Journal Article

High-level expression of recombinant glucose dehydrogenase and its application in NADPH regeneration

Liu, Ying, Jing, Keju, Cen, Peilin, Xu, Zhinan

2007

Overview

Abstract 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.

• Publisher: Oxford University Press,Springer
• Subject: Amino Acid Sequence / Bacillus megaterium / Bacillus megaterium - enzymology / Bacillus megaterium - genetics / Biological and medical sciences / Biotechnology / Cloning, Molecular / Dehydrogenase / Dehydrogenases / E coli / Escherichia coli / Escherichia coli - genetics / Fundamental and applied biological sciences. Psychology / Gene expression / Genetic Engineering / Glucose / Glucose Dehydrogenases - biosynthesis / Glucose Dehydrogenases - chemistry / Glucose Dehydrogenases - genetics / Molecular Sequence Data / NADP - chemistry / Recombinant Proteins - biosynthesis / Recombinant Proteins - chemistry / Recombinant Proteins - genetics / Sequence Homology, Amino Acid / Studies