胰蛋白酶水解全酪蛋白反应过程中的分析

将高效凝胶排阻 (HPSEC)技术与水解度 (DH)概念相结合 ,对酪蛋白 胰蛋白酶水解体系的酶解反应过程进行分析 ,得到定量表征复杂酶解反应进程和不同DH值时多样性酶解产物相对分子质量分布的二维图线 ;依据蛋白质结构信息 ,结合HPSEC实验谱图 ,对胰蛋白酶作用于酪蛋白时的酶解断裂位点进行剖析 ,初步推断反应历程 ,并得到理论酶解肽段的相对分子质量分布图及酶解物中活性多肽酪蛋白磷酸肽 (CPPs)肽谱。     

胰蛋白酶水解全酪蛋白反应过程中的分析

 将高效凝胶排阻 (HPSEC)技术与水解度 (DH)概念相结合 ,对酪蛋白 胰蛋白酶水解体系的酶解反应过程进行分析 ,得到定量表征复杂酶解反应进程和不同DH值时多样性酶解产物相对分子质量分布的二维图线 ;依据蛋白质结构信息 ,结合HPSEC实验谱图 ,对胰蛋白酶作用于酪蛋白时的酶解断裂位点进行剖析 ,初步推断反应历程 ,并得到理论酶解肽段的相对分子质量分布图及酶解物中活性多肽酪蛋白磷酸肽 (CPPs)肽谱。     

大环三胺1,4,7-三氮杂环癸烷锌(Ⅱ)配合物催化水解作用研究

在25℃、离子强度I=0.10(KNO3)条件下,采用pH电位滴定法测定了大环三胺配体1,4,7-三氮杂环癸烷(TACD)与Zn(Ⅱ)离子的配位平衡常数,讨论了配体与金属离子的配位情况.利用分光光度法,在pH=7～9范围内[2×10-4mol.L-1三羟甲基氨基甲烷(tris)为缓冲溶液]研究了配合物在对硝基苯酚乙酸酯(NA)水解中的催化动力学行为,得到了NA酯的水解速率常数kcat.结果表明,催化水解速率对底物(NA)及配合物浓度均呈一级反应,水解反应遵循速率方程v=(kcat.cZn2++kOH-.cOH-+…).在中性和弱碱性条件下,配合物对NA酯的水解具有很好的催化作用,当pH=9.19时,催化速率常数达3.420×10-2mol-1.L.s-1;催化反应受酸碱平衡控制.     

类球红杆菌羰基还原酶不对称催化还原性能与底物结构的关系

 将类球红杆菌 (Rhodobacter sphaeroides) 全细胞及其分离得到的羰基还原酶用于不对称催化还原多种潜手性酮类化合物, 通过比较产物的收率、ee 值、酶活力以及酶学动力学常数 Km, 探讨了类球红杆菌的催化还原性质与底物结构的关系. 结果表明, 对于类球红杆菌全细胞不对称催化还原苯乙酮衍生物, 产物 ee 值的变化遵循 Prelg 规则, 产物收率与底物苯环及侧链上取代基团的性质有关; 对于脂肪酮催化还原, 产物收率随底物链长的增加和分子量的增大而降低, 随支链数目的增加而升高, 产物 ee 值的变化也遵循 Prelg 规则. 利用羰基还原酶不对称催化还原潜手性酮类化合物发现, 对于芳香酮类化合物, 酶对 α 位为强电负性基团的底物专一性较强; 对于脂肪酮类化合物, 酶对五碳脂肪酮的专一性较高. 利用酶直接催化还原反应产物的 ee 值均为 99% 左右, 表明酶较全细胞有更高的立体选择性.     

含苯并咪唑基配体的锌配合物的晶体结构和催化对硝基苯磷酸酯水解的活性研究

合成和表征了一种磷酸脂酶模型化合物[Zn(L1)(L2)].H2O(L1=二(2-苯并咪唑亚甲基)胺,L2=5-二甲基萘胺-1-磺酰甘氨酸),并用对硝基苯磷酸酯(PNPP)作为反应底物测试了它的催化活性。研究结果表明锌配合物为扭曲的三角双锥构型,它催化PNPP的水解符合米氏方程模型。在不同的条件下测试了几种反应动力学参数Vmax、Km和kcat,发现锌配合物在35℃和pH=8.5时有最大的催化活性。     

化学修饰木瓜蛋白酶的酶学性质研究

采用邻苯二甲酸酐(PA)对木瓜蛋白酶进行了化学修饰,通过三硝基苯磺酸法、紫外光谱、荧光光谱及FT-IR光谱对修饰效果进行了初步表征,采用动力学方法考察了pH和温度对修饰酶水解活性和稳定性的影响,并计算了一系列动力学和热力学参数.实验结果表明:PA对木瓜蛋白酶的平均氨基修饰度为43%,未对酶的活性基团-SH发生修饰,修饰酶较原酶的紫外吸收峰和最大荧光发射峰均发生蓝移,紫外吸收强度降低、荧光强度增大;PA修饰未改变木瓜蛋白酶的最适反应温度,但将其最适反应pH由7.0提高到8.5,且酶活力也提高了约20%;PA修饰有效提高了酶的催化水解效率和酶与底物的亲和力,如40℃、最适pH条件下修饰酶的转化数kcat(3.03 s-1)和亲和力kcat/Km(1.70 s.L.g-1)均较原酶的(2.28 s-1、1.15 s.L.g-1)高,修饰酶催化水解反应的活化能Ea(25.4 kJ.mol-1)较原酶的(29.3 kJ.mol-1)低;PA修饰还明显提高了酶的pH稳定性和热稳定性,半衰期t1/2延长,酶分子的热变性活化能Ea,d由77.0 kJ.mol-1提高到94.5 kJ.mol-1.可见PA化学修饰法是一种有效改善木瓜蛋白酶的催化性质和稳定性的方法.     

丁二酸酐修饰对漆酶稳定性和除酚效率的影响(英文)

采用(NH4)2SO4分步沉淀法对诺维信中国公司生产的漆酶制剂DeniLiteIIS进行了纯化,并用丁二酸酐(SA)对纯化酶进行了化学修饰,运用三硝基苯磺酸法、紫外光谱法及荧光光谱法对修饰效果进行了初步表征,比较了天然酶和修饰酶的pH稳定性、热稳定性及除酚效率.结果表明,修饰酶的平均氨基修饰度为85%,其紫外吸收峰和荧光发射峰均出现蓝移,而且紫外吸收减小、荧光强度增加.尽管采用SA化学修饰未能改变漆酶的最适反应温度,但使其最适反应pH值由4.5提高到5.5,并且使酶活提高60%.与天然酶相比,修饰酶的pH稳定性和热稳定性更高,催化效率(kcat)和酶与底物的亲和力(kcat/Km)分别提高了53%和122%,对邻、间和对苯二酚的除酚效率分别提高了48%,57%和18%.这预示着这些修饰漆酶可望应用于工业生产和酚类污染废水的治理.     

含新型亲核体大环多胺锌(II)配合物对水解酶的模拟研究

本文探讨苯酚功能化的大环多胺配体6-(2'-羟基-3', 5'-二溴)-苄基-1, 4, 8, 11-四氮杂环十四烷(L)锌(II)配合物作为水解酶模拟物催化4-硝基苯酚醋酸酯(NA)水解的动力学。研究表明催化水解速率对NA及配合物浓度皆呈一级反应。水解速率遵循速率方程V=(kcat[Zn]+kOH[OH^-]+k0)[NA]。其二级反应速率常数kcat在一定范围内随着pH值的增加而增加, kcat的最大值(k)和kOH分别为0.12, 8.55mol^-^1.L.s^-^1。k0为NA的溶剂解速率常数, 其值为1.122×10^-^5s^-^1(298K, I=0.10,0.02mol.L^-^1tris缓冲溶液)。kcat值较以前报道的配合物更大, 催化活性更高。显示配位酚羟基可作为一种新型亲核体有效地催化NA的水解。配合物对NA水解的催化作用受酸碱平衡控制。根据实验结果提出了催化反应的机理。     

含新型亲核体大环多胺锌(II)配合物对水解酶的模拟研究

本文探讨苯酚功能化的大环多胺配体6-(2'-羟基-3', 5'-二溴)-苄基-1, 4, 8, 11-四氮杂环十四烷(L)锌(II)配合物作为水解酶模拟物催化4-硝基苯酚醋酸酯(NA)水解的动力学。研究表明催化水解速率对NA及配合物浓度皆呈一级反应。水解速率遵循速率方程V=(kcat[Zn]+kOH[OH^-]+k0)[NA]。其二级反应速率常数kcat在一定范围内随着pH值的增加而增加, kcat的最大值(k)和kOH分别为0.12, 8.55mol^-^1.L.s^-^1。k0为NA的溶剂解速率常数, 其值为1.122×10^-^5s^-^1(298K, I=0.10,0.02mol.L^-^1tris缓冲溶液)。kcat值较以前报道的配合物更大, 催化活性更高。显示配位酚羟基可作为一种新型亲核体有效地催化NA的水解。配合物对NA水解的催化作用受酸碱平衡控制。根据实验结果提出了催化反应的机理。     

贝壳状革耳菌漆酶酶活测定方法分析

酶活测定方法中所用的反应底物、反应条件以及酶活单位定义对漆酶酶活的测定结果有很大的影响。在对贝壳状革耳菌 (Panusconchatus)漆酶酶活测定方法的研究中发现 ,该酶与 2 ,2’ -连氮 -双 ( 3-乙基并噻- 6 -磺酸 ) (ABTS)反应的最适pH值为 3.0 ,而与紫丁香醛连氮、2 ,6-二甲氧基苯酚和邻甲联苯胺反应的最适pH值均为 4.0。上述底物与Panusconchatus漆酶反应的Km (米氏常数 )分别为 0 .0 1 1 6(mmol·L) - 1 ,1 4.1 0 95 (mmol·L) - 1 ,0 .1 0 1 1 (mmol·L) - 1 ,0 .1 41 5 (mmol·L) - 1 。该酶与ABTS、2 ,6-二甲氧基苯酚和邻甲联苯胺反应均表现为零级反应 ,而与紫丁香醛连氮反应时只在一定范围的酶浓度以及反应时间内才表现为零级反应。     

Surface enzyme kinetics for biopolymer microarrays: a combination of Langmuir and Michaelis-Menten concepts

Real-time surface plasmon resonance (SPR) imaging measurements of surface enzymatic reactions on DNA microarrays are analyzed using a kinetics model that couples the contributions of both enzyme adsorption and surface enzyme reaction kinetics. For the case of a 1:1 binding of an enzyme molecule (E) to a surface-immobilized substrate (S), the overall enzymatic reaction can be described in terms of classical Langmuir adsorption and Michaelis-Menten concepts and three rate constants: enzyme adsorption (k(a)), enzyme desorption (k(d)) and enzyme catalysis (k(cat)). In contrast to solution enzyme kinetics, the amount of enzyme in solution is in excess as compared to the amount of substrate on the surface. Moreover, the surface concentration of the intermediary enzyme-substrate complex (ES) is not constant with time, but goes to zero as the reaction is completed. However, kinetic simulations show that the fractional surface coverage of ES on the remaining unreacted sites does reach a steady-state value throughout the course of the surface reaction. This steady-state value approaches the Langmuir equilibrium value for cases where k(a)[E] > k(cat). Experiments using the 3' --> 5' exodeoxyribonuclease activity of Exonuclease III on double-stranded DNA microarrays as a function of temperature and enzyme concentration are used to demonstrate how this model can be applied to quantitatively analyze the SPR imaging data.     

Optimization of a microwave-coupled enzymatic digestion process to prepare peanut peptides

The best enzyme to prepare peanut peptides, papain, coupled with microwave irradiation was selected from five common proteases according to the results of the yield of peanut peptides [nitrogen solution index (NSI) in trichloroacetic acid (TCA), TCA-NSI] and the degree of hydrolysis (DH). The main factors that influenced the microwave-coupled enzymatic digestion method were optimized by response surface analysis. The optimal conditions obtained were as follows: microwave extraction time, 9.5 min; power, 600 W; substrate concentration, 4%; enzymatic reaction temperature, 50 °C; enzyme quantity, 6,500 U/g; pH, 7.1 (phosphate buffer, 0.05 mol/L). Under these conditions, TCA-NSI was 62.00% and DH was 25.89%, which is higher than that obtained with either protease hydrolysis or microwave hydrolysis alone.     

Kinetic Parameters of Choline Esterase-Catalyzed Hydrolysis in the Presence of the Antigen-Antibody Immune Complex

The influence of antigen-antibody immune complexes (with the immune pairs of Candida albicans and Phytophtora infestans antigens and the corresponding antibodies) on the catalytic activity of immobilized choline esterase in the enzyme immunosensor was studied. The antigen-antibody immune complex can act as effector of choline esterase, suppressing or enhancing its catalytic activity depending on the ratio of the components of the biospecific interaction (antibodies, antigen, enzyme, and substrate). The kinetic parameters of hydrolysis of butyryl thiocholine iodide, a specific substrate of choline esterase (apparent Michaelis constants, maximal reaction rates, activation and inhibition constants) were calculated at various concentrations of the substrate and antigen and at various dilutions of the antibodies. The types and kinds of the observed effects were determined. The effect of pH on the kinetic parameters of the enzymatic reaction was studied. Various pathways, depending on conditions, were suggested for choline esterase-catalyzed hydrolysis of butyryl thiocholine iodide in the presence of the antigen-antibody immune complex.     

A Two-Step,One-Pot Enzymatic Method for Preparation of Duck Egg White Protein Hydrolysates with High Antioxidant Activity

Biocatalytic hydrolysis reactions were designed for preparation of bioactive hydrolysate of duck egg white protein (DEWP) employing two enzymes in one pot. Firstly, the fresh DEWP was thermal treated at 95 °C, for 40 min at pH 10, to effectively deactivate enzyme inhibitors thus facilitating the following two-step enzymatic hydrolysis. Compared with single-enzyme processes, the two-step enzymatic procedures showed much higher reaction efficiency. The first enzymatic step (in the presence of Alcalase or hydrolase SEEP) allowed to hydrolyze DEWP with degree of hydrolysis (DH) of 8.8–10 % and soluble peptide yield (SEP) of 60.5–70.2 % in a short period (4 h). The second enzymatic step (in the presence of Trypsin or Alcalase) gave a further degradation of DEWP with DH and SEP being more than 26.2 % and 90.4 %, respectively. The final hydrolysates exhibited high antioxidant activity in an evident DH dependent manner. The hydrolysates achieved by sequential addition of the proteinase SEEP and Alcalase at DH value 21 % gave the highest antioxidant activity, which was mainly ascribed to the changes in the amino acid compositions that the contents of some key amino acids and total hydrophobic amino acids were significantly improved by the enzymatic hydrolysis.     

Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D: -xylopyranose (4NPX) and p-nitrophenyl-alpha-L: -arabinofuranose (4NPA), and it was found that the ratio k (cat)/K (m) 4NPA/k (cat)/K (m) 4NPX was approximately 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k (cat)/K (m) muA/k (cat)/K (m) muX was approximately 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K (i) of 6.8 +/- 0.62 mM and xylose K (i) of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t (1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling

One of the limiting factors restricting the effective and efficient bioconversion of softwood-derived lignocellulosic residues is the recalcitrance of the substrate following pretreatment. Consequently, the ensuing enzymatic process requires relatively high enzyme loadings to produce monomeric carbohydrates that are readily fermentable by ethanologenic microorganisms. In an attempt to circumvent the need for larger enzyme loadings, a simultaneous physical and enzymatic hydrolysis treatment was evaluated. A ball-mill reactor was used as the digestion vessel, and the extent and rate of hydrolysis were monitored. Concurrently, enzyme adsorption profiles and the rate of conversion during the course of hydrolysis were monitored. α-Cellulose, employed as a model substrate, and SO₂-impregnated steam-exploded Douglas-fir wood chips were assessed as the cellulosic substrates. The softwood-derived substrate was further posttreated with water and hot alkaline hydrogen peroxide to remove >90% of the original lignin. Experiments at different reaction conditions were evaluated, including substrate concentration, enzyme loading, reaction volumes, and number of ball beads employed during mechanical milling. It was apparent that the best conditions for the enzymatic hydrolysis of α-cellulose were attained using a higher number of beads, while the presence of air-liquid interface did not seem to affect the rate of saccharification. Similarly, when employing the lignocellulosic substrate, up to 100% hydrolysis could be achieved with a minimum enzyme loading (10 filter paper units/g of cellulose), at lower substrate concentrations and with a greater number of reaction beads during milling. It was apparent that the combined strategy of simultaneous ball milling and enzymatic hydrolysis could improve the rate of saccharification and/or reduce the enzyme loading required to attain total hydrolysis of the carbohydrate moieties.     

Enzymatic hydrolysis of protein: Mechanism and kinetic model

The bioreaction mechanism and kinetic behavior of protein enzymatic hydrolysis for preparing active peptides were investigated to model and characterize the enzymatic hydrolysis curves. Taking into account single-substrate hydrolysis, enzyme inactivation and substrate or product inhibition, the reaction mechanism could be deduced from a series of experimental results carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations and temperatures based on M-M equation. An exponential equation dh/dt = aexp(-bh) was also established, where parameters a and b have different expressions according to different reaction mechanisms, and different values for different reaction systems. For BSA-trypsin model system, the regressive results agree with the experimental data, i.e. the average relative error was only 4.73%, and the reaction constants were determined as K _m = 0.0748 g/L, K _s = 7.961 g/L, k _d = 9.358/min, k ₂ = 38.439/min, E _a = 64.826 kJ/mol, E _d = 80.031 kJ/mol in accordance with the proposed kinetic mode. The whole set of exponential kinetic equations can be used to model the bioreaction process of protein enzymatic hydrolysis, to calculate the thermodynamic and kinetic constants, and to optimize the operating parameters for bioreactor design. __________ Translated from Journal of Tianjin University, 2005, 38(9) (in Chinese)     

Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance

Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.     

Computer simulation of the steady state currents at enzyme doped carbon paste electrodes

The plate-gap model of porous enzyme doped electrode has been proposed and analyzed. It was suggested that reaction diffusion conditions in pores of bulk electrode resemble particular conditions in thin gap between parallel conducting plates. The model is based on the diffusion equations containing a nonlinear term related to the Michaelis–Menten kinetic of the enzymatic reaction inside gap. Steady state current was calculated for the wide range of given parameters and substrate concentrations. All dependences of current on substrate concentration were approximated by hyperbolas in order to obtain “apparent” parameters (maximal currents and apparent Michaelis constants) of modelled biosensors. Simple approximate relationships between given and apparent parameters were derived. The applicability of theoretical plate-gap model was tested for the case of carbon paste electrodes which were doped with PQQ – dependent glucose dehydrogenase. It was found, that soluble glucose dehydrogenase based biosensors exhibit characteristic features of the theoretical plate-gap biosensors.     

Kinetics and mechanism of electron transfer reactions. Osmium(VIII) catalyzed oxidation of mannitol by hexacyanoferrate(III) in aqueous alkaline medium

The kinetics of electron transfer from mannitol to hexacyanoferrate(III), catalyzed by osmium(VIII), has been studied in alkaline medium. The substrate order is complex, whereas it is one with respect to the catalyst. The rate is independent of the concentration of oxidant. Also, the rate increases with increasing concentration of hydroxide ion in a complex manner. A kinetic rate law corresponding to the proposed mechanism has been suggested as follows: