Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP).
To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Kmvalue. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.
a Key Laboratory for Bio-resources and Eco-Environment of the Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences , Sichuan University , Chengdu , P.R. China.|b Department of Biochemistry, School of Basic Medical Sciences , Hubei University of Medicine , Shiyan, Hubei , P.R. China.
Hong-Yan
HY
Zhao
a Key Laboratory for Bio-resources and Eco-Environment of the Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences , Sichuan University , Chengdu , P.R. China.
Li-Ying
LY
Wu
a Key Laboratory for Bio-resources and Eco-Environment of the Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences , Sichuan University , Chengdu , P.R. China.
Gang
G
Liu
a Key Laboratory for Bio-resources and Eco-Environment of the Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences , Sichuan University , Chengdu , P.R. China.
Hong
H
Feng
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013696:Temperature
D001426:Bacterial Proteins; D010450:Endopeptidases; C087822:alkaline protease
Bacillus pumilus; alkaline protease; cold activity; site-directed mutagenesis; thermostability