Screeing of New cold-active enzymes and their applications

冷适应（嗜冷和嗜冷）微生物与中温微生物的区别在于它们在低温下生长的能力。它们具有特殊的能量传递机制和调节细胞内代谢和代谢的机制。它们还具有细胞膜和细胞壁的结构和功能组分的稳定性，以及功能性冷活性酶。冷活性酶的特征在于与嗜中温酶相比，其最佳活性向较低温度移动。低温下的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。本文讨论了从鲑鱼肠中分离的一种冷活性蛋白酶及其源菌的特性，并讨论了其与冷适应特性的关系。雪土、鲑鱼肠和蟹肠中新分离细菌的淀粉酶、脂肪酶和蛋白酶特性 ...更多信息 肠已经被研究过了。一种淀粉酶，一种脂肪酶和三种蛋白酶的特征在于它们的表观最佳活性向低温的转变和它们的活化能值的降低。发现的酶在高于最适温度（30 ℃至40 ℃）的温度下迅速失活。结果表明，这些酶具有低温活性。从鲑鱼肠中分离到一株产低温蛋白酶能力最强的细菌，经16 S rRNA分析鉴定为Flavobacterium balustinum，其最适生长温度为20 ℃，但在10 ℃时产蛋白酶量高于20 ℃。研究了冷适应细菌的受体氧化还原酶，并对其进行了电化学表征，以应用于L-谷氨酸传感器。该酶在50 ℃时活性最高，在5 ℃时活性最高达40%，其最适温度低于牛肝中温NAD依赖的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，因为它与已知的L-谷氨酸氧化还原酶的来源和辅酶不同。然而，与来自牛肝的L-谷氨酸脱氢酶等嗜温酶相比，可以充分地知道该酶是否是冷活性酶。在L-氨基酸中，L-谷氨酸的氧化速率最高。纯化的酶既不依赖于NAD+，也不依赖于NADP+，但它与几种氧化还原染料反应。与1-甲氧基-5-甲基吩嗪硫酸甲酯（1-methoxy PMS）的反应速率是与NBT的反应速率的8倍。发现氧化还原染料的还原速率随着氧化还原电位的增加和分子量的减小而增加。氧化酶和NAD非依赖性氧化酶的酶活性可以通过用NBT活性染色来检测。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