有机聚合物整体材料共价键合制备毛细管固定化胰蛋白酶反应器;对酶反应浓度与反应时间等参数进行了系统研究,利用荧光显微镜、扫描电子显微镜和拉曼光谱对酶反应器进行了表征;采用微径液相色谱法对酶反应器的活性和动力学参数进行了系统评价;将制备的酶反应器用于20种中药黄酮化合物中胰蛋白酶抑制剂的筛选;通过分子对接手段进一步考察胰酶与抑制剂之间的作用机制。结果 16种黄酮化合物具有不同程度的胰蛋白酶抑制作用,其中,漆黄素的抑制效果最佳,抑制率为(62.9±1.2)%,分子对接结果进一步说明了漆黄素与胰蛋白酶之间的作用机制。结论建立了一种简单、有效的胰蛋白酶抑制剂的筛选方法,可进一步推广应用。     

青霉素酰化酶的固定化与应用新进展

青霉素酰化酶被广泛应用于半合成抗生素及中间体的制备、手性药物的拆分和多肽合成等方面。高效固定青霉素酰化酶能提高酶对温度、pH值、溶剂极性等方面的适用性和反复使用的稳定性，将成为拓宽青霉素酰化酶在工业中应用的必然选择和关键。本文主要介绍了青霉素酰化酶固定化技术的进展，讨论了不同固定化技术的特点和固定化酶在非水相体系中的催化作用，并展望了固定化青霉素酰化酶的发展前景。     

一种新的固定化酶方法——微粒明胶吸附交联青霉酰化酶

微粒明胶有较强的吸附作用,且分子中含大量氨基,因此,可用戊二醛一类交联剂将酶和明胶载体进行交联,经交联后,不仅酶和明胶牢固地结合,且明胶本身失去了水溶性,从而得到理想的固定化.采用0.18～0.32mm细度的明胶,按明胶:酶液1:0.5(w/v)吸附后,以适量的25%戊二醛交联30分钟后即可得到分散性良好的微粒明胶固定化酶.活力回收率为20～30%.采用此种固定化酶,裂解10%青霉素,6-APA收率可达84%以上.固定化酶在缓冲液中低温存放一年后,活力仍保留90%以上,在柱和搅拌罐两种反应器中,酶的半寿期分别为730小时和248小时.     

壳聚糖及其衍生物在固定化酶中的应用进展

综述了壳聚糖及其衍生物在固定化酶中的应用进展，重点介绍了以各种形态的壳聚糖及其衍生物作栽体通过多种固定化方法制备固定化酶的研究及应用；指出了壳聚糖及其衍生物作为固定化酶的载体具有固定化方法多样、简单易行、生物相容性好、来源丰富等优点。     

单宁酶的固定化及性质研究

比较几种固定化载体，确定以壳聚糖为载体，用戊二醛作交联剂制得固定化单宁酶。壳聚糖用量0．1g，用3％戊二醛5ml交联4h，然后加入酶58．4u，于4℃反应4h，固定化酶活回收率可达73％。单宁酶经固定化后，热稳定性、pH稳定性及最适温度均有所提高，最适pH降低。     

Liquid chromatographic separation of phenolic drugs using catalytic detection: Comparison of an enzyme reactor and enzyme electrode

A catalytic detection system based on the use of immobilized tyrosinase and two different transducers (photometric and electrochemical) is described. Comparison between tyrosinase immobilized in a packed-bed reactor and at the surface of a graphite electrode is discussed in terms of sensitivity in a flow injection system. The enzyme electrode configuration gives the highest sensitivity for the quantitation of dopamine. For the immobilized tyrosinase reactor with photometric detection the range for dopamine is linear up to 0.75 mM (136 micrograms ml-1) and the immobilized tyrosinase reactor with electrochemical detection and the tyrosinase electrode extends this dynamic range to 1 mM (181 micrograms ml-1). Liquid chromatographic separation and post-column detection using the tyrosinase electrode is shown for spiked samples of serum.     

Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase

The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.     

Lipases for biocatalysis: development of a chromatographic bioreactor

The development of a new chromatographic reactor based on immobilized Candida rugosa lipase (CRL) is described. The chromatographic system has been used to evaluate the rate differences by which the product enantiomers of esterolytic reactions catalyzed by immobilized CRL are obtained. The method has been applied to a series of racemic 2-aryloxyalkanoic acids and isosteric analogous methyl esters and to some non-steroidal antiinflammatory drugs 2-arylpropanoic acids methyl esters in order to study the structure effects on reaction rate and enantioselectivity. Lipase from C. rugosa has been non-covalently and covalently immobilized on HPLC chromatographic silica supports to develop an immobilized enzyme reactor (IMER). The reactor was connected through a switching valve to an analytical reversed-phase column, which was used for the on-line determination of the hydrolysis rate by peak area integration. The enantiomeric excess of the hydrolytic reaction products was determined off-line on a CSP utilizing immobilized penicillin G acylase (PGA-CSP).     

固定化谷氨酸脱羧酶转化γ-氨基丁酸的研究

目的用固定化谷氨酸脱羧酶转化制备γ-氨基丁酸(GABA)。方法以海藻酸钠与明胶为协同作用载体包埋固定化酶,考察影响固定化酶活性的因素,获得固定化酶的优化条件,并将酶应用于制备GABA。结果海藻酸钠、明胶、氯化钙和戊二醛浓度分别为2.5%,1.0%,3.0%和0.3%,硬化时间4 h,固定化酶的相对活性最高,酶活回收率达到65.3%。以谷氨酸为底物,固定化谷氨酸脱羧酶为催化剂,采用填充床反应器连续制备GABA,连续反应60 h,酶相对活性仍保持在初始值的76.9%,谷氨酸转化率为98.7%。结论该固定化谷氨酸脱羧酶可应用于生物转化法连续制备GABA。     

Liquid chromatographic separation and stereoselective detection of l- and d-amino acids with catalytic reaction detection using immobilized enzymes

A post-column LC detection system is described for the stereoselective detection of l- and d-amino acids. The effluent of the LC column passes an immobilized enzyme reactor (IMER) containing either l- or d-amino acid oxidase. The hydrogen peroxide formed in this reactor by the oxidation of the amino acids is then transported to a second IMER containing horse-radish peroxidase. With the addition of 4-aminophenazone and dichlorophenolsulphonyl chloride to the carrier, the hydrogen peroxide is reacted to form a red-coloured complex which is detected in a flow-through photometric cell at 514 nm. Applications to the analysis of amino acids in bovine and human sera are described.     

用固定化细胞生产D-氨基酸的研究

目的优化CPU 0 4 30 1菌株的发酵条件 ,提高酶活力 ,并建立D 氨基酰化酶酶活力的测定方法。方法对CPU 0 4 30 1菌株培养的碳源、氮源进行优化 ,同时观察温度、pH值、离子含量等对D 氨基酰化酶酶活力测定的影响。结果CPU 0 4 30 1经固定化后于 37℃反应 2 0h得产品 4 .78g ,纯度达 99.5 7%。结论CPU 0 4 30 1菌株经优化后 ,D 氨基酰化酶的产量和活力均明显提高 ,同时建立了准确、灵敏的酶活力测定方法。     

A new arginase enzymatic reactor: development and application for the research of plant-derived inhibitors

This work was dedicated to the development of a new micro immobilized enzyme reactor (IMER) by using an in situ procedure. Arginase was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12mm × 3mm i.d.) previously derivatized with glutaraldehyde. The activity of this IMER was investigated by inserting this micro-IMER in a HPLC system. The effect of the arginase inhibitors was evaluated by the simultaneous injection of each inhibitor with the nitro guanidino benzene (NGB) substrate. The relative IC50 values were found in agreement with those derived by the conventional spectrometric method. This arginase micro-IMER system was also used to study the effects of plant-derived products on the arginase activity. The pet ether extract from the stem bark of the plant Ficus glomerata Roxob. and the procyanidin oligomers of cocoa and chocolate inhibit the arginase activity. Our results confirmed the direct effect of some plant extracts on the arginase activity and their interest in therapies for treating several NO-dependent smooth disorders.