基于密码子优化的FAD依赖葡萄糖脱氢酶在毕赤酵母中的高效表达及酶学性质  

Expression and Enzymatic Characterization of Codon-optimized FAD-dependent Glucose Dehydrogenase in Pichia pastoris

作　　者：董聪 高庆华 王玥 罗同阳 DONG Cong;GAO Qing-hua;WANG Yue;LUO Tong-yang(Institute of Microbiology,Hebei Academy of Sciences,Baoding 071051)

机构地区：[1]河北省科学院微生物研究所

出　　处：《生物技术通报》2019年第7期114-120,共7页Biotechnology Bulletin

基　　金：河北省科学院科技计划项目(17202)

摘　　要：旨在获得表达量高的FAD依赖的葡萄糖脱氢酶。通过FAD依赖的葡萄糖脱氢酶密码子优化,人工合成基因片段,构建重组表达载体pMD-GDH,转化毕赤酵母X33菌株后利用甲醇诱导培养实现分泌表达。结果显示,经试管水平筛选阳性转化子获得一株酶活高且稳定的重组菌株,在10 L发酵罐培养时经过136 h诱导培养,酶活达到257 600 U/L。酶学性质分析表明,以葡萄糖为底物时最适温度和pH分别为55℃和7.0,在50℃下处理150 min仍有70%的活性,在pH 4-7范围内,37℃保温4 h,FAD-GDH仍能保持50%以上的活性。金属离子Cu2+对酶活抑制作用比较大。FAD-GDH的底物专一性较好,以葡萄糖为最适底物。经毕赤酵母密码子偏好性优化实现了FAD依赖的葡萄糖脱氢酶在毕赤酵母中的高效表达,为应用于血糖检测提供理论依据。The objective of this work is to obtain high-yield FAD-dependent glucose dehydrogenase(FAD-GDH).First,by optimizing the codons of FAD-GDH gene according to the codon preference of Pichia pastoris,the gene fragment was artificially synthesized,then the recombinant vector pMD-GDH containing FAD-GDH e gene was constructed and transferred into P.pastoris(X33),and the secretory expression by methanol induction was achieved.Results showed that a stable recombinant strain with high enzymatic activity was obtained by screening positive transformants at test-tube level.In the 10 L fermenter after 136 h induction culture,enzyme activity reached 257 600 U/L.The analysis of enzymatic characterization demonstrated that the optimal pH and temperature while using glucose as substrate were 7.0 and 55℃,respectively.The initial enzyme activity still remained 70%after 150 min treatment at 50℃.In the range of pH 4-7,FAD-GDH still retained over 50%activity after incubated at 37℃for 4 h.Cu2+presented relatively large inhibition to enzyme activity.FAD-GDH harbored a relative high substrate specificity and D-glucose was the optimal substrate.The efficient expression of FAD-GDH in P.pastoris is achieved by optimizing the codon preference of P.pastoris,which provides a theoretical basis for the detection of blood glucose.

关 键 词：FAD依赖的葡萄糖脱氢酶 密码子优化 重组毕赤酵母 酶学性质 

分 类 号：TS2[轻工技术与工程—食品科学与工程]