Overexpression of Pseudomonas putida D-Hydantoinase Gene in Escherichia coli and Pseudomonas putida

• Main Authors: Yi-Lan Jih, 紀怡蘭
• Other Authors: Yi-Hsiung Tseng
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/51584723004793110287

碩士 === 國立中興大學 === 植物學系 === 83 === Pseudomonas putida D-hydantoinase gene ( dht ) was cloned into arious expression vectors to investigate the optimal condition for the production of D-hydantoinase ( DHT ) by Escherichia coli and Pseudomonas putida. The dht gene was prepared by polymerase chain reaction and cloned into expression vectors pET-21b, pBluescript II KS+, pSP72, pSP73 and pMMB66EH, respectively. This gene could be expressed in E.coli and P. putida under the control of T7 or tac promoter. The highest DHT activity was obtained by using pET21b expression vector and expressing the dht gene in E. coli. The SDS-PAGE analysis revealed that about 30 % of cellular protein were DHT. Optimization of the growth and induction conditions was performed in order to increase the enzyme production. The recombinant E. coli was found to produce maximal cell mass in the Basal medium containing glycerol as carbon source. The dht gene was expressed at high level in E. coli in the presence of 5 mM lactose as an inducer. After 3 to 5 h incubation in Basal medium and 5 h induction by lactose, the DHT activity reached 170 U/l which was about 17-fold higher than that of gene donor strain.The recombiant P. putida was also found to produce maximal cell mass in the Basal medium. The dht gene was expressed at high level in P. putida in the presence both 1mM IPTG and 0.2 % hydantoin as inducers. After 6 h incubation in Basal medium and 2 h induction by IPTG and hydantoin, the DHT activity reached 124 U/l.