Enhanced thermostability of keratinase by computational design and empirical mutation
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Keratinases are proteolytic enzymes capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. To enhance the thermostability of Bacillus licheniformis BBE11-1 keratinase, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Use of the algorithm in combination with molecular modification of homologous subtilisin allowed the introduction of four amino acid substitutions (N122Y, N217S, A193P, N160C) into the enzyme by site-directed mutagenesis, and the mutant genes were expressed in Bacillus subtilis WB600. The quadruple mutant displayed synergistic or additive effects with an 8.6-fold increase in the t1/2 value at 60 °C. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase. These results provide further insight into the thermostability of keratinase and suggest further potential industrial applications.
KeywordsKeratinase PoPMuSiC Thermostability Site-directed mutagenesis Bacillus licheniformis
This project was financially supported by the National Natural Science Foundation of China (No. 30900013), the National High Technology Research and Development Program of China (863 Program, 2011AA100905), the National Key Technology R&D Program in the 12th Five year Plan of China (2011BAK10B03), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1135), and the National High Technology Research and Development Program of China (863 Program, 2011AA100901). We are also grateful to Professor Byong Lee for his helpful discussion and revision.
- 9.Gupta R, Sharma R, Beg QK (2012) Revisiting microbial keratinases: next generation proteases for sustainable biotechnology. Crit Rev Biotechnol. doi:10.3109/07388551.2012.685051
- 13.Liu B, Zhang J, Li B, Liao X, Du G, Chen J (2012) Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing. World J Microbiol Biotechnol. doi:10.1007/s11274-012-1237-5