1996 Fiscal Year Final Research Report Summary
Screeing of New cold-active enzymes and their applications
| Project/Area Number |
07459010
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
広領域
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| Research Institution | Japan Advanced Institute of Science and Technology, Hokuriku |
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Principal Investigator |
TAMIYA Eiiti JAIST Material Science Professor, 材料科学研究科, 教授 (60179893)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAGUTI Toshifumi JAIST Material Science Research Associate, 材料科学研究科, 助手 (10272999)
MURAKAMI Yuji JAIST Material Science Research Associate, 材料科学研究科, 助手 (70272995)
YOKOYAMA Kenji JAIST Material Science Associate Professor, 材料科学研究科, 助教授 (80242121)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Proteuse / Redox enzyme / Psychophilic enzyme / Ccldactive bacteria / Biosensor / Glutamate dehydrogenase |
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| Research Abstract |
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
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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
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