摘要
头孢菌素C(CPC)酰化酶可用于一步酶法合成7-氨基头孢烷酸(7-ACA)。对来自Pseudomonas sp.Strain SE83的高活性CPC酰化酶突变基因进行基于毕赤酵母偏好密码子优化,获得基因SECA。选取毕赤酵母内源信号肽DSE4,构建表达菌G/DSECA,首次在毕赤酵母中分泌了CPC酰化酶。与酿酒酵母α-交配因子前导肽(α-factor)相比,DSE4介导SECA的分泌表达效果更优,其介导SECA表达的胞内、外单位体积酶活分别比α-factor高65%和44%。分别以DSE4和α-factor为信号肽构建β-半乳糖苷酶表达菌G/DSEL与G/MFL,进一步考察DSE4介导异源蛋白的分泌效果,G/DSEL的胞内外单位体积酶活也均高于G/MFL。
Cephalosporin C （CPC） acylase is the key enzyme for the production of 7-amino- cephalosporanic acid （7-ACA） in one-step enzyme process. The improved CPC acylase mutant gene from Pseudomonas sp. Strain SE83 was codon optimized according to Pichia pastoris codon bias and named as SECA. An endogenous signal sequence DSE4 was used as the signal peptide and mediated the secretion of SECA in recombinant P. pastoris strain G/DSECA for the first time. In comparion with the control strain G/MFCA harbouring the similar expression cassette except that the signal sequence DSE4 was replaced by α-factor, 65% and 44% higher CPC acylase activity in intracellular and extracellular were obtained in G/ DSECA,respectively. Recombinant strains G/DSEL and G/MFL containing ffgalactosidase （lacZ） as the report gene were constructed to further investigate the secretion efficiency of DSE4, and G/DSEL showed higher intracellular and extracellular β-galactosidase activity than G/MFL did.
出处
《华东理工大学学报（自然科学版）》
CAS
CSCD
北大核心
2015年第4期449-454,共6页
Journal of East China University of Science and Technology
基金
教育部新世纪优秀人才支持计划(NCET-11-0639)
