| Project/Area Number |
05453167
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
ODA Junichi Inst.for Chem.Research, Kyoto Univ., Professor, 化学研究所, 教授 (50027041)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Hiroaki Inst.for Chem.Research, Kyoto Univ., Instructor, 化学研究所, 助手 (90204487)
HIRATAKE Jun Inst.for Chem.Research, Kyoto Univ., Instructor, 化学研究所, 助手 (80199075)
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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 |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1994: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1993: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | Catalytic antibody / Pseudomonas lipase / Ester hydrolysis / Gene cloning / Chaperon-like protein / Molecular modeling / 活性アルギニン残基 / 可変領域 / 生成物阻害 / 触媒性残基 / 巻き戻しタンパク質 / リパーゼ活性化 |
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| Research Abstract |
The purpose of this study is to delineate the reaction mechanisms and mode of action of two catalytically-related proteins-a Pseudomonas lipase and an esterolytic antibody, and their applications to preparative molecular transformations.
Monoclonal antibodies were generated against a phosphonate transitionstate analogue for ester hydrolysis.One of the antibodies was found to catalyze the hydrolysis of a racemic ester with high stereoselsecitivy. The reaction, however, was almost stoichiometric due to strong product inhibition. A series of chemical modification of this antibody revealed that one Arg residue in the antibody combining site was essential to the activity.In fact, one Arg was found in the antibody combining site (CDR3) by cloning the gene encoding the variable domain. The same Arg was also found to play a dominant role in product inhibition by charge interaction with a negatively charged product, because the antibody experienced much less product inhibition with a carbonate ester, which undergoes decarboxylation upon hydrolysis to yield a neutral alcohol as the final product. The antibody exhibited at least 100 turnovers without any loss of activity with this substrate.
A gene encoding a lipase was cloned from a Pseudomonas sp.and was overproduced in E.coli. The lipase, however, was produced as a non-active form of inclusion bodies. We found another gene (lip B) located right after the lipase gene, and cloned and overproduced the gene in E.coli.The hitherto unknown protein produced was found to have a chaperon-like activity, assiting the refolding of a denatured lipase in vitro to restore the lipase activity. The action of Lip B was unique to the parent lipase, suggesting a private chaperonin to the Pseudomonas lipase.
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