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1.
酶分子化学修饰研究进展   总被引:4,自引:0,他引:4  
酶是高效生物催化剂,在工业和临床医药上应用广泛。但由于酶是蛋白质,稳定性差,且在生物体内有较强的免疫原性,因而严重制约了其应用。对酶分子进行化学修饰是提高其稳定性和降低免疫原性的有效途径。简要介绍几种改进酶催化特性的方法、酶分子修饰效果的分析与评价、酶通过化学修饰获得的新性质及其原理、酶化学修饰的研究动态等。  相似文献   

2.
酶的分子设计、改造与工程应用   总被引:4,自引:0,他引:4  
酶工程的研究已经发展到分子水平 ,在体外通过基因工程、化学、物理等手段改造酶分子结构与功能 ,大幅提高了酶分子的进化效率和催化效率 ,生产有价值的非天然酶。对酶工程学若干“热点”和前沿课题的研究、应用进行了概述 ,分析了国际上酶工程研究及应用技术、手段、方法 ,包括体外分子进化、核酶和抗体酶的设计、酶分子的定向固定化技术、酶蛋白分子的化学修饰、融合酶、人工合成及模拟酶等技术 ,并展望了酶工程的技术进步和应用的新进展。  相似文献   

3.
本文评论了酶化学修饰在酶工程中的应用潜力,介绍了这方面的最新进展。事实证明,只要选择的化学修饰剂及修饰方法合适,有可能在较大范围内改变酶的性质,如稳定性和溶解度、酶催化活力和选择性等,从而创造天然酶所不具备的优良特性,扩大酶的应用范围。  相似文献   

4.
色氨酸残基在内切葡聚糖酶分子中的作用   总被引:13,自引:0,他引:13  
内切葡聚糖酶的化学修饰研究表明:色氨酸残基可能位于活性位点,与底物结合有关.荧光光谱测定指出该酶的荧光几乎都来自色氨酸残基,酶分子中色氨酸微环境对pH变化非常敏感,降低pH导致了酶分子构象发生了较大变化,配基结合使酶分子色氨酸微环境产生了改变,引发了与pH诱导不同的构象变化.  相似文献   

5.
豆壳过氧化物酶的盐酸胍变性与化学修饰研究   总被引:2,自引:0,他引:2  
研究了盐酸胍对豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC1.11.1.7)构象与活力的影响,发现去辅基SHP的盐酸胍变(复)性及荧光变化关系与SHP全酶分子的盐酸胍变(复)性及荧光变化关系明显不同。应用过碘酸氧化法去除SHP分子表面糖链,研究糖链去除对酶性质的影响,则证实了SHP分子表面的糖链去除导致酶热稳定性下降。应用不同的蛋白质侧链修饰剂对SHP进行化学修饰则表明,巯基、酪氨酸和色氨酸残基为酶活力非必需,而羧基、组氨酸和精氨酸残基为酶活力所必需。  相似文献   

6.
铜锌超氧化物歧化酶(Cu, Zn-SOD)表面的赖氨酸经化学修饰后, 酶的稳定性显著提高. 赖氨酸被修饰后, 酶的电荷结构遂发生变化, 从而影响到酶分子电场. 使用FDPB方法(有限差分法求解Poission-Boltzman方程)计算了酶修饰前后的静电场变化, 以及对维持酶的结构稳定起重要作用的Cu, Zn配位结构的影响.结果表明, Cu, Zn配位体的两级离解常数在酶修饰后分别约下降103, 106.  相似文献   

7.
嗜水气单胞菌胞外蛋白酶的化学修饰   总被引:8,自引:1,他引:8  
 蛋白酶是嗜水气单胞菌 (Aeromonashydrophila)的重要致病因子 .为研究其结构与功能之间的关系 ,用DEPC、EDC、PMSF、N AI等 9种化学修饰剂处理嗜水气单胞菌J 1株胞外蛋白酶ECPase54,然后检测残余酶活力 ,借以研究酶分子中氨基酸侧链基团与酶活性中心的关系 .结果表明 ,羧基、丝氨酸、ε 氨基、胍基等残基与酶活性无关 ;半胱氨酸残基与酶活性也无直接关系 ;而色氨酸、组氨酸、酪氨酸残基侧链以及二硫键的化学修饰引起酶活性的大幅度的下降 ,说明色氨酸、组氨酸、酪氨酸残基以及二硫键是酶活力所必需的基团  相似文献   

8.
用九种化学修饰剂研究了大肠杆菌AS1.357 L-天门冬酰胺酶分子中的五种不同氨基酸侧链基团与催化活性的关系。结果说明,渡酶活力与硫氧墓完全无关;与色氨酸、精氨酸和组氨酸亦无直接联系;而酪氨酸残基和羧基的修饰引起酶活力急剧下降。其中酪氢酸残基巳被证实是该酶活力的必需基团,处于该酶分子的活性部位。  相似文献   

9.
用化学修饰剂NEM、二甲基溴化锍、EDC、DEPC、TNM、对硝基苯乙二醛、PMSF、TNBS对芽孢杆菌B23产生的甘露聚糖酶M an23进行化学修饰,并测定修饰反应的动力学参数关系。结果显示半胱氨酸、色氨酸(1个)和谷氨酸(或天冬氨酸)残基(2个)是酶活性的必需基团;组氨酸、酪氨酸、精氨酸、丝氨酸和赖氨酸残基均为非必需基团。双向电泳结果显示酶蛋白分子具有一个链内二硫键(Cys90-Cys110)。荧光光谱测定结果显示该酶最大吸收峰为336 nm。底物作用导致酶的发射光谱发生蓝移,说明色氨酸残基位于酶蛋白分子内部的疏水区。  相似文献   

10.
蛋白质药物的聚乙二醇修饰   总被引:2,自引:0,他引:2  
聚乙二醇被广泛应用于蛋白质药物的化学修饰,修饰后的蛋白质药物的性质发生多方面变化,如:增加了此类药物的溶解度和稳定性,耐酶水解的能力增强,减弱或消除免疫原性,增加药物在体内的半衰期等。近年来,聚乙二醇修饰剂的种类和修饰方法发展较快,蛋白质分子上的氨基、巯基、羧基均成为化学修饰的研究对象。本文综述了聚乙二醇修饰的原理与方法,修饰方法的比较与优化,修饰后产品的鉴定与检测。  相似文献   

11.
Three enzymes used in cancer chemotherapy (asparaginases from Escherichia coli and Erwinia carotovora and glutaminase from Achromobacter) were each reacted with four amino specific reagents (ethyl acetimidate, O-methylisourea, succinic anhydride, and formaldehyde/sodium borohydride). The half-lives of the modified enzymes measured in the blood of rats showed that guanidation, acetimidation and reductive alkylation were more likely to increase the persistence of the native enzymes than succinylation. However, the improvement in the persistence of any one enzyme after any one modification could not be predicted from the results with the others. It was concluded that changes in persistence caused by each modification were due to the different effects on the tertiary structure of each native enzyme. The advantages of chemical modification for increasing the persistence of enzymes over other methods such as encapsulation or aggregation are discussed.  相似文献   

12.
Chemical modification of enzymes for enhanced functionality.   总被引:6,自引:0,他引:6  
The explosion in commercial and synthetic applications of enzymes has stimulated much of the interest in enhancing enzyme functionality and stability. Covalent chemical modification, the original method available for altering protein properties, has now re-emerged as a powerful complementary approach to site-directed mutagenesis and directed evolution for tailoring proteins and enzymes. Glutaraldehyde crosslinking of enzyme crystals and polyethylene glycol (PEG) modification of enzyme surface amino groups are practical methods to enhance biocatalyst stability. Whereas crosslinking of enzyme crystals generates easily recoverable insoluble biocatalysts, PEGylation increases solubility in organic solvents. Chemical modification has been exploited for the incorporation of cofactors onto protein templates and for atom replacement in order to generate new functionality, such as the conversion of a hydrolase into a peroxidase. Despite the breadth of applicability of chemically modified enzymes, a difficulty that has previously impeded their implementation is the lack of chemo- or regio-specificity of chemical modifications, which can yield heterogeneous and irreproducible product mixtures. This challenge has recently been addressed by the introduction of a unique position for modification by a site-directed mutation that can subsequently be chemically modified to introduce an unnatural amino acid sidechain in a highly chemo- and regio-specific manner.  相似文献   

13.
In the study of chemical modification of enzymes and other biologically active proteins, plots of fractional residual activity as a function of number of groups modified per enzyme molecule are often used to establish a correlation between the chemical modification and enzyme inactivation reactions and to determine the stoichiometry of the modification reaction. This paper presents a critical examination of the underlying theoretical framework of such graphs. Whereas these plots are usually presented as linear functions, it is shown here that the general equation describing the relationship between inactivation and modification contains an exponential term; therefore, in the general case, the plot is actually a curve. It is suggested that caution be exercised in the interpretation of such plots and that equations such as those derived in the text be used to fit theoretical curves to the data, in order to maximize the information gained from chemical modification experiments.  相似文献   

14.
The influence of chemical modification on the initial specific activity, residual activity, and deactivation kinetics of various enzymes is analyzed using a series mechanism. This straightforward multistate sequential model presented is consistent with the enzyme deactivation data obtained from different fields. The enzymes are placed in five different categories depending on the effect of chemical modification on initial specific activity and residual activity or stability. Wherever possible, structure-function relationships are described for the enzymes in the different categories. The categorization provides one avenue that leads to further physical insights into enzyme deactivation processes and into the enzyme structure itself.  相似文献   

15.
In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions. A combination of molecular genetics and chemical modification has been used for protein engineering.  相似文献   

16.
半乳甘露聚糖胶酶法改性研究进展   总被引:2,自引:0,他引:2  
由于半乳甘露聚糖的水溶液在低浓度下仍具高黏性以及它的凝胶性质,因此在工业上具有很多重要的应用。半乳甘露聚糖聚糖的酶法改性主要包括脱去支链和切断主链两种方式。相对于化学改性来说,酶法改性具有易控制、反应条件温和等很多优点,因此成为改变半乳甘露聚糖分子结构以获得所需特性的最具潜力的改性方法。α-半乳糖苷酶和 β-甘露聚糖酶是半乳甘露聚糖改性和水解中最常用的酶。简要介绍了有关这两种酶的来源和新型制备菌株的近期研究概况。在医药和食品等工业中,酶法改性后的半乳甘露聚糖具有很广阔的应用前景。  相似文献   

17.
New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance.

The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu+2) introduced p-aminobenzamidine (pABA- Cu+2: guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu+2 preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (Kcat, Km, Kcat/Km), optimum pH and temperature of the trypsin-pABA-Cu+2 complex was also investigated.  相似文献   

18.
The role of tryptophan, methionine, and histidine residues in mitochondrial aspartate aminotransferase from beef kidney has been established by using N-bromosuccinimide, 2-hydroxy-5-nitrobenzylbromide, and tetraiodofluoresceine as specific chemical modifiers of the amino acid residues of the enzyme. Since N-bromosuccinimide promotes extensive inactivation of the enzyme and the chemical modification of 1.65 tryptophan and 3 methionine residues per enzymes protomer, 2-hydroxy-5-nitrobenzylbromide modifies once more 1.65 tryptophan residues per enzyme protomer but induces only 10% inactivation of the enzyme. Tetraiodofluoresceine exerts a 40% inactivation of the enzyme which is due to the chemical modification of 5.8 histidine res in  相似文献   

19.
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