Biotechnology Bulletin ›› 2017, Vol. 33 ›› Issue (8): 186-191.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0168

• Research Report • Previous Articles     Next Articles

Recombinant Expression of NADPH-Dependent Mannitol Dehydrogenase and Transformation Conditions of Mannitol

FENG Zhi-mei, ZHAO Ya-tong, LIU Ye-xue, LU Fu-ping, LI Yu   

  1. Key Laboratory of Industrial Microbiology,Ministry of Education,College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457
  • Received:2017-03-06 Online:2017-08-01 Published:2017-08-01

Abstract: This work is to analyze the heterogeneous expression of NADPH-dependent mannitol dehydrogenase and the transformation of fructose for laying a foundation of constructing mannitol synthetic way. The recombinant strain BL21(DE3)/pET28a-mdh of expressing mannitol dehydrogenase was constructed and induced,and the target protein was purified with His-tag. Mannitol production from mannitol dehydrogenase transforming fructose was analyzed by HPLC. As results,the recombinant strain was constructed successfully and the mannitol dehydrogenase was purified. The activity of the purified mannitol dehydrogenase was 270 U/mL. The conditions of fructose transforming to mannitol by purified mannitol dehydrogenase were optimized,and the optimal conditions were as follows:300 g/L substrate fructose,pH5.8,9 mmol/L NADPH and incubation at 40℃. Under the optimized conditions,the transformation rate of mannitol reached 97.4%. In conclusion,the NADPH-dependent mannitol dehydrogenase was expressed successfully in Escherichia coli,providing the basis for studying the metabolism regulation of mannitol synthesis in E. coli.

Key words: mannitol dehydrogenase, NADPH, mannitol, Escherichia coli, purification