雅致放射毛霉羧肽酶Y的基因克隆、表达与酶学性质研究

Cloning, Expression, and Characterization of Carboxypeptidase Y from Actinomucor elegans PEP001

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（proCPY）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proCPY，产量为151.20±10.20 mg/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:

(CPY) was isolated and purified from Actinomucor elegans PEP001 after in vitro activation, and was then cloned and characterized. A strain of fermenting fungi, screened and identified from fermented bean curd, was named Actinomucor elegans PEP001. A partial length of the CPY gene was cloned using degenerate primers, which were designed based on conserved sequence of homologous fungal CPY, and the end sequence of 5’ and 3’ were obtained by rapid amplification of cDNA ends (RACE). The full length of the cloned CPY gene was 1557 bp, encoding 518 amino acids. The recombinant proCPY was expressed in Escherichia coli Rosetta (DE3) as inclusion bodies, and the proCPY was successfully expressed in Pichia pastoris GS115 as well, with a yield of 151.20±10.20 mg/L. Pure mature CPY was obtained using SDS-PAGE after in vitro activation. The optimal reaction pH and temperature of mature CPY was found to be 6.0 and 45 ℃, respectively. Mature CPY had high stability at 40 ℃, but was inactivated at 60 ℃. It was found to be stable at pH 4.0~7.0. Carboxypeptidase Y from A. elegans PEP001 was for the first time successfully cloned, expressed, and characterized. The results of this study lay the foundation for future research.