Molecular and biochemical characterization of a thermostable keratinase from Bacillus altitudinis RBDV1


A thermostable keratinase designated as KBALT was purified from Bacillus altitudinis RBDV1 from a poultry farm in Gujarat, India. The molecular weight of the native KBALT (nKBALT) purified using ammonium sulfate and ion exchange and gel permeation chromatography with a 40% yield and 80-fold purification was estimated to be ~ 43 kDa. The gene for KBALT was successfully cloned, sequenced and expressed in Escherichia coli. Recombinant KBALT (rKBALT) when purified using a single step Ni–NTA His affinity chromatography achieved a yield of 38.20% and a 76.4-fold purification. Comparison of the deduced amino acid sequence of rKBALT with known proteases of Bacillus species and inhibitory effect of PMSF suggest that rKBALT was a subtilisin-like serine protease. Both native and rKBALT exhibited higher activity at 85 °C and pH 8.0 in the presence of Mg2+, Mn2+, Zn2+, Ba2+ and Fe3+ metal ions. Interestingly, 70% of their activity was retained at temperatures ranging from 35 to > 95 °C. The keratinolytic activity of both nKBALT and rKBALT was enhanced in the presence of reducing agents. They exhibited broad substrate specificity towards various protein substrates. KBALT was determined for its kinetic properties by calculating its Km (0.61 mg/ml) and Vmax (1673 U/mg/min) values. These results suggest KBALT as a robust and promising contender for enzymatic processing of keratinous wastes in waste processing plants.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Altschul S, Madden TL (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402.

  2. Anitha TS, Palanivelu P (2012) Research article production and characterization of keratinolytic protease(s) from the fungus, Aspergillus parasiticus. Fungal Genom Biol 2(2):87–93

  3. Bressollier P, Letourneau F et al (1999) Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Appl Environ Microbiol 65(6):2570–2576

  4. Farag AM, Hassan MA (2004) Purification, characterization and immobilization of a keratinase from Aspergillus oryzae. Enzyme Microb Technol 34:85–93

    CAS  Article  Google Scholar 

  5. Felix CR (2002) Heterologous production of Aspergillus fumigatus keratinase in Pichia pastoris. World J Microbiol Biotechnol 18:563–568

  6. Gödde C et al (2005) Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli. Appl Environ Microbiol 71(7):3951–3958.

  7. Gopinath SCB et al (2015) Biotechnological aspects and perspective of microbial keratinase production. Biomed Res Int 2015(2015):140726.

  8. Gradisar H et al (2005) Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl Environ Microbiol 71(7):3420–3426

  9. Guo J, Ma Y (2008) High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris. Protein Expr Purif 58:301–308

    CAS  Article  Google Scholar 

  10. Gupta MR, Ramnani P (2006) Microbial keratinases and their prospective applications: an overview. Appl Microbiol Biotechnol 70:21–33

  11. Gupta S, Nigam A, Singh R (2015) Purification and characterization of a Bacillus subtilis keratinase and its prospective application in feed industry. Acta Biol Szeged 59(2):197–204

    Google Scholar 

  12. Hu H et al (2013) Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes. Biotechnol Lett 35(2):239–244

  13. Abdel-Naby MA (2017) Structural characterization, catalytic, kinetic and thermodynamic properties of Keratinase from Bacillus pumilus FH9. Int J Biol Macromol 105(1):973–980.

    CAS  Article  Google Scholar 

  14. Baweja M, Singh PK (2017) Cost effective characterization process and molecular dynamic simulation of detergent compatible alkaline protease from Bacillus pumilus strain MP27. Process Biochem 58:199–203

    CAS  Article  Google Scholar 

  15. Kumar EV et al (2011) A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent. Bioprocess Biosyst Eng 34(4):403–409

    Article  Google Scholar 

  16. Kumar V et al (2016) Recent developments in systems biology and metabolic engineering of plant–microbe interactions. Front Plant Sci 7:1–12

    Google Scholar 

  17. Lange L, Huang Y, Busk PK (2016) Microbial decomposition of keratin in nature-a new hypothesis of industrial relevance. Appl Microbiol Biotechnol 100:2083–2096

    CAS  Article  Google Scholar 

  18. Lin X et al (1992) Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain Bacillus licheniformis strain. Appl Environ Microbiol 58(10):3271–3275.

    CAS  Google Scholar 

  19. Lin X et al (1997) Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis. J Ind Biotechnol 19(2):134–138

    CAS  Article  Google Scholar 

  20. More SS et al (2013) Purification and properties of a novel fungal alkaline keratinase from Cunninghamella echinulata. Turk J Biochem 38(1):68–74

  21. Nayaka S (2013) Purification and characterization of keratinase from hair-degrading Streptomyces albus. Int J Bioassays 2(3):599–604

  22. Onifade AA (1998) A review: potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresour Technol 66:1–11

    CAS  Article  Google Scholar 

  23. Peña-montes C et al (2008) Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans. Appl Environ Microbiol 78:603–612

    Google Scholar 

  24. Pushpam PL, Rajesh T, Gunasekaran P (2011) Identification and characterization of alkaline serine protease from goat skin surface metagenome. AMB Express 1(1):3.

  25. Radha S, Gunasekaran P (2007) Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. J Appl Microbiol 103:1301–1310

    CAS  Article  Google Scholar 

  26. Radha S, Gunasekaran P (2009) Purification and characterization of keratinase from recombinant Pichia and Bacillus strains. Protein Expr Purif 64(1):24–31.

    CAS  Article  Google Scholar 

  27. Rajput R, Sharma R, Gupta R (2010) Biochemical characterization of a thiol-activated, oxidation stable keratinase from Bacillus pumilus KS12. Enzyme Res 2010:132148.

  28. Saibabu V, Niyonzima FN, More SS (2013) Isolation, partial purification and characterization of keratinase from Bacillus megaterium. Int Res J Biol Sci 2(2):13–20

    Google Scholar 

  29. Sareen R, Bornscheuer UT, Mishra P (2005) Cloning, functional expression and characterization of an alkaline protease from Bacillus licheniformis. Biotechnol Lett 9(5):1901–1907

    Article  Google Scholar 

  30. Selvam K, Vishnupriya B (2012) Biochemical and molecular characterization of microbial keratinase and its remarkable applications. Int J Pharm Biol Arch 3(2):267–275

    Google Scholar 

  31. Sharma R, Gupta R (2010) Extracellular expression of keratinase Ker P from Pseudomonas aeruginosa in E. coli. Biotechnol Lett 32:1863–1868

    CAS  Article  Google Scholar 

  32. Sivakumar T, Shankar T, Vijayabaskar P et al (2012) Optimization for keratinase enzyme production using Bacillus thuringiensis TS2. Acad J Plant Sci (AJPS) 5(3):102–109

  33. Sivakumar T, Shankar T, Ramasubramanian V (2012b) Purification properties of Bacillus thuringiensis TS2 keratinase enzyme. Am Eurasian J Agric Environ Sci 12(12):1553–1557

    CAS  Google Scholar 

  34. Zhang J, Li B (2013) 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 29(5):825–832

Download references


Authors are thankful to P.G. Department of Biosciences, Sardar Patel University for providing facilities to conduct research.

Author information



Corresponding author

Correspondence to R. B. Subramanian.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pawar, V.A., Prajapati, A.S., Akhani, R.C. et al. Molecular and biochemical characterization of a thermostable keratinase from Bacillus altitudinis RBDV1. 3 Biotech 8, 107 (2018).

Download citation


  • Bacillus altitudinis
  • Keratinase
  • Protease
  • Heterologous expression