Optimization of the Host/Vector System and Culture Conditions for Production of Penicillin Acylase in Escherichia coli

• Main Authors: BAO-YUAN KUO, 郭寶元
• Other Authors: C. PERRY CHOU
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/41149160282513710681

碩士 === 逢甲大學 === 化學工程學系 === 88 === The effect of SecB chaperone on production of periplasmic penicillin acylase in Escherichia coli was investigated. It appears that formation of PAC required the function of SecB chaperone and the amount of SecB required was at a basal level. The secB mutant was defective in production of PAC, and the impairment could be complemented by extrachromosomally supplementing SecB in trans. The function of SecB chaperone usually resulted in an increase in the amount of PAC precursors without enhancing PAC activity. In addition,most of the PAC precursors were located in the periplasm, suggesting that formation of active PAC was likely limited by periplasmic processing steps. Culture performance for the production of penicillin acylase in a bioreactor was investigated using HB101 or ATCC11105 as the host and pCLL2902, pCLL3201, or pTrcKnPAC2902 as the expression plasmid. We observed that the production of PAC by HB101 harboring pCLL3201 was, similar to ATCC11105, induced by phenyl acetic acid (PAA) and catabolically repressed by glucose, whereas the production of PAC by HB101 harboring pCLL2902 did not require PAA for induction and was not repressed by glucose. PAC activity of HB101 harboring pCLL2902 was significantly higher than that of HB101 harboring pCLL3201. There was no significant effect of host or carbon source on the production of PAC using pCLL2902.The production of PAC by HB101 harboring pTrcKnPAC2902, in which the pac gene expression was controlled by the trc promoter system, was about the same as that by HB101 harboring pCLL2902, when the culture was appropriately induced with isopropyl β-D-thiogalactopyranoside (IPTG). Therefore, the use of both pCLL2902 and pTrcKnPAC2902 may be feasible for industrial applications. However, optimization of IPTG induction for HB101 harboring pTrcKnPAC2902 might be required, since formation of inclusion bodies tends to limit the production of PAC in some cas