Enhanced thermostability of keratinase by computational design and empirical mutation

Fermentation, Cell Culture and Bioengineering


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.


Keratinase 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.


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Copyright information

© Society for Industrial Microbiology and Biotechnology 2013

Authors and Affiliations

  1. 1.Key Laboratory of Industrial Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  2. 2.National Engineering Laboratory for Cereal Fermentation TechnologyJiangnan UniversityWuxiChina
  3. 3.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  4. 4.School of BiotechnologyJiangnan UniversityWuxiChina

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