Immobilized Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Biotransformation of Penicillin G into Deacetoxycephalosporin G

• Main Authors: Hsieh Chuan-Cheng, 謝全城
• Other Authors: Chia-Li Wei
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/55900120779355654337

碩士 === 國立嘉義大學 === 生化科技學系研究所 === 101 === Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), which is an intermediate for cephalosporin synthesis. In this paper, in order to reuse and increase the stability of DAOCS, the chitin binding domain tagged DAOCS (CB-DAOCS) gene was expressed in E. coli cells and used for purification and immobilization onto chitin beads at the same time. The optimum binding temperature (30℃), time (1 hour) and cell extracts concentration (1 mg/ml) of CB-DAOCS immobilized onto chitin beads were firstly examined. The optimum concentrations of cofactors (2 mM ascorbate, 0.5 mM FeSO4 and 0.5 mM α-ketoglutarate) were also examined. However, the recycling analyses showed that the residual activities of the 2nd-cycled CB16 (wild-type) and CB3 (thermal stability mutant N268K) CB-DAOCSs were 17% and 24%. Since the decreased activities might be due to the iron oxidation, chelators or other chemical compounds, such as EDTA, citrate, triton X-100, tween 20, n-decane, urea and ATP were used to remove iron oxide. However, none of them showed improved activity in the recycling tests. Finally, the immobilized CB-DAOCSs were reacted with cell extracts containing corresponding CB-DAOCS at 30℃for 1 hour during each activity assays. The residual activities of CB16, CB2 (thermal stability mutant H244R), CB3, CB59 (C155Y/Y184H/V275I/C281Y) and CB59-5 (C155Y/Y184H/V275I/ C281Y/I305M) showed 97-67%, 65-55%, 82-70%, 96-75% and 97-63% during 2-4, 2-5, 2-5, 2-6 and 2-6 cycles, respectively. This study offers a possible way for bioproduction of 7-ADCA by DAOCS immobilized on chitin beads.