1997 Fiscal Year Final Research Report Summary
Research for the molecular evolution of the adaptive mutants obtained after continuous culture of cumene degrader on biphenyl.
| Project/Area Number |
08660089
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
OMORI Toshio The University of Tokyo, Biotechnology Research Center, Professor, 生物生産工学研究センター, 教授 (20011984)
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| Project Period (FY) |
1996 – 1997
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| Keywords | cumene / biphenyl / Pseudomonas / serine hydrolase / adaptation |
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| Research Abstract |
Cumene ( isopropylbenzene ) is an aromatic hydrocarbon with a chemical structure close to those of toluene and biphenyl. P.fluorescens IP01 can grow on cumene or toluene as a sole carbon and energy source but not on biphenyl. We surmised that the growth substrate specificity of strain IP01 is ascribed to the substrate specificity of the meta-cleavage compound hydrolase (CumD ).
In this study, we attempted to determine the residue responsible for the subatrate specificity in CumD by means of site-directed mutagenesis. Amino acid sequence comparison between CumD and other hydrolases led us to identify several conserved residues likely to have a functional role in the catalytic center of CumD,that is, three amino acids, Ser^<103>, Asp^<224>, His^<252>, were found to be arranged in a sequential order similar to that in serine hydrolases. Substitutions of each of these three residues by Ala decreased enzymatic activity on hydrolisis of meta-cleavage compound of cumene (HOMODA ) below 5%. The
… More
se results suport the hypothesis that Ser^<103>, Asp^<224>, His^<252> constitute the catalytic triad of CumD.Substitution of Ile^<256> by Trp and that of Trp^<143> by Phe increased enzymatic activity on hydrolysis of meta-cleavage compound of biphenly ( HOPDA ). This result suggests that Ile^<256> and Trp^<143> are the key residues to alter the substrate specificity of CumD so as to hydolase HOPDA.It was also indicated that substitution of Val^<227> by Ile increased enzymatic activity on hydrolysis of HOMODA.This result suggests that Val^<227> is Iocated nearby the catalytic triad topologically, and is involved in the substrate-binding site in CumD.
We obtained 25 mutants degrading efficiently biphenyl after continuous culture of IP01 on biphenyl containing a small amount of cumene. In these mutants, it was predicted that substrate specificity of CumD had been altered as a result of mutation for the cumD gene, but any mutation in cumD was not detected in sequence analysis. Comparison of growth of 5 mutants ( selected from 25 mutants ) with that of wild-type IP10 indicates that the activity of a series of enzymes in the cumene degradation pathway, or mechanism of transcriptional control of the cum gene, may have altered in the 5 mutants. Less
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