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

doi:10.13560/j.cnki.biotech.bull.1985.2018-0941

摘要/Abstract

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。

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

Abstract: 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.

Key words: FAD-dependent glucose dehydrogenase, codon optimization, recombinant Pichia pastoris, enzymatic characterization

董聪, 高庆华, 王玥, 罗同阳. 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[J]. 生物技术通报, 2019, 35(7): 114-120.

DONG Cong, GAO Qing-hua, WANG Yue, LUO Tong-yang. Expression and Enzymatic Characterization of Codon-optimized FAD-dependent Glucose Dehydrogenase in Pichia pastoris[J]. Biotechnology Bulletin, 2019, 35(7): 114-120.

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