1997 Fiscal Year Final Research Report Summary
Reaction Mechanisms and Application of New Bacterial Enzymes
| Project/Area Number |
08044218
|
|---|
| Research Category |
Grant-in-Aid for international Scientific Research
|
| Allocation Type | Single-year Grants |
|---|
| Section | Joint Research |
|---|
| Research Field |
応用微生物学・応用生物化学
|
|---|
| Research Institution | Toyama Prefectural University |
|---|
Principal Investigator |
ASANO Yasuhisa Toyama Prefectural University, Biotechnology Research Center, Professor, 工学部, 教授 (00222589)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ヘイデン ブロナ ダブリン大学, 生化学科, 博士研究員
アーテミュイク ピーター シェフィールド大学, 分子生物, 博士研究員
ライス デイビット・W シェフィールド大学, 分子生物, 教授
KOMEDA Hidenobu Toyama Prefectural University, Biotechnology Research Center, Associate Professo, 工学部, 助手 (50285160)
KATO Yasuo Toyama Prefectural University, Biotechnology Research Center, Associate Professo, 工学部, 助教授 (20254237)
DAIRI Tohru Toyama Prefectural University, Biotechnology Research Center, Associate Professo (70264679)
|
|---|
| Project Period (FY) |
1996 – 1997
|
| Keywords | Amino Acid Dehydrogenase / Opine Dehydrogenase / X-ray Crystallographic Studies / Site Directed Mutagenesis / Molecular Cloning / Leucine Dehydrogenase / Phenylalanine Dehydrogenase / Methylaspartate Ammonia-Lyase |
|---|
| Research Abstract |
(1) Phenylalanine dehydrogenase (PheDH) : PheDHs from Bacillus sphaericus, B.badius, Sporosarcina ureae were purified from Escherichia coli transformants in large scales and sent to Prof.Engel (University College Dublin, Ireland) and Prof.Rice (University of Sheffield, U.K.) for X-ray crystallography. Based on informations of X-ray crystallography of glutamate dehydrogenase, chimeric enzymes of PheDH with altered substrate specificities were constructed.
(2) Methylaspartate ammonia-lyase (MAL) : MALs from Enterobacteria such as Enterobacter, Citrobacter, Proteus were purified to homogeneities and their enxymological properties were analyzed in detail. MAL from Citrobacter amalonaticus strain YG-1002 was digested with endo peptidases and N-terminal amino acid sequences were determined. Based on the information, DNA probes were synthesized. The gene for the enzyme was cloned form the genomic library of c.amalonaticus strain YG-1002 by PCR and Southern hybridization. The sequence of the gene was compared with that of a strict anaerobe Clostridium tetanomorphum. MAL was crystallized and sent to Prof.Rice for studies of X-ray crystallography.
(3) Opine dehydrogenase (ODH) : The gene for ODH from Arthrobacter sp.IC was cloned and expressed in E.coli. The enzyme was purified in a large scale and sent to Prof.Rice, and on its X-ray studies were started. The first structure of a (D,L) superfamily member, N-(1-D-Carboxylethyl)-L-norvaline dehydrogenase from Arthrobacter sp.strain 1C,has been solved to 1.8* resolution and the location of the bound coenzyme determined.
(4) Results were presented in some international academic meetings.
|
