氮沉降增加对森林凋落物分解酶活性的影响

氮沉降增加对森林凋落物分解酶产生的影响在世界范围受到关注。综述了凋落物分解酶的种类、影响酶的因素、酶的生态学意义和土壤酶研究技术的研究发展趋势。根据森林凋落物底物性质的不同,将凋落物分解酶分为纤维素分解酶类、木质素分解酶类、蛋白水解酶类和磷酸酶类。目前普遍认为,氮沉降增加,磷酸酶类活性随之增加,其它三类酶活性未呈现规律性变化。此外,还对氮沉降增加与土壤酶之间关系的研究前景进行了探讨。     

嗜热酶的耐热机理

酶蛋白在高温下的不稳定性是影响其广泛应用的主要瓶颈,嗜热酶因为独特的性质而被作为热稳定研究的极好材料。了解嗜热酶的热稳定性机制,对于采用酶工程定向设计、改造酶具有重要的意义。嗜热酶的热稳定性并不是由单一因素决定的,氨基酸组成、氢键、离子对、二硫键等都是影响嗜热酶热稳定性的重要因素。相对于嗜温酶,嗜热酶更多地采用寡聚体的形式。     

Release of anchored membrane enzymes by lipoamidase

Lipoamidase is found to be able to release various membrane-anchored enzymes from the membrane compartment of pig brain. Released enzymes revealed their intact enzyme activities in the soluble fraction. Lipoamidase could release at least two types of anchored enzymes, i.e. glycosyl-phosphatidylinositol-bonded and myristoylated enzymes, but not integral membrane bound enzymes. The reaction was competitively inhibited by lipoyllysine. This releasing mechanism found in the membranes may play important roles in the secretory mechanism of extracellular enzymes and also in the cellular signal-transduction system through topological changes in cellular enzymes.     

Variation in Constraint Versus Positive Selection as an Explanation for Evolutionary Rate Variation Among Anthocyanin Genes

It has been argued that downstream enzymes in metabolic pathways are expected to be subject to reduced selective constraint, while upstream enzymes, particularly those at pathway branch points, are expected to exhibit more frequent adaptive substitution than downstream enzymes. We examined whether these expectations are met for enzymes in the anthocyanin biosynthetic pathway in Ipomoea. Previous investigations have demonstrated that downstream enzymes in this pathway have substantially higher rates of nonsynonymous substitution than upstream enzymes. We demonstrate here that the difference in rates between the most upstream enzyme (CHS) and the two most downstream enzymes (ANS and UFGT) is explained almost entirely by differences in levels of selective constraint. Adaptive substitutions were not detected in any of these genes. Our results are consistent with suggestions that constraint is greater on enzymes with greater connectivity.     

Tricarboxylic acid cycle enzymes following thiamine deficiency

Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the alpha-ketoglutarate dehydrogenase complex (KGDHC) (-21.5%) and PDHC (-10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase (STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter brain function in neurodegenerative disorders.     

Predicting functional associations from metabolism using bi-partite network algorithms

Background  

Metabolic reconstructions contain detailed information about metabolic enzymes and their reactants and products. These networks can be used to infer functional associations between metabolic enzymes. Many methods are based on the number of metabolites shared by two enzymes, or the shortest path between two enzymes. Metabolite sharing can miss associations between non-consecutive enzymes in a serial pathway, and shortest-path algorithms are sensitive to high-degree metabolites such as water and ATP that create connections between enzymes with little functional similarity.     

Production,characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.     

Immunochemical comparisons of ribulosebisphosphate carboxylases using antisera to tobacco and spinach enzymes

Ribulosebisphosphate carboxylase molecules from over 50 species of angiosperms and gymnosperms have been compared by quantitative microcomplement fixation, using antisera prepared against tobacco and spinach enzymes. There were close antigenic similarities between tobacco enzyme, enzymes from other members of the Solanaceae, and enzymes from members of the Nolanaceae, Cuscutaceae, and Convolvulaceae. There were relatively close similarities between spinach enzyme and enzymes from two other members of the Chenopodiaceae. There were relatively great differences between tobacco enzyme, spinach enzyme, and most other enzymes tested. The enzymes from most of the angiosperms tested were as different from tobacco enzyme and almost as different from spinach enzyme as were the enzymes from the gymnosperms.     

Hepatocytes: the powerhouse of biotransformation

Liver is the most important organ involved in biotransformation of xenobiotics. Within the main organisational unit, the hepatocyte, is an assembly of enzymes commonly classified as phase I and phase II enzymes. The phase I enzymes principally cytochrome P450 catalyse both oxidative and reductive reactions of a bewildering number of xenobiotics. Many of the products of phase I enzymes become substrates for the phase II enzymes, which catalyse conjugation reactions making use of endogenous cofactors. As xenobiotic metabolising enzymes are responsible for the toxicity of many chemicals and drugs, testing the role of the biotransformation enzymes and the transporters within the hepatocyte is critical. New methodologies may be able to provide information to allow for better in vitro to in vivo extrapolation of data.     

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.     

Role of microbial enzymes in flavor development in foods

There are four main sources of enzymes in foods—these being the inherent enzymes, enzymes from microbial contaminants, enzymes elaborated by microorganisms added to foods, and specific enzymes added to foods. This study primarily deals with the latter two sources of enzymes in food. Although both plants and animals serve as sources of enzymes, they are not as economical or versatile sources as are enzymes obtained from microorganisms. In the meat industry, proteases are used to tenderize muscle and to obtain flavor precursors. In the preparation of cured meat products such as sausages, lipases, and proteases from bacterial cultures are utilized. Similarly, proteases and lipases are used in the dairy industry to develop flavor compounds. Proteases and amylases also have applications in the baking and milling industries where they are used to produce precursors for the nonenzymatic browning reactions. Carbohydrases such as amylase, amyloglucosidase, and glucose isomerase have found usage in the starch and syrup industry for the production of high dextrose and high fructose syrups. Other enzymes such as glucose oxidase, pectinase, and naringinase are of value to the wine and fruit juice industries. A better understanding of the mode of action of enzymes as well as the mechanisms of development of flavor compounds will further enhance the use of microbial enzymes to develop specific and desired flavors in foods.     

Structural and immunological relationships among mammalian arylsulfatase A enzymes

Structural and immunological properties of numerous arylsulfatase A enzymes (EC 3.1.6) were examined in order to assess the relationships among these enzymes in animals. Arylsulfatase A enzymes from all animals bind to a Concanavalin A-Sepharose column, consistent with the conclusion that they are all glycoproteins. At pH 7.5 the apparent mol. wts of the enzymes are 80-182 kDa, while at pH 4.5 the mammalian arylsulfatase A enzymes dimerize and exhibit apparent mol. wts in the range of 297-348 kDa, but the enzymes from opossum and other lower classes of animals do not aggregate at pH 4.5. The mammalian arylsulfatase A enzymes, which aggregate at pH 4.5, also bind to rabbit liver arylsulfatase A monomers immobilized on an Affi-Gel 10 matrix. The arylsulfatase A enzymes that were studied all exhibit the anomalous kinetic behavior regarded as characteristic of these enzymes. However, not all of the inactivated enzymes are reactivated by sulfate ions. Goat antiserum raised against homogeneous rabbit liver arylsulfatase A cross-reacts with all of the mammalian enzymes in Ouchterlony gel diffusion experiments, whereas the enzymes from lower classes of animals do not cross-react. Quantitative immunoprecipitation experiments demonstrate that the mammalian enzymes are very similar to each other, with greater than 60% primary sequence homology indicated, while arylsulfatase A from opossum and other lower classes of animals show only a partial immunological similarity with the mammalian enzymes. Taken together, the data suggest that the active site of the enzyme and the structural features of the protein are highly conserved during the evolution of the enzyme molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toward a systems biology perspective on enzyme evolution

Large superfamilies of enzymes derived from a common progenitor have emerged by duplication and divergence of genes encoding metabolic enzymes. Division of the functions of early generalist enzymes enhanced catalytic power and control over metabolic fluxes. Later, novel enzymes evolved from inefficient secondary activities in specialized enzymes. Enzymes operate in the context of complex metabolic and regulatory networks. The potential for evolution of a new enzyme depends upon the collection of enzymes in a microbe, the topology of the metabolic network, the environmental conditions, and the net effect of trade-offs between the original and novel activities of the enzyme.     

Covalent immobilization of enzymes within micro-aqueous organic media

Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes.This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly.     

极端酶的研究进展

极端酶具有超常的生物学稳定性,能够在极端温度、pH、压力和离子强度下表现出生物学活性,因此极端酶为生物催化和生物转化提供了良机.新的极端物种的发现、基因组序列的确定及基因工程技术的应用,加快了发现和制备新酶的进程.蛋白质工程和定向进化技术进一步改善酶的活性和特异性,促进了极端酶的工业应用.对极端酶的研究加深了人们对酶稳定性机制的理解,丰富了分子进化理论.     

酶的定向固定化方法及其对酶生物活性的影响

固定化酶可以提高酶的稳定性,但通常酶通过酶分子上的多个赖氨酸残基随意固定在载体上,这样会使酶的活性显著下降,采用定向固定化酶不仅可以提高酶的稳定性,而且可以保存它的活性。综述了定向固定化酶的几种方法,比较了定向固定化和随意固定化对酶活性的影响。另外,还叙述了酶的活性位点结构变化的自旋共振波谱(EPR)检测。     

大鵟消化系统蛋白水解酶种类和活性分析

采用蛋白水解酶复性电泳(G-PAGE)技术对大(Buteo hemilasius)消化系统5种器官腺胃、胰脏、十二指肠、空肠、大肠蛋白水解酶的种类和性质进行了研究,以期为研究野生鸟类的分类地位、系统演化提供基础资料,结果表明,①受pH值的影响和制约,大消化系统蛋白水解酶的活性在碱性、中性与酸性条件下递减;②在酸性条件下,45 ku蛋白水解酶存在于除腺胃外的各受检器官;③pH 7.0时,腺胃、胰脏酶谱相似,均含有683、5、342、0 ku的蛋白水解酶;④pH 8.0时,空肠和十二指肠的蛋白水解酶种类最多、活性最强,分别检出8种和7种蛋白水解酶。总之,pH值对蛋白水解酶的活性有明显的制约作用,46、41ku蛋白水解酶随着pH值的增高而失去活性,为酸性蛋白水解酶,250、2064、5 ku蛋白水解酶随着pH值的增高活性逐渐增强,为碱性蛋白水解酶。十二指肠和空肠的蛋白水解酶种类多、活性强,可能为蛋白质消化的主要场所。     

Effect of age and diet composition on activity of pancreatic enzymes in birds

Digestive enzymes produced by the pancreas and intestinal epithelium cooperate closely during food hydrolysis. Therefore, activities of pancreatic and intestinal enzymes processing the same substrate can be hypothesized to change together in unison, as well as to be adjusted to the concentration of their substrate in the diet. However, our knowledge of ontogenetic and diet-related changes in the digestive enzymes of birds is limited mainly to intestinal enzymes; it is largely unknown whether they are accompanied by changes in activities of enzymes produced by the pancreas. Here, we analyzed age- and diet-related changes in activities of pancreatic enzymes in five passerine and galloanserine species, and compared them with simultaneous changes in activities of intestinal enzymes. Mass-specific activity of pancreatic amylase increased with age in young house sparrows but not in zebra finches, in agreement with changes in typical dietary starch content and activity of intestinal maltase. However, we found little evidence for the presence of adaptive, diet-related modulation of pancreatic enzymes in both passerine and galloanserine species, even though in several cases the same birds adaptively modulated activities of their intestinal enzymes. In general, diet-related changes in mass-specific activities of pancreatic and intestinal enzymes were not correlated. We conclude that activity of pancreatic enzymes in birds is under strong genetic control, which enables evolutionary adjustment to typical diet composition but is less adept for short term, diet-related flexibility.     

Metabolic enzyme diversity in different human thyroid cell lines and their sensitivity to gravitational forces

Many cancer cells show unique protein expression patterns. We used proteome technology including MS, free flow isoelectric focusing and Western blotting to determine current concentrations of metabolic enzymes in healthy and malignant human thyroid cells. Three different types of human thyroid cells were investigated after they had been cultured under equal conditions. MS revealed high quantities of glycolytic enzymes and moderate quantities of citric acid cycle enzymes in malignant FTC-133 cells with abnormal LDH B-chains, high quantities of glycolytic enzymes and marginal quantities of citric acid cycle enzymes in normal HTU-5 cells, and low quantities of glycolytic enzymes and marginal quantities of citrate cycle enzymes in malignant CGTH-W1 cells with abnormal LDH A-chains. When an alteration of gene expression activity was challenged physically by removing gravity forces, the concentrations of various glycolytic enzymes were changed in normal and malignant thyroid cells. However, the changes varied among the different cell types. Different cellular alignment of the enzymes could be one reason for the cell type-specific behavior as demonstrated by histological analysis of alpha-enolase.