Open AccessB 12 ‐independent glycerol dehydratase and its reactivase from Clostridia butyricum : Optimizing cloning by uniform design logic
Author(s) -
Jiang Wei,
Wang Shizhen,
Yang Zhongli,
Fang Baishan
Publication year - 2015
Publication title -
engineering in life sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 57
eISSN - 1618-2863
pISSN - 1618-0240
DOI - 10.1002/elsc.201400217
Subject(s) - clostridia , clostridium butyricum , cloning (programming) , glycerol , computational biology , biology , computer science , biochemistry , genetics , bacteria , programming language
Glycerol dehydratase (GDHt) is a key and rate‐limiting enzyme in the biological process of producing 1, 3‐propanediol from glycerol. Using a uniform design to get the optimal system of amplifying dha B 1 B 2 by PCR, a new GDHt and its reactivase were obtained from Clostridia butyricum . SDS‐PAGE showed that the relative molecular weights of the dha B 1 B 2 expression products were about 88 and 35 kDa, which confirmed the coexpression of dha B 1 B 2 in Escherichia coli BL21. The best inducing conditions, 0.1 mM IPTG, 28°C, and 11‐h induction time, were obtained by the uniform design and regression analysis. By anaerobic inducible expression in E. coli BL21, the activity of GDHt activity was six times higher than in C. butyricum (2.37 U/mL), and its specific activity was 36.3 U/mg. These results and methods would be useful for effective PCR amplification on a long gene fragment, high‐efficiency expression of the recombinant enzyme, and enzymatic production of 1, 3‐propanediol.