竞争性抑制的非稳态酶动力学布尔函数图论研究

以非稳戊酶动力学的布尔函数图形方法,来研究一类竞争性抑制的非稳态酶动力学问题,推导出此类反应的百稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类竞争性抑制酶反应体系的非稳态酶动力学问题。     

Immobilization of hemoglobin on chitosan films as mimetic peroxidase

Hemoglobin (Hb) was immobilized on the chitosan films using glutaraldehyde as a bifunctional agent. Atomic force microscopy (AFM) was used to examine the film surface in order to image the presence of Hb and Fourier transform infrared spectroscopy (FT-IR) was detected to elucidate the structural change of the immobilized Hb. The influences of several immobilization parameters were investigated, the optimum concentration of glutaraldehyde, pH and binding time were determined as 0.7%, 4.5 and 6 h, respectively. The enzymatic assay indicates that the immobilized Hb showed a higher thermal stability than that of free Hb, and the catalytic activity in organic solvents was also enhanced.     

The Effect of Cations on the Amidase Activity of Human Tissue Kallikrein: 1-Linear Competitive Inhibition by Sodium,Potassium, Calcium and Magnesium. 2-Linear Mixed Inhibition by Aluminium

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases.     

Amphitrite ornata dehaloperoxidase: enhanced activity for the catalytically active globin using MCPBA

Dehaloperoxidase (DHP) from Amphitrite ornata is the only heme-containing, hydrogen peroxide-dependent globin capable of oxidatively dehalogenating halophenols to yield the corresponding quinones. To ascertain that this enzymatic activity is intrinsic to DHP, we have cloned and expressed the enzyme in Escherichia coli. We also find that an alternate oxygen atom donor, meta-chloroperbenzoic acid, gives appreciably higher activity than hydrogen peroxide. Under optimal turnover conditions (large peroxide/peracid excess), after an initial burst of activity, DHP appears to become trapped in a non-catalytic state (possibly Compound II) and is unable to fully convert all halophenol to product. However, full substrate conversion can be achieved under more physiological conditions involving a much smaller excess of oxygen atom donor. Parallel studies have been carried out using horseradish peroxidase and myoglobin to calibrate the activity of DHP versus typical peroxidase and globin proteins, respectively.     

Kinetic behaviour of proenzymes activation in the presence of different inhibitors for both activating and activated enzymes

In the present paper, a kinetic analysis of a general model for proenzyme activation, where the activating enzyme and also the activated one are reversibly inhibited in two steps by two different inhibitors, has been performed. The cases in which both inhibitors are the same, or in which the inhibition is irreversible (only one or the two inhibition routes) are treated as particular cases of the general model. In addition, the kinetic behaviour of many other proenzyme activation systems involving inhibition, particular cases of the reaction scheme under study, can be obtained. The total number of particular cases for the general model under study is 370, so this approach offers to the scientific community working in limited proteolysis regulation for the first time a method based on general solutions which only needs to be specified to their concrete problem of zymogen activation. Finally, new adimensional parameters are introduced, allowing the knowledgement, in the case that any of the inhibition routes is irreversible, the relative weight of both activation and irreversible inhibition routes.     

两种生物状态肠道益生菌的粘附和粘附拮抗效应的对比研究

目的:研究活菌和灭活菌两种生物状态的肠道主要益生菌--德氏乳杆菌、双歧杆菌和肠球菌对肠黏膜上皮细胞粘附性及其对肠道几种常见病原菌的粘附拮抗效应.方法:用光镜和电镜技术分析了两种生物状态的三种益生菌对肠黏膜上皮细胞的粘附指数,通过排除实验、竞争实验和替代实验研究了两种生物状态益生菌对侵袭性大肠埃希菌、产毒性大肠埃希菌和痢疾志贺菌的粘附拮抗效应,应用平板扩散法观察了三种益生菌的代谢乏液对上述肠道病原菌的抑制能力.结果:德氏乳杆菌和肠球菌的灭活状态较活菌状态对肠黏膜上皮细胞的粘附性显著增高,双歧杆菌经灭活后对细胞的粘附性与活菌相比差异无显著性,两种生物状态的三种益生菌对肠道致病菌均具有粘附拮抗作用.滤过后的德氏乳杆菌、双歧杆菌和肠球菌的代谢乏液对侵袭性大肠埃希菌、产毒性大肠埃希菌和痢疾志贺菌均具有较明显的抑制作用,经42℃、65℃和100℃加热不影响德氏乳杆菌和双歧杆菌代谢乏液的抑菌作用.结论:灭活状态的德氏乳杆菌、双歧杆菌和肠球菌是具有潜在开发价值的微生态制剂.     

Crystal structure of Leishmania major peroxidase and characterization of the compound i tryptophan radical

The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp(208) in LmP and Trp(191) in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp(191) forms a stable cationic radical after reaction with H(2)O(2) to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp(208) in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp(208) using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of β structure near Trp(191) and Trp(208), respectively, which is absent in APX. This region provides stability to the Trp(191) radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp(208) forms a stable cationic radical. In LmP conversion of the Cys(197) to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase.     

Inhibition of microbial β-N-acetylhexosaminidases by 4-deoxy- and galacto-analogues of NAG-thiazoline

NAG-thiazoline is a well-established competitive inhibitor of two physiologically relevant glycosidase families—β-N-acetylhexosaminidases (GH20) and β-N-acetylglucosaminidases (GH84). Based on the different substrate flexibilities of these enzyme groups, we designed and synthesized the 4-deoxy derivative of NAG-thiazoline aiming at the selective inhibition of GH20 β-N-acetylhexosaminidases. One GH84 and two GH20 microbial glycosidases were employed as model enzymes for the inhibition assays. Surprisingly, the new compound 4-deoxy-thiazoline exhibited no activity inhibition with either of the enzyme families of interest. Unlike with the substrates, the 4-hydroxyl group of the inhibitor’s sugar ring seems to be crucial for binding the inhibitor to the active sites of these enzymes.     

Enzyme inhibitors and other bioactive compounds from marine actinomycetes

Several enzyme-inhibitor-producing actinomycetes were isolated from various samples collected from the marine environment and characterized. Most of them produced novel compounds that are useful in medicine and agriculture. Actinomycete strain no. 18, which produces antibiotics against Gram-positive bacteria only in the presence of seawater, was isolated from sediment sampled from neritic sea water and characterized. The production of antibiotics was observed at seawater concentrations ranging from 60 to 110% (v/v). Thus, the production was seawater-dependent. The production of tetrodotoxin (TTX), known otherwise as puffer fish toxin, was investigated in various actinomycetes collected from the marine environment. Of 10 isolates from various sea areas, 9 produced TTX as judged by their retention times on high-performance liquid chromatography (HPLC). To our knowledge, this is the first report of actinomycetes from the marine environment that produce TTX.     

Characterization of Trametes versicolor laccase for the transformation of aqueous phenol

Laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor was thoroughly characterized in terms of its catalytic stability and its effectiveness as a biocatalyst under various reaction conditions when using phenol as a model substrate. This enzyme demonstrated high or moderate degrees of stability at pHs from 5 to 8 at 25 degrees C and at temperatures from 10 to 30 degrees C at pH 6. Exponential decay expressions were successfully used to model laccase inactivation when incubated under various conditions of pH and temperature. Phenol transformation was optimum at pH 6, but significant transformation was observed over a pH range of 4-7, provided that sufficient laccase was present in the reacting solution. Partial inactivation of laccase was observed during the oxidation of phenol, even under conditions of optimal stability (pH 6 and 25 degrees C).     

Expression of human β-N-acetylhexosaminidase B in yeast eases the search for selective inhibitors

Human lysosomal β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases are dimeric enzymes catalysing the cleavage of terminal β-N-acetylglucosamine and β-N-acetylgalactosamine residues from a broad spectrum of glycoconjugates. Here, we present a facile, robust, and cost-effective extracellular expression of human β-N-acetylhexosaminidase B in Pichia pastoris KM71H strain. The prepared Hex B was purified in a single step with 33% yield obtaining 10 mg of the pure enzyme per 1 L of the culture media. The enzyme was used in the inhibition assays with the known mechanism-based inhibitor NAG-thiazoline and a wide variety of its derivatives in the search for specific inhibitors of the human GH20 β-N-acetylhexosaminidases over the human GH84 β-N-acetylglucosaminidase, which was expressed, purified and used in the inhibition experiments as well. Moreover, enzyme-inhibitor complexes were analysed employing computational tools in order to reveal the structural basis of the results of the inhibition assays, showing the importance of water-mediated interactions between the enzyme and respective ligands. The presented method for the heterologous expression of human Hex B is robust, it significantly reduces the costs and equipment demands in comparison to the expression in mammalian cell lines. This will enhance accessibility of this human enzyme to the broad scientific community and may speed up the research of specific inhibitors of this physiologically important glycosidase family.     

水体酚类化合物污染对水稻幼苗生长的影响

用０、５０、１００和１５０×１０－６浓度的苯酚溶液处理水稻幼苗,本实验的目的是探讨苯酚对水稻幼苗生长的影响。实验结果如下：苯酚抑制水稻幼苗生长,且随着浓度的增加,其抑制作用越明显。１５０ｐｐｍ是抑制作用最强的浓度。经苯酚处理后的水稻幼苗,其根和茎的生长都抑制了,而鲜重、干重、水分含量和叶绿素含量则明显减少,硝酸还原酶和过氧化物酶活性也下降了。     

Reactivity study on microperoxidase-8

The catalytic activity of the microperoxidase-8/H₂O₂ system toward tyramine and 3-(4-hydroxyphenyl)propionic acid has been determined in acetate buffer, pH 5.0. Operating with a strong excess of hydrogen peroxide, the rate-determining step of the reaction was substrate oxidation. Owing to the fast microperoxidase-8 degradation, only the very initial phase of the reactions were analyzed. The reaction rates follow a substrate saturation behavior, with turnover numbers [k_cat=26±1 s^–1 for 3-(4-hydroxyphenyl)propionic acid and k_cat=22±1 s^–1 for tyramine] that were similar for the two substrates. In contrast, the K_M values indicated a reduced affinity for the catalyst active species by the positively charged phenol, probably due to repulsive interaction with the protonated N-terminal microperoxidase-8 amino group. The reactivity of the catalyst active species was studied upon incubation of microperoxidase-8 with a small excess hydrogen peroxide, followed by reaction with the phenolic substrates. The kinetic analysis showed that more than two active species are accumulated. The species responsible for the faster reactions was present in solution as a minor fraction. The active intermediate which accumulated in a larger amount (intermediate III) has a reduced substrate oxidation activity. Comparison of this activity with the kinetic constants obtained under turnover experiments shows that intermediate III is not involved in the microperoxidase-8 catalytic cycle. The active species of the catalytic process are intermediates I and II, which in the absence of substrate rapidly convert to intermediate III.Abbreviations ABTS 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) - HPA 3-(4-hydroxyphenyl)propionic acid - HRP horseradish peroxidase - MP-8 microperoxidase-8     

Cadmium Inhibition of a Structural Wheat Peroxidase

The major peroxidase from 15-day-old wheat plants was purified to homogeneity by FPLC ion exchange and molecular exclusion chromatography. It consists of a single polypeptide of M₁ 37, 500 according to gel filtration and SDS-PAGE and has a pI of 7.0. Kinetics of pyrogallol peroxidation showed that the enzyme follows the accepted mechanism for peroxidase, with kinetic constants k₁= 4.4 × 10⁶ M^-1 s^-1 and k₃ = 8.6 × 10⁵ M^-1s^-1. The effect of different metal ions was assayed on peroxidase activity. None of the ions used had any effect on enzyme activity, except for Cd(II), which was an inhibitor. This was an unexpected and novel finding for a peroxidase. The kinetics of pyrogallol peroxidation at different concentrations of Cd(II) have been studied and a mechanism for Cd(II) inhibition proposed. The results obtained could explain, in part, cadmium-induced oxidative stress.     

Effect of enzyme additions on methane production and lignin degradation of landfilled sample of municipal solid waste

Operation of waste cells as landfill bioreactors with leachate recirculation is known to accelerate waste degradation and landfill gas generation. However, waste degradation rates in landfill bioreactors decrease with time, with the accumulation of difficult to degrade materials, such as lignin-rich waste. Although, potential exists to modify the leachate quality to promote further degradation of such waste, very little information is available in literature. The objective of this study was to determine the viability of augmenting leachate with enzymes to increase the rate of degradation of lignin-rich waste materials. Among the enzymes evaluated MnP enzyme showed the best performance in terms of methane yield and substrate (lignin) utilization. Methane production of 200 mL CH₄/g VS was observed for the MnP amended reactor as compared to 5.7 mL CH₄/g VS for the control reactor. The lignin reduction in the MnP amended reactor and control reactor was 68.4% and 6.2%, respectively.     

The effect of sulfate reduction on the thermophilic (55°C) methane fermentation process

Summary The continuously operated suspended growth anaerobic contact system was utilized to estimate the effect of sulfate reduction on the thermophilic (55°C) methane fermentation process. Results indicated that reduction in methanogenesis in the presence of sulfate was due to two separate, but related, processes;i.e. competitive and sulfide inhibition. Although prevention of competitive inhibition would be difficult under normal fermenter operation, sulfide inhibition could be minimized by environmental selection of sulfide tolerant microbial populations through biomass recycle and pH control. Stable fermenter operation was achieved at soluble sulfide concentrations as high as 330 mg/l soluble sulfide. Using batch fermenters, a maximum thermophilic sulfate reduction rate of 3.7 mg SO₄ ^2––S/g volatile solids (VS)-day was estimated. The importance of reporting sulfate reduction rates on a biomass basis is demonstrated by a simple population adjustment kinetic model.This research study was conducted at the Department of Agricultural Engineering, Cornell University, Riley Robb Hall, Ithaca, NY 14853, U.S.A.