鸭肝脂肪酸合成酶的NADPH底物抑制及作用动力学

己知动物脂肪酸合成酶的底物乙酰辅酶Ａ和丙二酰辅酶Ａ具有竞争性双底物抑制的乒乓机制。实验发现鸭肝脂肪酸合成酶的第三个底物ＮＡＤＰＨ也具有底物抑制,并研究了它的规律及与ＮＡＤＰＨ有关的稳态动力学。发现对于该酶的全反应,增加丙二酰辅酶Ａ浓度,降低环境盐浓度,均使ＮＡＤＰＨ底物抑制减少。但以ＮＡＤＰＨ作底物的酮酰还原和烯酰还原二步单独反应以及包含四步单独反应的乙酰乙酰辅酶Ａ还原反应都无ＮＡＤＰＨ底物抑制现象。ＮＡＤＰＨ底物抑制对丙二酰辅酶Ａ为竞争性,丙二酰辅酶Ａ底物抑制对ＮＡＤＰＨ为非竞争性。在全反应中ＮＡＤＰＨ和丙二酰辅酶Ａ之间发现为乒乓机制,在乙酰乙酰辅酶Ａ还原反应中,两个底物ＮＡＤＰＨ和乙酰乙酰辅酶Ａ之间则表现为序列反应机制。降低环境盐浓度使ＮＡＤＰＨ和丙二酰辅酶Ａ之间的乒乓机制向序列机制转化。在全反应中,ＮＡＤＰ产物抑制相对ＮＡＤＰ为竞争性,对丙二酰辅酶Ａ为非竞争性。     

胆碱脱氢酶的动力学性质

本文对增溶胆碱脱氢酶的稳态初速度及产物抑制动力学做了。底物胆碱和ＰＭＳ的相互影响：变化一个底物的浓度,另一个底物的Ｋｍ及Ｖｍａｘ均变化。该酶的产物三甲胺 地 制表现为对底物胆碱非竞争性而地ＰＭＳ竞争性,在胆碱饱和的情况下,三甲胺乙醛对酶的抑制仍表现为对ＰＭＳ竞争性。这些结果表明增溶胆碱脱氢酶的催化机制搂双底物双产物乒乓机制。１－ＰＣ与９－ＡＣ对增溶胆碱脱氢酶均有抑制作用,且均为混和型抑制,Ｋ１分别     

简单节杆菌转化氢化可的松的△’一脱氢反应动力学II．固定化简单节杆菌细胞转化氢化可的松的△’-脱氢反应动力学

本文报道了简单节杆菌固定化细胞转化氢化可的松过程中固定化细胞颗粒度与有效系数的关系和底物初始浓度与有效系数及扩散系数的关系。随着固定化细胞颗粒度的增大,有效系数减小,固定化细胞颗粒的边长与西勒模数成正比,有效系数和扩散系数都随底物初始浓度的增加而减少。氧化可的松在固定化细胞中扩散系数的数量级为IO…m。／％固定化细胞转化氢化可的松的反应过程曲线与自由细胞的相似。用前文[1]中的产物抑制反应动力学方程(9)进行拟合时,其计算值与实验值相符。     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

简单节杆菌转化氢化可的松的△-脱氢反应动力学I．简单节杆菌BY-2-13自由细胞转化氢化可的松的△-脱氢反应动力学

本文研究了简单节杆菌(Arthrobacter simplex) By-2-13转化氢化可的松为氢化泼尼松韵△一脱氢反应动力学。其中包括溶解底物的底物浓度对反应速度的影响,反应初速度与底物浓度的关系;固体悬浮液中底物总浓度对反应速度的影响,反应初速度与底物浓度的关系;酶量对反应的影响以及产物的抑制作用等。溶解底物和固体悬浮液底物的反应初速度与底物浓度的关系都符合简单米氏方程,米氏常数Km分别为0．33mg／mJ和29．41n,g／ml,而两者的反应过程曲线均与简单米氏方程不符。无论在较低或较高浓度的固体悬浮液底物转化过程中,产物对A’。脱氢反应都呈现抑制作用。由此建立了该反应的反应动力学模型及相应的动力学方程,井经线性化后回归得出反应的动力学参数。当反应时间小于‘(达到约85％的转化率所需要的时间)时,用此动力学方程拟合所得的数据与实验测定值相符。     

鸭肝脂酸合成酶的NADPH底物抑制及作用动力学

已知动物脂肪酸合成酶的底物乙酰辅酶Ａ和丙二酰辅酶Ａ具有竞争性双底物抑制的乒乓机制。实验发现鸭肝脂肪酸合成酶的第三个底物ＮＡＤＰＨ也具有底物抑制,并研究了它的规律及与ＮＡＤＰＨ有关的稳态动力学。发现对于该酶的全反应,增加丙二酰辅酶Ａ浓度,降低环境盐浓度,均使ＮＡＤＰＨ底物抑制减少。但以ＮＡＤＰＨ作底物的酮酰还原和烯酰还原二步单独反应以及包含四步单独反应的乙酰乙酰辅酶Ａ还原反应都无ＮＡＤＰＨ底物抑制现     

透性化嗜酸乳杆菌细胞转化亚油酸为共轭亚油酸的反应动力学

本实验旨在研究透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的反应动力学。探讨了细胞浓度、底物浓度、反应体系pH值和温度等因素对生物转化共轭亚油酸反应速度的影响;建立了透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的动力学模型。结果表明,透性化嗜酸乳杆菌细胞有利于共轭亚油酸的生物转化,最适细胞浓度、pH值和反应温度分别为10×1010ufc/mL、4.5和45℃;生物转化共轭亚油酸存在底物抑制现象,当亚油酸的浓度为0.6mg/mL时,反应速度达到最大值17.8μg/(mL·min)。在低亚油酸浓度下,反应初始阶段的反应规律与经典米氏方程相符,而在高亚油酸浓度下,存在底物抑制现象。在最适反应条件下建立了动力学模型,模型基本反映了共轭亚油酸的生物转化特性。     

啤酒废水二相厌氧消化动力学研究

本工作分阶段研究了啤酒废水厌氧消化反应特性。酸化初始速度很快,ｐＨ下降至４．０以下时,酸化产物对酸化菌代谢活性具有显著的抑制效应。系统ｐＨ值的大小对甲烷化过程中底物降解速率、产气速率和产气质量均有显著影响。ｐＨ６．５以上时,高浓度底物不构成底物抑制。底物浓度低于５００ｍｇ／Ｌ,甲烷化速率明显下降。合理控制预酸化程度以及甲烷化反应器的进料速率是提高厌氧消化处理效率,维持系统稳定性的关键措施。     

银离子对辣根过氧化物酶的影响

实验研究Ag 对HRP的影响对检测银的污染有重要意义。以ABTS[2,2-连氮-双-(3-乙基苯并噻唑-6-磺酸)]和H2O2为底物,在pH值5.0的条件下,用分光光度法考察了Ag 存在下的辣根过氧化物酶催化氧化反应。Ag 对辣根过氧化物酶的催化活性显示出抑制作用,并进一步分别探讨了对两种底物的抑制类型和对酶结构的影响。结果表明Ag 对底物H2O2而言,对酶的抑制效应属于反竞争性抑制类型,抑制常数Ki=14.83mmol/L;对底物ABTS而言,对酶的抑制效应属于非竞争性抑制,抑制常数Ki=16.139mmol/L。不同浓度Ag 分别与酶作用后,测定酶的内源荧光光谱。光谱结果表明Ag 影响酶活性的同时也影响酶的构象。     

来源于西梅的(R)-醇腈酶促立体选择性转氰合成(R)-酮醇腈

在水/有机溶剂双相反应体系中,研究了来源于西梅的(R)-醇腈酶催化酮与丙酮醇腈合成(R)-酮醇腈的立体选择性转氰反应．系统探讨了不同酶源、酶粉颗粒大小、底物浓度、两底物配比、酶浓度和底物结构对转氰反应的影响．结果发现西梅醇腈酶能高效催化三甲基硅酮与丙酮醇腈的立体选择性转氰．酶粉颗粒大小以直径0.3～0.45 mm为优,底物浓度以21 mmol/L左右为佳,底物丙酮醇腈与三甲基硅酮摩尔浓度比以2∶1为宜,酶浓度以60.9 g/L左右为好．西梅醇腈酶对3, 3-二甲基-2-丁酮几乎没有催化活性,而对其硅结构类似物三甲基硅酮却具有非常高的立体选择性和催化活性,在上述优化反应条件下反应24 h的底物转化率和产物光学纯度均高达99%以上,表明底物中的硅原子对西梅醇腈酶的催化活性有非常显著的促进作用．     

Effectiveness factors for substrate and product inhibition

The transformation technique of Na and Na (Math. Biosci., 6 , 25, 1970) is extended to convert boundary-value problems involving the steady-state diffusion equation for spherical immobilized enzyme particles exhibiting substrate and product inhibition to initial-value problems. This allows a study of the influence of external mass transfer resistances on the effectiveness factors. It also considerably reduces the number of calculations required to investigate the effect of changes in the kinetic parameters on the overall rate of reaction. The existence of multiple steady states for substrate inhibition kinetics in spherical catalyst particles is illustrated and a criterion for uniqueness of steady states is developed. Effectiveness factors for competitive and noncompetitive product inhibition increase with increasing value of the Sherwood number for the substrate and increasing value of the ratio of substrate to product effective diffusivities within the particle.     

Theoretical analysis of intrinsic reaction kinetics and the behavior of immobilized enzymes system for steady-state conditions

Mathematical modeling of immobilized enzymes under different kinetics mechanism viz. simple Michaelis–Menten, uncompetitive substrate inhibition, total competitive product inhibition, total non-competitive product inhibition and reversible Michaelis–Menten reaction are discussed. These five kinetic models are based on reaction diffusion equations containing non-linear terms related to Michaelis–Menten kinetics of the enzymatic reaction. Modified Adomian decomposition method is employed to derive the general analytical expressions of substrate and product concentration for all these five mechanisms for all possible values of the parameters Φ_S (Thiele modulus for substrate), Φ_P (Thiele modulus for product) and α (dimensionless inhibition degree). Also we have presented the general analytical expressions for the mean integrated effectiveness factor for all values of parameters. Analytical results are compared with the numerical results and also with the limiting case results, which are found to be good in agreement.     

Design of a nonuniformly distributed biocatalyst

The properties of a nonuniformly distributed biocatalyst, where the active enzymes are immobilized on the exterior or the interior portions o a solid support, are compared with those of a conventional biocatalyst which is uniformly distributed in a spherical geometry. To investigate the performance of nonuniformly distributed biocatalysts their effectiveness factors are computed and compared for six different enzyme distribution configurations: one-half core, one-half shell, one-third center space, one-third middle annulus, one-third outer shell, and the uniformly distributed. According to the results of numerical analysis, the biocatalyst performance of the exterior "shell" configuration is always far more effective for the immobilized enzymes with positive order reaction kinetics such as Michaelis-Menten and competitive product inhibition. However, in the case of negative order enzymatic reaction kinetics such as substrate inhibition, the interior "core" configuration of the biocatalyst can render far greater enzyme utilization efficiency.     

The choline transport system of erythrocytes. Distribution of the free carrier in the membrane

A method is described, based on the kinetics of transport, for determining the equilibrium distribution of the carrier site on the inner and outer surfaces of the cell membrane, and this method is applied to the choline carrier of human erythrocytes. This method depends on measurement of flux ratios for both entry and exit, i.e., the transport rates of a low concentration of labeled substrate into a solution which contains either no substrate or a saturating concentration of unlabeled substrate. The concentrations of inward-facing and outward-facing carrier are found to be nearly equal, and therefore the 5-fold difference in choline affinity on the inner and outer surfaces of the membrane cannot be explained by an unequal carrier distribution. It is also shown that both reorientation and dissociation of the carrier-substrate complex are far more rapid than reorientation of the free carrier.     

Kinetic studies of alpha-galactosidase-containing mold pellets on PNPG hydrolysis.

Little is known about techniques for applying untreated microbial cells containing enzymes directly to industrial processes as a biocatalyst. The kinetic behavior of alpha-galactosidase-containing spherical pellets which are formed naturally under given conditions in a submerged culture of Mortierella vinacea was studied on the hydrolysis of PNPG (p-nitrophenyl-alpha-D-galactopyranoside). The effect on intraparticle diffusion on the overall reaction rate was assessed by the use of an effectiveness factor, which was calculated by the approximate solution to the equation derived from the mass balance within a pellet. The experimental effectiveness factors were found to be represented as a single function of the modified Thiele modulus, including such parameters as pellet size, enzyme concentration in the pellet, and substrate concentration. As the diffusional effect became more significant, the marked substrate inhibition as seen for a free enzyme disappeared gradually. The effect of product inhibition on the pellets was much weaker than that for a free enzyme at a given substrate concentration. In the region of diffusion controlled reaction, it was found that the rate is proportional to the square root of the enzyme concentration in the pellet. In addition, similarly to what was reported previously for a free enzyme,the reaction in a batch system was found to be approximately representable as simple first-order kinetics in which the rate constant was dependent on the initial substrate concentration.     

Evaluation of the effectiveness factor along immobilized enzyme fixed-bed reactors: Design of a reactor with naringinase covalently immobilized into glycophase-coated porous glass

A design equation is presented for packed-bed reactors containing immobilized enzymes in spherical porous particles with internal diffusion effects and obeying reversible one-intermediate Michaelis-Menten kinetics. The equation is also able to explain irreversible and competitive product inhibition kinetics. It allows the axial substrate profiles to be calculated and the dependence of the effectiveness factor along the reactor length to be continuously evaluated. The design equation was applied to explain the behavior of naringinase immobilized in Glycophase-coated porous glass operating in a packed-bed reactor and hydrolyzing both p-nitrophenyl-alpha-L-rhamnoside and naringin. The theoretically predicted results were found to fit well with experimentally measured values.     

A Fortran Program for Evaluation of the Effectiveness Factor of Biocatalysts in the Presence of External and Internal Diffusional Limitations

A Fortran program called SPEFF for evaluation of the effectiveness factor of immobilized enzyme preparations of spherical form in the presence of external and internal mass transfer resistances is described, and a listing of the program is given. Enzyme distribution in the bioparticle may be uniform or nonuniform. In the latter case the enzyme distribution is approximated by fifth-order polynomial. In the program differential equations are replaced by the system of non-linear algebraic equations, and the latter are solved by Newton iteration technique. The program is developed for Michaelis-Menten kinetics with allowance for competitive product inhibition and substrate inhibition. After slight modifications the program can be used for computation of the effectiveness factor of a membrane with an immobilized enzyme, or in the case when the enzyme kinetics are more complex. A typical run on a PDP-11/45 computer took 10-20 seconds. A typical computation time in the case of IBM-compatible TURBO PC was 15-30 seconds.     

General theory of determining intraparticle active immobilized enzyme distribution and rate parameters

A general theory is presented in this article for determining the intrinsic rate constants for the main reaction and deactivation reaction, the effective diffusivity of the substrate, and the active enzyme distribution within porous solid supports from deactivation study of a continuous stirred-basket reactor (CSBR). For the parallel deactivation five reaction kinetics are considered: (a) Michaelis-Menten, (b) substrate inhibition, (c) product inhibition (competitive), (d) product inhibition (anticompetitive), and (e) zero-order kinetics. The experimental results of the system of hydrogen-peroxide-immobilized catalase on controlled-pore glass particles are analyzed to demonstrate the application of the theory developed for parallel deactivation of active immobilized enzyme (IME). For series deactivation only first-order kinetics is treated, and a numerical procedure is proposed to deter mine the rate parameters and the internal active enzyme distribution. The experimental data of the system of glucose-immobilized glucose oxidase on silica-alumina and controlled-pore glass particles are used to verify the theory.     

Theory of the kinetics of reactions catalyzed by enzymes attached to the interior surfaces of tubes

A theoretical treatment is given of the kinetics of reactions catalyzed by enzymes attached to the inner surface of a tube, through which the substrate solution passes. A utilization factor, the ratio of the actual reaction rate to that in the absence of diffusional effects, is defined. A numerical procedure is proposed and numerical and approximate solutions for the utilization factor are given for five kinetic conditions: (a) Michaelis-Menten behavior, (b) substrate inhibition, (c) product inhibition (competitive), (d) product, inhibition (non-competitive), and (e) product inhibition (anticompetitive). When the enzyme chemically attached to a tube obeys a Michaelis-Menten relationship, criteria for insignificant and significant diffusional effects are proposed.     

Kinetic study of the oxidase reaction of ceruloplasmin

The effects of ionic strength, buffer composition and pH on the oxidase activity of ceruloplasmin isolated from human donor blood were studied. The steady-state kinetics of ceruloplasmin-catalyzed oxidation of organic substrates (pyrocatechine, adrenaline, rho-phenyldiamine) and Fe(II) was analyzed. The relationship between the initial reaction rate and Fe(II) concentration is described by the Michaelis--Menten kinetics, that for organic substrates--by substrate activation or by a scheme which implicates the existence of two catalytic centers in the enzyme molecule. The inhibition of adrenaline oxidation by its reaction product, adrenochrome, is competitive. The reactions studied were shown to occur via the formation of a ternary complex.