重组葡萄糖脱氢酶的酶学性质及其偶联辅酶再生

doi:10.3969/j.issn.1673-1689.2014.09.003

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重组葡萄糖脱氢酶的酶学性质及其偶联辅酶再生
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                        10.3969/j.issn.1673-1689.2014.09.003
                    
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Enzymatic Characterization and Coenzyme Regeneration of a Recombinant Glucose 1-Dehydrogenase

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为解决生物催化氧化还原反应中辅酶循环问题，人工合成密码子优化后的嗜酸热源体Thermoplasma acidophilum葡萄糖脱氢酶基因Sygdh，于E. coli BL21（DE3）中表达，粗酶液经镍柱亲和层析获得纯化的重组葡萄糖脱氢酶SyGDH。SDS-PAGE显示相对分子质量为41.0 kDa。酶学性质分析表明：该酶的最适pH值为7.5，在pH 6.0~8.0稳定；最适反应温度为40 ℃，在 55 ℃以下稳定；最适条件下其比活性达4.5 U/mg；Zn2+对其有明显激活作用；该酶对NADP+的亲和力大于NAD+且对大多数有机溶剂有良好的耐受性；对D-葡萄糖的Km和Vmax值分别为28.2 mmol/L和6.5 U/mg。在葡萄糖脱氢酶与羰基还原酶偶联构建的NADPH辅酶循环体系中，以4-氯乙酰乙酸乙酯为底物，羰基还原酶催化产物4-氯-3-羟基丁酸乙酯的产率为99.0%，是未添加葡萄糖脱氢酶时产物产率的3.11倍，表明重组SyGDH具有为生物催化氧化还原反应提供辅酶NADPH再生的能力。

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A codon-optimized gene(named Sygdh) encoding a glucose 1-dehydrogenase of Thermoplasma acidophilum was cloned into the plasmid pET28a and functionally expressed in Escherichia coli. The expressed enzyme was purified to homogeneity using Ni-NTA affinity chromatography with an apparent molecular weight of 41.0 kDa by SDS-PAGE analysis and characterized using the model substrate of D-glucose. The purified recombinant SyGDH was 4.5 U/mg at pH 7.5 and 45 ℃, and highly stable at pH 6.0~8.0 and 55 ℃ or below. It was activated by Zn2+, but strongly inhibited by Cu2+ and not significantly affected by other metal ions tested and EDTA. The Km and Vmax of recombinant SyGDH towards D-glucose were 28.2 mmol/L and 6.5 U/mg, respectively. The results showed that SyGDH exhibits much higher affinity with NADP+ than NAD+ and remarkably resists to several organic solvents. Furthermore, two recombinant strains E. coli BL21/pET28a-S1 and E. coli BL21/pET28a-Sygdh, which include recombinant NADPH-dependent carbonyl reductase and recombinant SyGDH, respectively, were mixed and used for the asymmetric reduction of Ethyl 4-chloroacetoacetate to Ethyl-4-chloro-3-hydroxybutyrate in the coenzyme cycle system. The product yield reached 99.0%, which is 3.11 fold as high as that of reaction without adding recombinant SyGDH strain, confirming that the recombinant SyGDH could be used as NADPH regenerator in biocatalysis oxidation and reduction system.

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余涛,胡蝶,邬敏辰,汪俊卿,冯峰,顾颖.重组葡萄糖脱氢酶的酶学性质及其偶联辅酶再生[J].食品与生物技术学报,2014,33(9):910-916.

YU Tao, HU Die, WU Minchen, WANG Junqing, FENG Feng, GU Ying. Enzymatic Characterization and Coenzyme Regeneration of a Recombinant Glucose 1-Dehydrogenase[J]. Journal of Food Science and Biotechnology,2014,33(9):910-916.

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在线发布日期: 2014-10-19
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