Zhang Botao, Zhang Rongzhen, Wang Lei, Xu Yan
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Wei Sheng Wu Xue Bao. 2013 Jun 4;53(6):561-8.
A gene encoding solvent-resistant glucose dehydrogenase was cloned from Bacillus sp. YX-1 and expressed in Escherichia coli. The recombinant enzyme was then characterized.
The glucose dehydrogenase gene was amplified from Bacillus sp. YX-1 genome according to its conserved sequences in Bacillus sp. The recombinant enzyme was over-expressed in E. coli and purified by HisTrap HP affinity chromatography. The purified enzyme were characterized.
The glucose dehydrogenase gene contains an open reading frame of 786 bp encoding 261 amino acids. The maximum activity was observed at 45 degrees C and pH 8.0. The recombinant enzyme was highly resistant to several organic solvents. More than 90% of the activity was maintained when the enzyme was incubated in 50% cyclohexane, octane, decane at home temperature for 1 h. In addition, the enzyme displayed broad substrate spectrum and has catalytic activity for several sugars to afford reduced coenzymes. It exhibits similar capability to regenerate either NADH or NADPH with specific activity of 8.37 U/mg and 8.62 U/mg for NAD+ and NADP+.
The organic solvent-tolerant glucose dehydrogenase was explored successfully on the basis of bioinformatics analysis. The work supplied a new biocatalyst for the cofactor-regeneration during the reaction in organic phases catalyzed by oxidoreductases.
从芽孢杆菌属YX-1中克隆出编码耐溶剂葡萄糖脱氢酶的基因，并在大肠杆菌中表达，随后对重组酶进行表征。
根据芽孢杆菌属中的保守序列，从芽孢杆菌属YX-1基因组中扩增葡萄糖脱氢酶基因。该重组酶在大肠杆菌中过量表达，并通过HisTrap HP亲和层析进行纯化，对纯化后的酶进行表征。
葡萄糖脱氢酶基因包含一个786 bp的开放阅读框，编码261个氨基酸。在45℃和pH 8.0时观察到最大活性。该重组酶对几种有机溶剂具有高度抗性。当酶在室温下于50%的环己烷、辛烷、癸烷中孵育1小时时，超过90%的活性得以保持。此外，该酶具有广泛的底物谱，对几种糖类具有催化活性以产生还原型辅酶。它在再生NADH或NADPH方面表现出相似的能力，对NAD+和NADP+的比活性分别为8.37 U/mg和8.62 U/mg。
基于生物信息学分析成功探索出了耐有机溶剂的葡萄糖脱氢酶。该工作为氧化还原酶催化的有机相反应中的辅因子再生提供了一种新的生物催化剂。
