Mannanase Man23 mutant library construction based on a novel cell-free protein expression system.
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.


CONCLUSIONS
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.
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
Haiyan
H
Zhou
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
Jie
J
Yong
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
Han
H
Gao
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
Tong
T
Li
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
Hongshi
H
Xiao
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
Yongyao
Y
Wu
D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D044902:beta-Mannosidase
Brevibacillus brevis; cell-free protein synthesis; mannanase; rational design