| Project/Area Number |
05660109
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Kumamoto Institute of Technology |
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Principal Investigator |
MURAO Sawao Kumamoto Institute of Technology, Applied Microbial Technology, Professor, 工学部, 教授 (00081472)
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| Co-Investigator(Kenkyū-buntansha) |
OYAMA Hiroshi Kumamoto Institute of Technology, Applied Microbial Technology, Associated Prof., 工学部, 講師 (50221700)
SHIN Takeshi Kumamoto Institute of Technology, Applied Microbial Technology, Associated Prof., 工学部, 助教授 (50179066)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | kumamolysin / acid proteinase / thermostable / 耐熱性酸性プロテアーゼ |
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| Research Abstract |
In the course of our screening of an acid proteinase, insensitive to the known specific inhibitors, in thermophilic microorganism, we found a novel enzyme designated "Kumamolysin" in Bacillus novosp. MN-32. The present project is to elucidate the unique substrate specificity and primary structure of the enzyme from protein sequencing and/or coloning of the kumamolysin gene. The summary of experimental results are as follows.
(1) The unique character of kumamolysin is its substrate specificity : kumamolysin specificaly hydrolyzed the peptide bond of Leu (15) -Tyr (16) in oxidized insulin B chain. The subsite requirement was disclosed from synthetic peptide substrate, mimic to the sccile site in oxidized insulin B chain. The minimum length of substrate was determined to be hexapeptide (VEALYL).
(2) Primary structure : Partial amino acid sequence (total 100 residues) of kumamolysis was determined from tryptic, lysyl endopeptidase fragements by automated protein sequencer. From these results, DNA probe for cloning was designed. After hybridization, Sal I fragment of chromosomal DNA was deduced to contain kumamolysin gene. Insertion of this fragemnt in pUC18 to yield plasmid pK1. Subcloning of this plasmid was also carried out, and partial base sequence of kumamolysin gene was elucidated.
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