Enhanced thermostability of keratinase by computational design and empirical mutation

Fermentation, Cell Culture and Bioengineering

Abstract

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.

Keywords

Keratinase PoPMuSiC Thermostability Site-directed mutagenesis Bacillus licheniformis 

Notes

Acknowledgments

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.

References

  1. 1.
    Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201PubMedCrossRefGoogle Scholar
  2. 2.
    Barzegar A, Moosavi-Movahedi AA, Pedersen JZ, Miroliaei M (2009) Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: stability-determining roles of proline residues and loop conformations. Enzyme Microbiol Technol 45:73–79CrossRefGoogle Scholar
  3. 3.
    Brandelli A, Daroit DJ, Riffel A (2010) Biochemical features of microbial keratinases and their production and applications. Appl Microbiol Biotechnol 85:1735–1750PubMedCrossRefGoogle Scholar
  4. 4.
    Brayan PN, Rollence ML, Pantoliano MW, Wood J, Finzel BC, Gilliland GL, Howard AJ, Poulos TL (2004) Proteases of enhanced stability: characteization of a thermostable variant of subtilisin. Proteins 1:326–334CrossRefGoogle Scholar
  5. 5.
    Cabrita LD, Gilis D, Robertson AL, Dehouck Y, Rooman M, Bottomley SP (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci 16:2360–2367PubMedCrossRefGoogle Scholar
  6. 6.
    Chan MK, Mukund S, Kletzin A, Adams M, Rees DC (1995) Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase. Science 267:1463PubMedCrossRefGoogle Scholar
  7. 7.
    Dehouck Y, Kwasigroch JM, Gilis D, Rooman M (2011) PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics 12:151PubMedCrossRefGoogle Scholar
  8. 8.
    Fisher SJ, Blakeley MP, Cianci M, McSweeney S, Helliwell JR (2012) Protonation-state determination in proteins using high-resolution X-ray crystallography: effects of resolution and completeness. Acta Crystallogr D 68:800–809PubMedCrossRefGoogle Scholar
  9. 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
  10. 10.
    Gushterova A, Vasileva-Tonkova E, Dimova E, Nedkov P, Haertle T (2005) Keratinase production by newly isolated antarctic actinomycete strains. World J Microbiol Biotechnol 21:831–834CrossRefGoogle Scholar
  11. 11.
    Jaouadi B, Aghajari N, Haser R, Bejar S (2010) Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. Biochimie 92:360–369PubMedCrossRefGoogle Scholar
  12. 12.
    Jaouadi B, Ellouz-Chaabouni S, Rhimi M, Bejar S (2008) Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency. Biochimie 90:1291–1305PubMedCrossRefGoogle Scholar
  13. 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
  14. 14.
    Pace CN, Fu H, Fryar KL, Landua J, Trevino SR, Shirley BA, Hendricks MM, Iimura S, Gajiwala K, Scholtz JM, Grimsley GR (2011) Contribution of hydrophobic interactions to protein stability. J Mol Biol 408:514–528PubMedCrossRefGoogle Scholar
  15. 15.
    Pantoliano MW, Ladner RC, Bryan PN, Rollence ML, Wood JF, Poulos TL (1987) Protein engineering of subtilisin BPN′: enhanced stabilization through the introduction of two cysteines to form a disulfide bond. Biochemistry 26:2077–2082PubMedCrossRefGoogle Scholar
  16. 16.
    Pantoliano MW, Whitlow M, Wood JF, Dodd SW, Hardman KD, Rollence ML, Bryan PN (1989) Large increases in general stability for subtilisin BPN′ through incremental changes in the free energy of unfolding. Biochemistry 28:7205–7213PubMedCrossRefGoogle Scholar
  17. 17.
    Siezen RJ, Leunissen JA (1997) Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6:501–523PubMedCrossRefGoogle Scholar
  18. 18.
    Takagi H, Hirai K, Wada M, Nakamori S (2000) Enhanced thermostability of the single-Cys mutant subtilisin E under oxidizing conditions. J Biochem 128:585–589PubMedCrossRefGoogle Scholar
  19. 19.
    Tina KG, Bhadra R, Srinivasan N (2007) PIC: protein interactions calculator. Nucleic Acids Res 35:W473–W476PubMedCrossRefGoogle Scholar
  20. 20.
    Watanabe K, Kitamura K, Suzuki Y (1996) Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues. Appl Environ Microbiol 62:2066–2073PubMedGoogle Scholar
  21. 21.
    Williamson G, Vallejo J (1997) Chemical and thermal stability of ferulic acid esterase-III from Aspergillus niger. Int J Biol Macromol 21:163–167PubMedCrossRefGoogle Scholar
  22. 22.
    Yamamura S, Morita Y, Hasan Q, Rao SR, Murakami Y, Yokoyama K, Tamiya E (2002) Characterization of a new keratin-degrading bacterium isolated from deer fur. J Biosci Bioeng 93:595–600PubMedGoogle Scholar
  23. 23.
    Yang DF, Wei YT, Huang RB (2007) Computer-aided design of the stability of pyruvate formate-lyase from Escherichia coli by site-directed mutagenesis. Biosci Biotech Biochem 71:746–753CrossRefGoogle Scholar
  24. 24.
    Yang Y, Jiang L, Yang S, Zhu L, Wu Y, Li Z (2000) A mutant subtilisin E with enhanced thermostability. World J Microbiol Biotechnol 16:249–251CrossRefGoogle Scholar
  25. 25.
    Zhang SB, Pei XQ, Wu ZL (2012) Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger. Bioresour Technol 117:140–147PubMedCrossRefGoogle Scholar
  26. 26.
    Zhang SB, Wu ZL (2011) Identification of amino acid residues responsible for increased thermostability of feruloyl esterase A from Aspergillus niger using the PoPMuSiC algorithm. Bioresour Technol 102:2093–2096PubMedCrossRefGoogle Scholar
  27. 27.
    Zhao H, Arnold FH (1999) Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng 12:47–53PubMedCrossRefGoogle Scholar
  28. 28.
    Zyprian E, Matzura H (1986) Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system. DNA 5:219–225PubMedCrossRefGoogle Scholar

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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