Gene cloning and characterization of a solvent-resistant glucose dehydrogenase from Bacillus sp. YX-1

Abstract:

Abstract:［Objective］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．［Methods］ 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．［Results］The glucose dehydrogenase gene contains an open reading frame of 786 bp encoding 261 amino acids．The maximum activity was observed at 45℃ 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 + ．［Conclusion］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．

Key words:Keywords: glucose dehydrogenase，coenzyme regeneration，organic solvent-tolerant，substrate specificity