Gene regulation and protein characterization of collagenase from Vibrio parahaemolyticus
| Autor: | Huan-Yu Lin, 林奐妤 |
|---|---|
| Rok vydání: | 2002 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 90 Vibrio parahaemolyticus has been identified as a cause of acute gastroenteritis in 1950. It is an important diarrhoeal agent associated with seafood poisoning in Taiwan, Japan and many coastal areas. The vpc mRNA was observed in RNA harvested from cells grown under iron-limiting conditions. Under iron liming condition, the collagenase was detected by SDS-PAGE, Western blot, and 2D electrophoresis, and expressed from extracellular of V. parahaemolyticus. A 2,541-kb vpc gene fragment was ligated into pET-21a(+) in the same orientation as the T7 promoter, and the recombinant plasmid, pET-VPC, was transformed into E. coli BL21-CodonPlus (DE3)-RIL. The resulting transformant was grown in LB medium until the OD600 reached 0.4 ~ 0.6 and then induced by adding 0.1 mM IPTG. After 4 hr induction, the highest activity (30.29 U/mg) was detected in the culture supernatant. VPC-82 and VPC-63 was purified by ion-exchange chromatography Hiprep Q, gel filtration Sephacryl S-200, and strong ion-exchange chromatography Resource Q. The specific activities of the VPC-82 and VPC-63 against azocoll were 417.1 and 82.8 U/mg of protein respectively. Isoelectric focusing of the purified VPC-82 and VPC-63 revealed an isoelectric point of pH 4.62 and 4.72 respectively. The optimal temperature for VPC-82 and VPC-63 were both 40 ℃, and the optimal pH were 7.8. Type I, II, III, and IV collagen were hydrolyzed by VPC-82 and VPC-63. VPC-82 and VPC-63 have high activity against insoluble type I collagen and gelatin. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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