枯草芽孢杆菌头孢菌素C脱乙酰基酯酶在大肠杆菌中的诱导及组成型表达

摘要

本研究构建了8种诱导型质粒和16种组成型质粒,试图实现枯草芽孢杆菌头孢菌素C脱乙酰基酯酶(CAH)基因在大肠杆菌中的高效异源表达.经过筛选,最终选择带有pUC19上的起始位点、SIL3基因、trp启动子以及间隔序列为B1的组成型表达质粒pB1-SIL3-K为最佳表达质粒.将PB1-SIL3-K转入Escherichia coli DH5α,重组菌在37 ℃发酵培养24 h,其OD值达到5.4,酶活达到了138.61 U/mL.进一步通过培养基组成、培养条件、放大过程等优化,在7 L发酵罐上,CAH最高酶活达到1 340 U/mL,为目前国内外文献报道最高水平.%In this work,8 inducible plasmids and 16 constitutive plasmids were constructed to fulfil the high-level heterogenous expression of cephalosporin-C deacetylase (CAH) gene in Escherichia coli.A constitutive expression plasmid pB1-SIL3-K,with a replication origin derived from pUC19 and B1 sequence between the SIL3 gene and trp promoter,was selected as the most efficient expression plasmid.The OD and CAH activity expressed by E.coli DH5α/pB1-SIL3-K attained 5.4 and 138.61 U/mL when cultivating at 37 ℃ for 24 h.Furthermore, 1 340 U/mL of CAH activity was obtained in a 7-L fermenter by optimizing the medium components,culture conditions and scale-up process,which is the highest CAH activity reported to date.