| Project/Area Number |
62560105
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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 |
発酵・醸造
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| Research Institution | Osaka University |
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Principal Investigator |
ATSUHIKO SHINMYO Osaka University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (30029235)
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| Co-Investigator(Kenkyū-buntansha) |
MITSUO TAKANO Osaka University, Faculty of Engineering, Professor, 工学部, 教授 (20029036)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1987: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Bacillus pumilus / xylanase / X-ray crystalline analysis / active site / 部位特異的変異 / X線解析 |
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| Research Abstract |
X-ray crystalline analysis of xylanase of Bacillus pumilus was done at 2.2 A^^゜ resolution. Xylanase molecule has a size of 30 x 40 x 35 A^^゜ and a cleft observed in the molecule was suggested to be the site of catalytic reaction. Since xylanase and lysozyme catalyze hydrolysis of -1,4-linkage of glycosidic bond, a catalytic mechanism might be similar in both enzymes. Chemical and heavy metal modification of xylanase suggested two acidic amino acid residues as catalytic site. On the other hand, functionally important residues in xylanase will be conserved in those from different origins. Among sequence alignments of 5 xylanases derived from Bacillus and fungi, Asp^<21>, Glu^<93>, Asp^<121> and Glu^<182> in B. pumilus xylanase were conserved acidic residues. Asp^<21> was not located in the cleft of the molecule. By taking account the distance between two amino acids, most possible pair as catalytic site was Glu^<93> and Glu^<182>.
Before site-directed mutation of the two residues, the region coding signal sequence of the structural gene of xylanase was replaced by the initiation codon using synthetic oligonucleotides. Restriction sites in the structural gene were also modified to create unique sites and ligated downstream of tac promoter in high expression vector in E. coli, pKP1500. Then, Glu^<93> and Glu^<182> were changed to Asp or Ser. The mutated enzymes were purified to homogeniety from E. coli cell extract. Activity of the mutated enzymes was not detected except the mutation of Glu^<93> to ASP. A little activity observed in Asp^<93> enzyme was the result of marked decrease of Vm value.
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