| Project/Area Number |
12575019
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 海外学術 |
|---|
| Research Field |
応用微生物学・応用生物化学
|
|---|
| Research Institution | KYOTO UNIVERSITY |
|---|
Principal Investigator |
ESAKI Nobuyoshi Inst. Chem. Res., Kyoto Univ., Professor, 化学研究所, 教授 (50135597)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KURIHARA Tatsuo Inst. Chem. Res., Kyoto Univ., Instructor, 化学研究所, 助手 (70243087)
YOSHIMURA Tohru Inst. Chem. Res., Kyoto Univ., Assoc. Prof., 化学研究所, 助教授 (70182821)
|
|---|
| Project Period (FY) |
2000 – 2001
|
| Project Status |
Completed (Fiscal Year 2001)
|
| Budget Amount *help |
¥12,600,000 (Direct Cost: ¥12,600,000)
Fiscal Year 2001: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2000: ¥7,700,000 (Direct Cost: ¥7,700,000)
|
|---|
| Keywords | psychrophilic bacteria / psychrochilic microorganisms / psychrotrophic microorganisms / Acinetobacter / cold-active enzymes / lipolytic enzymes / lipases / esterases / Acinetobactor / 好冷酵素 / プロテアーゼ |
|---|
| Research Abstract |
1. A psychrotrophic bacterium, Acinetobacter sp. strain no. 6, was isolated from Siberian tundra soil. The bacterium grew at 4℃ but did not grow at 37℃. We found that the cells produce extracellular lipolytic enzymes acting on triglycerides when they are cultivated at low temperatures. When cultivation was carried out at 4℃ with the LB medium, 60% of soybean oil (initial concentration, 1% w/v) was degraded in 7 days, indicating that the bacterium is useful in removing fats from waste water under cold environments.
2. The gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters was cloned from Acinetobacter sp. strain no. 6. The gene encoded a protein consisting of 258 amino acid residues. The enzyme showed a high sequence similarity to β-ketoadipate enol-lactone hydrolase. An overproduction system for the enzyme was constructed, and the enzyme was purified to homogeneity and characterized. The enzyme was monomeric and efficiently catalyzed hydrolysis of fatty acid vinyl esters with a short-chain acyl group. The enzyme also catalyzed transesterification, for example, between vinyl propionate and propanol yielding propyl propionate at 4℃. Thus the enzyme is useful for the synthesis of esters by transesterification using vinyl esters as an acyl donor.
3. We cloned a gene coding for a cold-active esterase from Acinetobacter sp. strain no. 6. The gene encoded a protein of 301 amino acid residues. We found weak phylogenetic relationship of the enzyme to the EST group of the esterase/lipase family. It preferentially catalyzed the hydrolysis of esters with short-chain acyl group and showed lower activation energy than those of mesophilic enzymes.
4. A novel vector for Acinetobacter sp. strain no. 6 was constructed, and an efficient transformation method for the psychrotrophic bacterium was established.
|
