SpringerLink
Log in

Isolation and Biochemical Properties of Extremophilic Keratinase from Bacillus cereus FC1365

  • Research Article
  • Published:
Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

Keratinases secreted by microorganisms belong to proteolytic enzymes with unique characteristics to efficiently degrade tough, fibrous, and hydrolysis resistant keratin proteins. In this study, the biochemical characterization of a highly thermoactive and alkalophilic keratinase enzyme (FCnase) was reported that was isolated from the soil samples procured from a Bengaluru-based poultry dump yard, India. The screened isolate was identified as Bacillus cereus FC1365 (# MN712509—accession number) by 16S rRNA gene sequencing. FCnase production was carried out in minimal salt media supplemented with feather meal, and partial purification of the extracellularly secreted enzyme was achieved by acetone precipitation method. 3.4-fold purification of the enzyme was attained with a maximum activity of 21.4 U/ml. The enzyme was optimally active at pH 10 and 70 ℃. Km and Vmax of the enzyme were 0.396 mg/ml and 2.86 U/ml, respectively. SDS-PAGE and Zymogram confirmed the molecular weight of the FCnase as 63 kDa. Metal ions like Ca2+ and Mg2+ enhanced the enzyme activity while the ethylenediaminetetraacetic acid inhibited it, proving it to be a metalloprotease. Efficient feather degradation was demonstrated by the isolated robust enzyme making it suitable for feather waste management and other industrial applications.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Agrahari S, Wadhwa N (2010) Degradation of chicken feather a poultry waste product by keratinolytic bacteria isolated from dum** site at Ghazipur poultry processing plant. Int J Poult Sci 9:482–489. https://doi.org/10.3923/ijps.2010.482.489

    Article  CAS  Google Scholar 

  2. Navone L, Speight R (2018) Understanding the dynamics of keratin weakening and hydrolysis by proteases. PLoS ONE 13:2608. https://doi.org/10.1371/journal.pone.0202608

    Article  CAS  Google Scholar 

  3. Vidmar B, Vodovnik M (2018) Microbial keratinases: enzymes with promising biotechnological applications. Food Technol Biotechnol 56(3):312–328. https://doi.org/10.17113/ftb.56.03.18.5658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kumawat TK, Sharma A, Sharma V, Chandra S. (2018) Keratin waste: the biodegradable polymers. In: Keratin. IntechOpen https://doi.org/10.5772/intechopen.79502

  5. Brandelli A, Daroit DJ, Riffel A (2010) Biochemical features of microbial keratinases and their production and applications. Appl Microbiol Biotechnol 85:1735–1750. https://doi.org/10.1007/s00253-009-2398-5

    Article  CAS  PubMed  Google Scholar 

  6. Papadopoulos M, El Boushy A, Roodbeen A, Ketelaars E (1986) Effects of processing time and moisture content on amino acid composition and nitrogen characteristics of feather meal Animal Feed. Sci Technol 14:279–290. https://doi.org/10.1016/0377-8401(86)90100-8

    Article  Google Scholar 

  7. Desai SS, Hegde S, Inamdar P, Sake N, Aravind MS (2010) Isolation of keratinase from bacterial isolates of poultry soil for waste degradation. Eng Life Sci 10:361–367. https://doi.org/10.1002/elsc.200900009

    Article  CAS  Google Scholar 

  8. Herzog B, Overy D, Haltli B, Kerr R (2014). Keratinases – A viable approach for hair removal and younger looking skin. In: Conference: maritime natural products https://doi.org/10.13140/2.1.4891.8403

  9. Deivasigamani B, Alagappan K (2008) Industrial application of keratinase and soluble proteins from feather keratins. J Environ Biol 29:933–936

    CAS  PubMed  Google Scholar 

  10. Ramakrishnan N, Sharma S, Gupta A, Alashwal BY (2018) Keratin based bioplastic film from chicken feathers and its characterization. Int J Biol Macromol 111:352–358. https://doi.org/10.1016/j.ijbiomac.2018.01.037

    Article  CAS  PubMed  Google Scholar 

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

    Google Scholar 

  12. Nagai Y, Nishikawa T (1970) Alkali solubilization of chicken feather keratin. Agric Biol Chem 34:16–22. https://doi.org/10.1080/00021369.1970.10859572

    Article  CAS  Google Scholar 

  13. Uddin ME, Ahmad T, Ajam M, Moniruzzaman M, Mandol D, Ray S, Sufian A, Rahman M, Hossain E, Ahammed R (2017) Thermotolerant extracellular proteases produced by Bacillus subtilis isolated from local soil that representing industrial applications. J Pure Appl Microbiol 11:733–741. https://doi.org/10.22207/JPAM.11.2.12

    Article  CAS  Google Scholar 

  14. Ghosh A, Chakrabarti K, Chattopadhyay D (2008) Degradation of raw feather by a novel high molecular weight extracellular protease from newly isolated Bacillus cereus DCUW. J Ind Microbiol Biotechnol 35:825–834. https://doi.org/10.1007/s10295-008-0354-5

    Article  CAS  PubMed  Google Scholar 

  15. Lo W-H, Too J-R, Wu J-Y (2012) Production of keratinolytic enzyme by an indigenous feather–degrading strain Bacillus cereus Wu2. J Biosci Bioeng 114:640–647. https://doi.org/10.1016/j.jbiosc.2012.07.014

    Article  CAS  PubMed  Google Scholar 

  16. Lateef A, Oloke J, Kana EG, Sobowale B, Ajao S, Bello B (2010) Keratinolytic activities of a new feather-degrading isolate of Bacillus cereus LAU 08 isolated from Nigerian soil. Int Biodeterior Biodegrad 64:162–165. https://doi.org/10.1016/j.ibiod.2009.12.007

    Article  CAS  Google Scholar 

  17. Tork S, Aly M, Nawar L (2010) Biochemical and molecular characterization of a new local keratinase producing Pseudomomanas sp. MS21. Asian J Biotechnol 2:1–13. https://doi.org/10.3923/ajbkr.2010.1.13

    Article  CAS  Google Scholar 

  18. Tamreihao K, Mukherjee S, Khunjamayum R, Devi LJ, Asem RS, Ningthoujam DS (2019) Feather degradation by keratinolytic bacteria and biofertilizing potential for sustainable agricultural production. J Basic Microbiol 59:4–13. https://doi.org/10.1002/jobm.201800434

    Article  CAS  PubMed  Google Scholar 

  19. Toyokawa Y, Takahara H, Reungsang A, Fukuta M, Hachimine Y, Tachibana S, Yasuda M (2010) Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce. Appl Microbiol Biotechnol 86:1867–1875. https://doi.org/10.1007/s00253-009-2434-5

    Article  CAS  PubMed  Google Scholar 

  20. Abdel-Fattah AM, El-Gamal MS, Ismail SA, Emran MA, Hashem AM (2018) Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1. J Genet Eng Biotechnol 16:311–318. https://doi.org/10.1016/j.jgeb.2018.05.005

    Article  PubMed  PubMed Central  Google Scholar 

  21. Gessesse A, Hatti-Kaul R, Gashe BA, Mattiasson B (2003) Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb Technol 32:519–524. https://doi.org/10.1128/AEM.62.8.2875-2882.1996

    Article  CAS  Google Scholar 

  22. Bressollier P, Letourneau F, Urdaci M, Verneuil B (1999) Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Appl Environ Microbiol 65:2570–2576. https://doi.org/10.1128/AEM.65.6.2570-2576.1999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Friedrich AB, Antranikian G (1996) Keratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales. Appl Environ Microbiol 62:2875–2882. https://doi.org/10.1128/AEM.62.8.2875-2882.1996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nam G-W, Lee D-W, Lee H-S, Lee N-J, Kim B-C, Choe E-A, Hwang J-K, Suhartono MT, Pyun Y-R (2002) Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbiol 178:538–547. https://doi.org/10.1007/s00203-002-0489-0

    Article  CAS  PubMed  Google Scholar 

  25. Abdel-Naby MA, El-Refai HA, Ibrahim MH (2017) Structural characterization, catalytic, kinetic and thermodynamic properties of Keratinase from Bacillus pumilus FH9. Int J Biol Macromol 105:973–980. https://doi.org/10.1016/j.ijbiomac.2017.07.118

    Article  CAS  PubMed  Google Scholar 

  26. Nigam V, Singhal P, Vidyarthi A, Mohan M, Ghosh P (2013) Studies on keratinolytic activity of alkaline proteases from halophilic bacteria. Int J Pharm Biol Sci 4:389–399. https://doi.org/10.1007/s12088-014-0477-5

    Article  CAS  Google Scholar 

  27. Kojima K, Nakata H, Inouye K (2014) Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability. Biochem Biophys Acta 1844:330–338. https://doi.org/10.1016/j.bbapap.2013.10.014

    Article  CAS  PubMed  Google Scholar 

  28. Balaji S, Kumar MS, Karthikeyan R, Kumar R, Kirubanandan S, Sridhar R, Sehgal P (2008) Purification and characterization of an extracellular keratinase from a hornmeal-degrading Bacillus subtilis MTCC (9102). World J Microbiol Biotechnol 24:2741–2745

    Article  CAS  Google Scholar 

  29. Wang S-L, Hsu W-T, Liang T-W, Yen Y-H, Wang C-L (2008) Purification and characterization of three novel keratinolytic metalloproteases produced by Chryseobacterium indologenes TKU014 in a shrimp shell powder medium. Bioresour Technol 99:5679–5686. https://doi.org/10.1016/j.biortech.2007.10.024

    Article  CAS  PubMed  Google Scholar 

  30. Gradišar H, Friedrich J, Križaj I, Jerala R (2005) Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl Environ Microbiol 71:3420–3426. https://doi.org/10.1128/AEM.71.7.3420-3426.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. R. Mouneesha and H. Sushma have contributed equally to this work.

Authors and Affiliations

  1. School of Basic and Applied Sciences, Dayananda Sagar University, Bengaluru, Karnataka, India

    K. G. Priyanka, R. Mouneesha, H. Sushma, A. Prakruti, H. K. Manjushree, Sunil S. More & Aneesa Fasim

Authors
  1. K. G. Priyanka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Mouneesha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. Sushma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Prakruti
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H. K. Manjushree
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sunil S. More
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Aneesa Fasim
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KGP, RM, HS (undergraduate students) performed experiments and collected review of literature. AP, HKM (Ph.D. students) performed experiments, analyzed, and wrote the manuscript. SSM reviewed and edited the manuscript. AF contributed to conceptualization, writing, and editing the manuscript.

Corresponding authors

Correspondence to Sunil S. More or Aneesa Fasim.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Significant Statement: The study describes the isolation, and identification of an alkalophilic and thermoactive keratinase producing microorganism that can be an eco-friendly and cost-effective alternative in feather waste management and production of feed by-products.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Priyanka, K.G., Mouneesha, R., Sushma, H. et al. Isolation and Biochemical Properties of Extremophilic Keratinase from Bacillus cereus FC1365. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 93, 721–729 (2023). https://doi.org/10.1007/s40011-023-01472-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40011-023-01472-5

Keywords

Search

Navigation