| Summary: | 碩士 === 逢甲大學 === 化學工程學系 === 89 === To produce penicillin acylase (PAC) by recombinant DNA technology in Escherichia coli (E. coli), the overproduction was often limited by periplasmic processing and inclusion bodies were formed at a large amount in the periplasm. This raises an important issue that, for the overproduction of recombinant proteins, not only the transcriptional and/or translational efficiency has to be increased but also a ‘balanced’ protein synthesis flux throughout various gene expression (i.e., transcription, translation, and posttranslational processing) and folding steps should be properly maintained to avoid the accumulation of polypeptide intermediates. In this study, we demonstrated the extracellular production of penicillin acylase (PAC) by coexpression of the brp gene encoding bacteriocin release protein (BRP) and the pac gene. The performance for the production and release of PAC was optimized by taking several culture parameters, including host, inducer concentration, and induction timing for brp expression, into consideration. The effect of PAC release on inclusion body formation was also investigated. It was observed that the amount of inclusion bodies was significantly increased by brp expression. The formation of inclusion bodies was not caused by over-accumulation of active PAC. In the second part of this study, we demonstrated the enhancement of recombinant penicillin acylase (PAC) production in E. coli by increasing the intracellular concentration of the periplasmic protease DegP. The amount of these periplasmic inclusion bodies was significantly reduced and PAC activity was significantly increased upon coexpression of DegP. The results suggest that DegP could in vivo assist the periplasmic processing though the enzyme was shown to be not absolutely required for the formation of active PAC in E. coli. The formation of PAC inclusion bodies should be primarily caused by limitation of the proteolysis on periplasmic PAC precursors. In addition, the steps limiting the production of PAC were identified.
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