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