Title
Mannanase Man23 Mutants Library Construction Basing on a Novel Cell-free Protein Expression System
Author
Haiyan Zhou, Jie Yong, Han Gao, Tong Li, Hongshi Xiao, Yongyao Wu
Year
2016
Journal
Journal of the Science of Food and Agriculture
Abstract
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 the rational design and mutations were introduced on loops around catalytic region. Brevibacillus brevisB16 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 multi-volume spectrophotometer. The mutant Man1606 gained from this system is a sextet which has mutations of N146G, S147H, S156P, T157Y, Q206S and T249H simultaneously on loop 6, 8 and 10. Man1606 showed higher activity and stability than those of Man23. The optimal temperature of Man1606 rose by 5 °C (from 55 °C to 60 °C) and the optimal pH increased slightly but its range became broader. This experiment demonstrated B. brevis cell-free system shortens the expression time and is an efficient tool for mannanase engineering.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry, Agricultural and environmental