Premium
Mannanase Man23 mutant library construction based on a novel cell‐free protein expression system
Author(s) -
Zhou Haiyan,
Yong Jie,
Gao Han,
Li Tong,
Xiao Hongshi,
Wu Yongyao
Publication year - 2017
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.8029
Subject(s) - mutant , directed evolution , enzyme , mutation , rational design , protein engineering , chemistry , biochemistry , biology , microbiology and biotechnology , genetics , gene
BACKGROUND Mannanases are important enzymes which are widely used as a tool in agriculture and food industries. To improve the performance of mannanase Man23, a mutant library was created with rational design, and mutations were introduced on loops around the catalytic region. The Brevibacillus brevis B16 cell‐free system which was created in this experiment provided the ability to express the mutant library efficiently. The activities of mutants were measured with a multi‐volume spectrophotometer. RESULTS The mutant Man1606 gained from this system is a sextet which has mutations of N146G , S147H , S156P , T157Y , Q206S and T249H simultaneously on loops 6, 8 and 10. Man1606 showed higher activity and stability than Man23. The optimal temperature of Man1606 rose by 5 °C (from 55 to 60 °C) and the optimal pH increased slightly but its range became broader. CONCLUSION This experiment demonstrated the B. brevis cell‐free system shortens the expression time and is an efficient tool for mannanase engineering. © 2016 Society of Chemical Industry