雅致放射毛霉羧肽酶Y的基因克

发布时间：2018-06-14 06:37

  本文选题：雅致放射毛霉 + 羧肽酶Y　； 参考：《现代食品科技》2017年01期

【摘要】：本研究从雅致放射毛霉中克隆、表达和纯化羧肽酶Y(CPY),体外激活后检测其酶学性质。本文筛选并鉴定了一株腐乳发酵菌株,命名为雅致放射毛霉PEP001(Actinomucor elegans PEP001)。以相近种族来源的真菌羧肽酶Y保守序列设计简并引物调取部分目的基因序列,利用末端扩增技术(RACE)分别获得3’与5’端基因全长,克隆获得羧肽酶Y基因,全长为1557 bp,编码518 aa。将其酶原(pro CPY)在大肠杆菌Rosetta中重组表达,产物为包涵体;在毕赤酵母GS115中成功分泌表达pro CPY,产量为151.20±10.20mg/L。通过体外激活与纯化,获得电泳纯的成熟羧肽酶Y。成熟羧肽酶Y最适pH为6.0,最适温度为45℃,在40℃下有较高稳定性,在60℃下很快失活,在pH 4.0~7.0下都有较高的稳定性。本文中雅致放射毛霉PEP001羧肽酶Y为首次报道,为进一步研究其应用打下一定基础。
[Abstract]:In this study, we cloned, expressed and purified carboxypeptidase Yapeptidase from Mucor radiorrhoeae, and detected its enzymatic properties after activation in vitro. In this paper, a sufu fermentation strain was screened and identified, named PEP001, Actinomucor elegans PEP001. A degenerate primer was designed with conserved sequence of fungal carboxypeptidase Y from similar ethnic origin to obtain partial target gene sequence. The complete length of 3 'and 5' terminal genes were obtained by terminal amplification technique, and the carboxypeptidase Y gene was cloned. The total length is 1557 BP, encoding 518 aa. The enzyme was expressed in Escherichia coli Rosetta and secreted successfully in Pichia pastoris GS115 with a yield of 151.20 卤10.20 mg / L. The mature carboxypeptidase Y. was obtained by in vitro activation and purification. The optimum pH of mature carboxypeptidase Y is 6.0, the optimum temperature is 45 鈩，

本文编号：2016486