2004 Fiscal Year Final Research Report Summary
Physiological functions of "aldoxime-nitrile pathway" in microorganisms and the use of enzymes responsible for the pathway to organic synthesis
| Project/Area Number |
15580066
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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 |
Applied microbiology
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| Research Institution | Faculty of Engineering, Toyama Prefectural University |
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Principal Investigator |
KATO Yasuo Toyama Prefectural University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (20254237)
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| Co-Investigator(Kenkyū-buntansha) |
ASANO Yasuhisa Toyama Prefectural University, Faculty of Engineering, Professor, 工学部, 教授 (00222589)
KOMEDA Hidenobu Toyama Prefectural University, Faculty of Engineering, Assistant Professor, 工学部, 助手 (50285160)
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| Project Period (FY) |
2003 – 2004
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| Keywords | aldoxime dehydratase / nitrile hydratase / nitrilase / Rhodococcus / Bacillus / dehydration / enzyme / screening |
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| Research Abstract |
We examined the following experiments in order to clarify 1) reaction mechanism of aldoxime dehydratase (Oxd) and 2) physiological function of "aldoxime-nitrile pathway" in microorganisms:
1. We constructed an overexpression system of nitrilase (Nit)-linked axd gene in a recombinant E. coli and obtained large quantities of Oxd protein (OxdB) in highly pure and homogenous form.
2. We speculated a reaction mechanism of OxdB by analyzing detailed characters and spectrophotometric analysis of the enzyme.
3. We purified several hundreds mg of OxdB from the recombinant E. coli strain grown under optimized conditions at (1) and applied for a preliminary crystallization studies.
4. We screened several strains having Oxd liked with nitrile hydratase (NHase) from our stock cultures and soil samples.
5. We highly purified NHase-linked Oxd (OxdRG) from Rhodococcus globerulus A-4, which has been isolated at (4), and compared its properties with the previously characterized Oxds.
6. We cloned oxd gene from thegenomic library of R. globerulus based on an information of N-terminal amino acid sequence of OxdRG. We clarified that the oxd gene coexisted with genes coding NHase, amidase, and their regulatory proteins and activators at the genome of the strain as to form gene clusters.
7. We cloned oxd gene homologue existing at an upstream of amidase gene of Rhodococcus erythropolis N-771, which had been isolated as NHase producer. We constructed the overexpression system of the Oxd (OxdRE) in the recombinant E. coli and optimized its overexpression system. Under the optimized conditions, the recombinant E. coli was cultivated and OxdRE was purified from the strain, characterized, and compared its properties with the known Oxds.
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