摘要
核糖核酸酶Ba(barnase)是一种产自解淀粉芽孢杆菌的胞外核糖核酸酶,具有强毒性,能降解RNA,在农业、医疗、制药等领域有广泛的应用前景.利用重组表达barnase的大肠杆菌为研究对象,以期使用kil基因改善分泌状况,并实现大肠杆菌的高密度培养以及蛋白的高产表达.通过把kil-Km分泌盒克隆入质粒载体pET-32a(+)-barnase-barstar使barnase更加容易分泌与纯化,并使用Riesenberg培养基对重组大肠杆菌发酵生产barnase的不同葡萄糖浓度底物反馈控制补料流加策略进行了研究.初步确定1.0 g/L葡萄糖底物反馈流加时,获得的barnase酶活最高,为7.14 kU/mL,是文献报道的1.88倍,此时细胞干质量浓度为22.93 g/L,其中细胞间质的酶活达到3.53 kU/mL.为今后的发酵bar-nase研究提供了参考.
RNase Ba（barnase） is a ribonuclease from Bacillus amyloliquefaciens with strong toxicity for degrading intracellular RNA.Barnase has broad application prospects in agriculture,clinical medicine,pharmaceutical,etc.In this study,the recombinant Escherichia coli harboring plasmid pET-32a（＋）-barnase-barstar-kil was used to fermentation for barnase.In the experiment,kil-Km secretion cassette was inserted for reducing the costs in downstream separation.Then the fermentation strategy,feedback control with different concentrations of glucose in fed-batch,was used to accumulate high production of barnase.When the concentration of glucose in medium was 1.0 g/L,the highest activity of barnase reached 7.14 kU/mL,which was 1.88 times that reported by the literatures,and the maximum cell dry mass concentration was 22.93 g/L,barnase activity in periplasmic increased to 3.53 kU/mL.This work gives a support to fermentation of barnase later.
出处
《厦门大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第1期88-92,共5页
Journal of Xiamen University：Natural Science
基金
福建省高等学校新世纪优秀人才支持计划
