Screeing of New cold-active enzymes and their applications

适应低温的微生物(嗜冷菌和嗜冷菌)与中温微生物的区别在于它们在低温下生长的能力。它们有特定的能量转导机制以及调节细胞内环境和新陈代谢的机制。它们还具有细胞膜和细胞壁的结构和功能成分的稳定性，以及功能性的冷活性酶)。与中温酶相比，冷活性酶的最适活性向较低温度移动是其特征。低温冷活性酶催化可以减少不稳定产物的降解，可以应用于低温生物传感器。对从鲑鱼肠道中分离的冷活性蛋白酶和来源细菌的特性进行了讨论。冰雪覆盖土壤、鲑鱼肠和蟹…中新分离细菌分泌的淀粉酶、脂肪酶和蛋白酶的性质对更多的肠道进行了研究。一种淀粉酶、一种脂肪酶和三种蛋白水解酶的特征是它们的表观最适活性向低温方向转移，其活化能值降低。发现的酶在高于最适温度(30ﾟC至40ﾟC)时迅速失活。结果表明，这些酶具有低温活性。经16Sﾟ分析，从鲑鱼肠道中分离到最具低温活性的蛋白酶产生菌为巴鲁斯汀黄杆菌。该菌的最适生长温度为20ﾟC，但在10ﾟC时产酶能力高于20℃。本研究对一种新型的L谷氨酸：受体氧化还原酶进行了研究，并将其用于L谷氨酸传感器。该酶在50ﾟC时酶活最高，5ﾟC时酶活最高达40%。该酶的最适温度低于依赖NAD的牛肝L谷氨酸脱氢酶。因此，该酶可归类为冷活性酶。纯化的酶确实是无与伦比的，因为它的来源和辅酶与已知的L谷氨酸氧化还原酶不同。但与L等牛肝谷氨酸脱氢酶等中温酶相比，该酶是否为冷活性酶可以得到充分的认识。L-谷氨酸的氧化速率是L-氨基酸中最高的。纯化的酶既不依赖NAD+，也不依赖NADP+，但与多种氧化还原染料反应。1-甲氧基-5-甲基吩嗪甲硫酸盐(1-甲氧基-5-甲基吩嗪甲硫酸盐)的反应速率是NBT的8倍，氧化还原染料的还原速率随氧化还原电位的增大和相对分子质量的减小而增大。氧化酶和NAD非依赖性脱氢酶的活性可以通过NBT活性染色来检测。较少

Cold-adapted (psychrophilic and psychrotrophic) microorganisms are distinguished from mesophiles by their ability to grow at low temperature. They have specific mechanisms for energy transduction and for the regulation of the intracellular environ-ment and metabolism. They have also stability of structural and functional components of the cell membrane and the cell wall, and functional cold-active enzymesl). The cold-active enzymes are characterized by a shift of the optimum activity towards lower temperature as compared with meso-philic enzymes. The cold-active enzyme catalysis at low temperature can reduce degradation of unstable products, and it can be applied to a biosensor at low temperature.The characterizations of a cold-active protease and the source bacterium isolated from salmon intestine are discussed in relation to the cold-adapted properties. The properties of amylase, lipase and protease excreted by newly isolated bacteria from snow-covered soil, salmon intestine and crab … More intestine have been investigated. One amylase, one lipase and three proteases have been characterized by shifts in their apparent optimal activities towards low temperatures and by reductions in their activation energy values. The discovered enzymes were rapidly inactivated at temperatures above the optimum (30ﾟC to 40ﾟC). The seresults suggest that the enzymes are cold-active. The best cold-active protease producer, isolated from salmon intestine, has been identified as Flavobacterium balustinum by the analysis of 16S rRNA.The optimum growth temperature of this bacterium was 20ﾟC but a higher amount of protease was present at 10ﾟC than 20ﾟC.In this study, a novel L-glutamate : acceptor oxidoreductase from cold-adapted bacterium was investigated and electrochemically characterized to apply to L-glutamate sensor. The enzyme activity reached maximum at 50ﾟC and showed 40 % of maximum at 5ﾟC.The optimum temperature of the enzyme activity was lower than that of mesophilic NAD-dependent L-glutamate dehydrogenase from beef liver. Therefore, this enzyme could be classified to a cold-active enzyme. The purified enzyme was really unparalleled, because it differed the origin and the co-enzyme from known L-glutamate oxido-reductases. However, whether this enzyme is cold-active enzyme or not can sufficiently know in comparison with mesophilic enzyme such as L-glutamate dehydrogenase from beef liver. The rate of L-glutamate oxidation was highest among L-amino acids. The purified enzyme showed dependence on neither NAD+ nor NADP+, though it reacted with several redox dyes. The reaction rate with 1-methoxy-5-methylphanazine methosulfate (1-methoxy PMS) was 8 times as large as with NBT.Reduction rates of the redox dyes were found to increase with the increase in the redox potential and with the decrease in molecular weight. Enzyme activity of oxidases and NAD-independent dehydrogenases can be detected by activity staining with NBT. Less