High-level expression of recombinant glucose dehydrogenase and its application in NADPH regeneration.
Two glucose dehydrogenase (E.C. 1.1.1.47) genes, gdh223 and gdh151, were cloned from Bacillus megaterium AS1.223 and AS1.151, and were inserted into pQE30 to construct the expression vectors, pQE30-gdh223 and pQE30-gdh151, respectively. The transformant Escherichia coli M15 with pQE30-gdh223 gave a much higher glucose dehydrogenase activity than that with the plasmid pQE30-gdh151. Thus it was used to optimize the expression of glucose dehydrogenase. An proximately tenfold increase in GDH activity was achieved by the optimization of culture and induction conditions, and the highest productivity of glucose dehydrogenase (58.7 U/ml) was attained. The recombinant glucose dehydrogenase produced by E. coli M15 (pQE30-gdh223) was then used to regenerate NADPH. NADPH was efficiently regenerated in vivo and in vitro when 0.1 M glucose was supplemented concomitantly in the reaction system. Finally, this coenzyme-regenerating system was coupled with a NADPH-dependent bioreduction for efficient synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoate.
Xu
Zhinan
Z
Department of Chemical Engineering and Bioengineering, Institute of Bioengineering, Zhejiang University, Hangzhou, 310027, Zhejiang Province, PR China. znxu@zju.edu.cn
Jing
Keju
K
Liu
Ying
Y
Cen
Peilin
P
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005948:Glucose Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D009249:NADP; D005948:Glucose Dehydrogenases