生物转化中的逆胶囊酶催化

作为生物催化剂的酶以其催化力强、范围广、专一性高,适合于温和条件、水溶液和低底物浓度中催化等特点而得到广泛应用。酶固定化技术的发展使酶催化的工业应用范围更为广阔,但酶促反应需要以水为反应介质,若反应物难溶于水或反应本身要求不能有大量水存在(如酯化反应),则传统的酶反应就难于实现。这些因素限制了酶在工业上的应用。     

关于酶促反应速度与酶反应的初速度的教学

酶促反应速度是酶促反应动力学中的一个基本概念。一定条件下 ,酶催化反应的活力大小要由该反应条件下酶促反应速度来反映和确定 :酶促反应速度越小 ,酶活力越小 ;反之 ,酶促反应速度越大 ,酶活力也越大。在研究底物浓度、酶浓度、温度、ｐＨ、激活剂、抑制剂等因素对酶活力的影响时 ,都必须测定酶促反应的速度。为了排除实验中存在的一些干扰 ,一个酶的酶促反应的速度要在酶促反应进行的初期测定 ,即测定所谓的酶促反应的初速度。在教学过程中 ,学生不易弄清楚酶促反应速度与酶促反应的初速度之间的关系。我们发现 :当一般地问起酶反应速度…     

分子信标方法研究HIV-1整合酶3'加工反应动力学

HIV-1整合酶催化病毒cDNA与宿主细胞基因组DNA的整合,是病毒在宿主细胞中增殖的一个关键酶.3'加工是整合酶催化整合过程的第一步反应,3'加工反应动力学的研究对整合酶催化机理研究和以整合酶为靶点的药物研发都具有重要意义.构建了野生型HIV-1整合酶重组质粒,在大肠杆菌BL21中诱导表达,通过对包涵体变性、复性,纯化得到了整合酶蛋白.基于分子信标原理,设计了荧光和淬灭基团标记的DNA底物,通过荧光信号实时监测3'加工反应,对酶反应的动力学性质进行研究.结果表明,纯化的整合酶蛋白具有较高的活性,酶反应表现出显著的Mg2+偏好性.酶动力学研究(Km=131.79 nmol/L,Kcat=0.0042 min-1)表明,该分子信标方法和设计的DNA底物可用于整合酶3'加工反应动力学研究以及酶反应性质的研究.     

超临界流体中酶催化的研究进展

阐述了充当反应介质的超临界流体、固定化酶的载体以及反应温度和压力对超临界流体中酶催化反应的影响 ,并介绍了超临界流体中酶催化反应在工业上的应用现状和发展前景。     

分子信标方法研究HIV-1整合酶3'加工反应动力学

HIV-1整合酶催化病毒cDNA与宿主细胞基因组DNA的整合,是病毒在宿主细胞中增殖的一个关键酶。 3'加工是整合酶催化整合过程的第一步反应,3'加工反应动力学的研究对整合酶催化机理研究和以整合酶为靶点的药物研发都具有重要意义。构建了野生型HIV-1整合酶重组质粒,在大肠杆菌BL21中诱导表达,通过对包涵体变性、复性,纯化得到了整合酶蛋白。 基于分子信标原理,设计了荧光和淬灭基团标记的DNA底物,通过荧光信号实时监测3' 加工反应,对酶反应的动力学性质进行研究。 结果表明,纯化的整合酶蛋白具有较高的活性,酶反应表现出显著的Mg²⁺偏好性。 酶动力学研究 (K_m = 131.79 nmol/L,K_cat = 0.0042 min ^-1) 表明,该分子信标方法和设计的DNA底物可用于整合酶3'加工反应动力学研究以及酶反应性质的研究。     

关于酶促反应速度与酶促反应的初速度的教学

酶促反应速度是酶促反应动力学中的一个基本概念。一定条件下，酶催化反应的活力大小要由该反应条件下酶促反应速度来反映和确定：酶促反应速度越小，酶活力越小；反之，酶促反应速度越大，酶活力也越大。在研究底物浓度、酶浓度、温度、pH、激活剂、抑制剂等因素对酶活力的影响时，都必须测定酶促反应的速度。为了排除实验中存在的一些干扰，一个酶的酶促反应的速度要在酶促反应进行的初期测定，即测定所谓的酶促反应的初速度。在教学过程中，学生不易弄清楚酶促反应速度与酶促反应的初速度之间的关系。我们发现：当一般地问起酶反应速度在何时测定时，绝大多数学生都会回答在酶促反应的初期；而当问起酶促反应动力学中的v-[S]曲线(图1)上的酶促反应初速度在哪里和为什么时，却有相当一些学生指出在曲线的原点处做切线，切线的斜率就是；有的则指出是与1/2vmax相应的位置的速度值；个别的则指出是与vmax相应的位置的速度值。只有极少数学生指出曲线上的每一点相应的速度值都是酶促反应初速度所在。实际上，正确的答案应为曲线上的每一点相应的速度值均为该底物浓度[S]处的酶促反应初速度值。     

高等植物体内1-氨基环丙烷-1-羧酸(ACC)的形成、转化和调节

本文介绍了ACC合成酶的纯化、性质及其酶促反应的机理;ACC转化反应的产物和机理、EFE酶的性质以及光和CO_2、自由基、CaM等对ACC转化的影响,并提出了ACC形成和转化的调控模式。     

元宝枫叶蛋白酶的动力学特征

酶促动力学是研究酶促反应的速度及各种因素如底物浓度、酶浓度、抑制剂、激活剂、温度、pH等对酶促反应速度影响的科学,是酶学研究中重要的内容。在一定条件下,酶促反应都有其特定的动力学参数,如Km值（米氏常数）和Vmax值（最大反应速率）等。Km是反映酶动力学性质的重要特征性常数,对某一酶促反应而言,在一定条件下都有特定的Km值,可以用来鉴别酶。Km值还可以判断酶的专一性和天然底物,     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。     

多酶共固定化反应体系的研究进展

近年来,生物催化为化学、生物学和生物工程学等领域提供了一种绿色研究工具,其中多酶体系在这些领域中的应用越来越受到关注,其克服了以往单个酶不能满足催化需求的局限性,同时多酶共固定化在级联反应过程中,可增加酶周围的反应物浓度,并将不同酶的催化特性结合起来,能排除干扰因素,从而提高酶的整体催化效率。对多酶共固定化反应体系的研究进展进行了综述,包括多酶反应体系的类别、共固定化技术的特点以及相关应用,并对共固定化多酶反应体系进行了展望。     

Evolutionary optimization of the catalytic efficiency of enzymes.

1. The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first-order rate constants in the mechanism at given concentrations of enzyme, substrate and product. 2. As long as on-velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion-controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first-order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions. 3. When magnitudes of on-velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant. 4. Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free-energy relationships for rate and equilibrium constants in the reaction mechanism.     

利用酶催化反应过程曲线确定动力学参数的新方法

本文提出了一个利用过程曲线确定酶催化反应动力学参数的新方法.利用这一方法,仅仅根据两条实验曲线就可以确定单底物酶催化反应的全部动力学参数,并且所有的图形都是(?)     

Integrated steady state rate equations and the determination of individual rate constants.

Integrated steady state rate equations have been used to determine the kinetic constants (Vs, Ks, Vp, and Kp) and rate constants (k1, k2, k3, and k4) of the reversible enzyme mechanism: (see article). The fumarase reaction has been used as a model to illustrate the procedures for determining these constants. In contrast to initial velocity studies, the values of the constants have been obtained by examining the enzyme reaction in only one direction rather than in both forward and reverse directions. To accomplish this, a new procedure is described for fitting data to integrated rate equations which eliminates problems encountered when data are analyzed graphically. The advantages of examining on enzyme reaction in one direction with these new procedures allow this method to be extended to the examination of enzymes with simple mechanisms where initial velocities are difficult to measure because either the substrate or product is not readily available, or because the reaction is not readily reversible.     

Mode of interaction between beta-lactam antibiotics and the exocellular DD-carboxypeptidase--transpeptidase from Streptomyces R39.

The exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39 is inhibited by beta-lactam antibiotics according to the same general scheme of reaction as the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61. However, the values for the kinetic constants involved in the reaction are very different for the two enzymes and provide an explanation for the observation that the R39 enzyme is more sensitive to beta-lactam antibiotics than the R61 enzyme. Further, particular beta-lactams influence the kinetic constants to different extents depending on the source of the enzyme, so that a physical basis for the spectrum of antibiotic activity against particular enzyme systems is provided.     

Human placental glutamate dehydrogenase - purification - kinetic and regulatory properties - physicochemical studies

Glutamate dehydrogenase (EC. 1.4.1.3) has been purified more than 9,000 times from human placental alcoholic subfractions as a homogenous protein of 55,155 daltons (subunit molecular weight). Kinetic constants for the reverse reaction (reductive amination of α-ketoglutarate) have been shown to be similar to those of the bovine liver enzyme, while the kinetic constants for the forward reaction were markedly different as well as some regulatory properties (lack of activation by ADP in the reverse reaction). The amino acid composition differs from the bovine liver enzyme composition. Furthermore, the tryptic peptide patterns of the placental enzyme and the human liver enzyme have been compared. Besides the low specific activity of this enzyme, the results indicate that human placental glutamate dehydrogenase is closely related to other mammalian glutamate dehydrogenases.     

The kinetics of inhibition of erythrocyte cholinesterase by monomethylcarbamates

1. The kinetics of the interaction of erythrocyte cholinesterase with 1-naphthyl N-methylcarbamate, 2-isopropoxyphenyl N-methylcarbamate and phenyl N-methylcarbamate were studied. Rate constants for inhibition and rate constants for spontaneous reactivation were determined. The calculated rate constants for spontaneous reactivation agreed well with those obtained experimentally. 2. The degree of inhibition obtained after preincubation of enzyme and inhibitor was found to be independent of both the substrate concentration and the dilution of the inhibited enzyme. 3. The reaction between the enzyme and the inhibitor was consistent with carbamates being regarded as poor substrates of cholinesterases. There was no evidence for the formation of a reversible complex between the enzyme and the carbamate.     

Performance of the continuous glucose isomerase reactor system for the production of fructose syrup

Glucose isomerase in the form of heat-treated whole-cell enzyme prepared from Streptomyces phaeochromogenus follows the reversible single-substrate reaction kinetics in isomerization of glucose to fructose. Based on the Kinetic constants determined and the mathematical model of the reactor system developed, the preformance of a plug-flow-type continuous-enzyme reactor system was studied experimentally and also simulated with the aid of a computer for the ultimate objective of optimization of the glucose isomerase reactor system. The enzyme decay function for both the enzyme storage and during the use in the continuous reactor, was found to follow the first-order decay kinetics. When the enzyme decay function is taken into consideration, the ideal homogeneous enzyme reactor kinetics provided a satisfactory working model without further complicatin of the mathematical model, and the results of computer simulation were found to be in good agreement with the experimental results. Under a given set of constraints the performance of the continuous glucose isomerase reactor system can be predicted by using the computer simulation method described in this paper. The important parameters studied for the optimization of reactor operation were enzyme loading, mean space time of the reactor, substrate feed concentration, enzyme decay constants, and the fractional conversion, in addition to the kinetic constants. All these parameters have significant effect on the productivity. Some unique properties of the glucose isomerization reaction and its effects on the performance of the continuous glucose isomerase reactor system have been studied and discussed. The reaction kinetics of glucose isomerase and the effects of both the enzyme loading and the changes in reaction rate within a continuous reactor on the productivity are all found to be of particular importance to this enzyme reactor system.     

Relationship between the lifetime of an enzyme-substrate complex and the properties of the molecular environment.

A theoretical discussion of the decomposition rate constants of enzyme substrate complexes is presented, based upon an enzyme model published earlier (Damjanovich &; Somogyi,1973). These rate constants are expressed by the aid of molecular parameters characteristic for the enzyme-substrate complexes and the molecules in the surrounding liquid phase.Both the exponential and pre-exponential factors of the expressions describing the composition rate constants contain parameters depending on the mass distribution of the reaction mixture in a specific way which is characteristic for the enzyme-substrate complex. The findings suggest a new kind of the enzyme regulation generated by the surrounding medium.     

Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.