Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

  • Ana Maria Mazotto
  • Ana Cristina N. de Melo
  • Andrew Macrae
  • Alexandre Soares Rosado
  • Raquel Peixoto
  • Sabrina M. L. Cedrola
  • Sônia Couri
  • Russolina B. Zingali
  • Ana Lúcia V. Villa
  • Leon Rabinovitch
  • Jeane Q. Chaves
  • Alane B. VermelhoEmail author
Original Paper


In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml−1). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15–140 kDa range, and B. cereus produced a keratinase of ~200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40–50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50–70°C and pH 7.0–11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.


Bacillus spp. Feather degradation Feather keratin Keratinase Peptidase 



We would like to thank the technical assistance of Denise da Rocha de Souza supported by fellowships grants from MCT/CNPq. This study was supported by grants from Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT/CNPq), Conselho de Ensino para Graduados e Pesquisas (CEPG/UFRJ), Fundação Oswaldo Cruz (FIOCRUZ), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Fundação Universitária José Bonifácio (FUJB).


  1. Bálint B, Bagi Z, Tóth A, Rákhely G, Perei K, Kovács KL (2005) Utilization of keratin-containing biowaste to produce biohydrogen. Appl Microbiol Biotechnol 69:404–410. doi: 10.1007/s00253-005-1993-3 CrossRefGoogle Scholar
  2. Barone JR, Schmidt WF, Liebner CFE (2005) Compounding and molding of polyethylene composites reinforced with keratin feather fiber I. Compos Sci Technol 65:683–692. doi: 10.1016/j.compscitech.2004.09.030 CrossRefGoogle Scholar
  3. Bernal C, Cairo J, Coello N (2006) Purification and characterization of a novel exocellular keratinase form Kocuria rosea. Enzyme Microb Technol 38:49–546. doi: 10.1016/j.enzmictec.2005.02.021 CrossRefGoogle Scholar
  4. Bertsch A, Coello N (2005) A biotechnological process for treatment and recycling poultry feathers as a feed ingredient. Bioresour Technol 96:1703–1708. doi: 10.1016/j.biortech.2004.12.026 CrossRefGoogle Scholar
  5. Brandelli A (2008) Bacterial Keratinases: useful enzymes for bioprocessing agroindustrial wastes and beyond. Food Bioprocess Technol 1:105–116. doi: 10.1007/s11947-007-0025-y CrossRefGoogle Scholar
  6. Cai C, Zheng X (2009) Medium optimization for keratinase production in hair substrate by a new Bacillus subtilis KD-N2 using response surface methodology. J Ind Microbiol Biotechnol 36:875–883. doi: 10.1007/s10295-009-0565-4 CrossRefGoogle Scholar
  7. Cai CG, Chen JS, Qi JJ, Yin Y, Zheng XD (2008) Purification and characterization of keratinase from a new Bacillus subtilis strain. J Zhejiang Univ Sci B 9(9):713–720. doi: 10.1631/jzus.B0820128 CrossRefGoogle Scholar
  8. Claus D, Berkeley RCW (1986) Genus Bacillus Cohn 1872. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG, Williams ST, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, 2nd edn. Williams & Wilkins, Baltimore, pp 1105–1141Google Scholar
  9. Dalev P, Ivanov I, Liubomirova A (1997) Enzymic modification of feather keratin hydrolysates with lysine aimed at increasing the biological value. J Sci Food Agric 73:242–244. doi: 10.1002/(SICI)1097-0010(199702)73:2<242:AID-JSFA712>3.0.CO;2-3 CrossRefGoogle Scholar
  10. Daroit DJ, Corrêa APF, Brandelli A (2009) Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus. Int Biodeterior Biodegradation 63:358–363. doi: 10.1016/j.ibiod.2008.11.008 CrossRefGoogle Scholar
  11. De Melo AC, Dornelas-Ribeiro M, Souza EP, Macrae A, Fracalanzza SEL, Vermelho AB (2007) Peptidase profiles from non-albicans Candida spp. isolated from the blood of a patient with chronic myeloid leukemia and another with sickle cell disease. FEMS Yeast Res 7:1004–1012. doi: 10.1111/j.1567-1364.2007.00269.x CrossRefGoogle Scholar
  12. El-Refai HA, AbdelNaby MA, Gaballa A, El-Araby MH, Abdel Fattah AF (2005) Improvement of the newly isolated Bacillus pumilus FH9 keratinolytic activity. Process Biochem 40:2325–2332. doi: 10.1016/j.procbio.2004.09.006 CrossRefGoogle Scholar
  13. Fraser RDB, Parry DAD (2008) Molecular packing in the feather keratin filament. J Struct Biol 162:1–13. doi: 10.1016/j.jsb.2008.01.011 CrossRefGoogle Scholar
  14. Giongo JL, Lucas FS, Casarin F, Heeb P, Brandelli A (2007) Keratinolytic proteases of Bacillus species isolated from the Amazon basin showing remarkable de-hairing activity. World J Microbiol Biotechnol 23:375–382. doi: 10.1007/s11274-006-9234-1 CrossRefGoogle Scholar
  15. Gosh 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. doi: 10.1007/s10295-008-0354-5 CrossRefGoogle Scholar
  16. Grazziotin A, Pimentel FA, de Jong EV, Brandelli A (2006) Nutritional improvement of feather protein by treatment with microbial keratinase. Anim Feed Sci Technol 126:135–144. doi: 10.1016/j.anifeedsci.2005.06.002 CrossRefGoogle Scholar
  17. Gupta R, Ramnani P (2006) Microbial keratinases and their prospective applications: an overview. Appl Microbiol Biotechnol 70:21–33. doi: 10.1007/s00253-005-0239-8 CrossRefGoogle Scholar
  18. Jones LB, Fontamini D, Jarvinen M, Pekkarinen A (1998) Simplified endoproteinase assays using gelatin or azogelatin. Anal Biochem 263:214–220. doi: 10.1006/abio.1998.2819 CrossRefGoogle Scholar
  19. Khardenavis AA, Kapley A, Purohit HJ (2009) Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383. Waste Manage 29:1409–1415. doi: 10.1016/j.wasman.2008.10.009 CrossRefGoogle Scholar
  20. Kojima M, Kanai M, Tominaga M, Kitazume S, Inoue A, Horikoshi K (2006) Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30–01. Extremophiles 10:229–235. doi: 10.1007/s00792-005-0491-y CrossRefGoogle Scholar
  21. Laemmli VK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  22. Lin X, Lee CG, Casale ES, Shin JCH (1992) Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis PWD-1. Appl Environ Microbiol 58:3271–3275Google Scholar
  23. Lopes BG, Santos AL, Bezerra CD, Wanke B, Dos Santos LM, Nishikawa MM, Mazotto AM, Kussumi VM, Haido RM, Vermelho AB (2008) A 25-kDa Serine Peptidase with Keratinolytic activity secreted by Coccidioides immitis. Mycopathol 166:35–40. doi: 10.1007/s11046-008-9116-1 CrossRefGoogle Scholar
  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:267–275Google Scholar
  25. Mabrouk MEM (2008) Feather degradation by a new keratinolytic Streptomyces sp. MS-2. World J Microbiol Biotechnol 24:2331–2338. doi: 10.1007/s11274-008-9748-9 CrossRefGoogle Scholar
  26. Macedo AJ, Beys da Silva WO, Termignoni C (2008) Properties of a non collagen-degrading Bacillus subtilis keratinase. Can J Microbiol 54:80–188CrossRefGoogle Scholar
  27. Massol-Deya AA, Odelson DA, Hickey RF, Tiedje JM (1995) Bacterial community fingerprinting of amplified 16S and 16–23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA). In: Akkermans ADL (ed) Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, pp– Scholar
  28. Matsui T, Yamada Y, Mitsuya H, Shigeri Y, Yoshida Y, Saito Y, Matsui H, Watanabe K (2009) Sustainable and practical degradation of intact chicken feathers by cultivating a newly isolated thermophilic Meiothermus ruber H328. Appl Microbiol Biotechnol 82:941–950. doi: 10.1007/s00253-009-1880-4 CrossRefGoogle Scholar
  29. Mazotto AM, Cedrola SML, Lins U, Rosado AS, Silva KT, Chaves JQ, Rabinovitch L, Zingali RB, Vermelho AB (2010) Keratinolytic activity of Bacillus subtilis AMR using human hair. Lett Appl Microbiol. doi: 10.1111/j.1472-765X.2009.02760.x
  30. Mohorcic M, Torkar A, Friedrich J, Kristl J, Murdan S (2007) An investigation into keratinolytic enzymes to enhance ungual drug delivery. Int J Pharm 332:196–201. doi: 10.1016/j.ijpharm.2006.09.042 CrossRefGoogle Scholar
  31. Nam GW, Lee D, Lee HS, Lee NJ, Kim BC, Choe EA, Hwang JK, Suhartono MT, Pyun YR (2002) Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbiol 178:538–547. doi: 10.1007/s00203-002-0489-0 CrossRefGoogle Scholar
  32. Nilegaonkar SS, Zambare VP, Kanekar PP, Dhakephalkar PK, Sarnaik SS (2007) Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour Technol 98:1238–1245. doi: 10.1016/j.biortech.2006.05.003 CrossRefGoogle Scholar
  33. Odetallah NH, Wang JJ, Garlich JD, Shih JC (2005) Versazyme supplementation of broiler diets improves market growth performance. Poult Sci 84:858–864. doi: 10.1002/9780470385869.ch12 Google Scholar
  34. Onifade AA, Al-Sane NA, Al-Musallan AA, Al-Zarban S (1998) A review: potential for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resource. Bioresour Technol 66:1–11. doi: 10.1016/S0960-8524(98)00033-9 CrossRefGoogle Scholar
  35. Papadopoulos MC (1986) The effect of enzymatic treatment on amino acid content and nitrogen characteristics of feather meals. Anim Feed Sci Technol 16:151–156. doi: 10.1016/0377-8401(86)90058-1 CrossRefGoogle Scholar
  36. Park GT, Son HJ (2009) Keratinolytic activity of Bacillus megaterium F7–1, a feather-degrading mesophilic bacterium. Microbiol Res 164:478–485. doi: 10.1016/j.micres.2007.02.004 CrossRefGoogle Scholar
  37. Park CH, Sang JL, Lee SJ, Lee SG, Lee WS, Byun SM (2004) Hetero- and autoprocessing of the extracellular metalloprotease (Mpr) in Bacillus subtilis. J Bacteriol 186:6457–6464. doi: 10.1128/JB.186.19.6457-6464.2004 CrossRefGoogle Scholar
  38. Pillai P, Archana G (2008) Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate. Appl Microbiol Biotechnol 78:643–650. doi: 10.1007/s00253-008-1355-z CrossRefGoogle Scholar
  39. Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635Google Scholar
  40. Riffel A, Lucas F, Heeb P, Brandelli A (2003) Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Arch Microbiol 179:258–265. doi: 10.1007/s00203-003-0525-8 Google Scholar
  41. Rozs M, Manczinger L, Vágvölgyi C, Kevei F (2001) Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis. FEMS Microbiol Lett 205:221–224. doi: 10.1111/j.1574-6968.2001.tb10951.x CrossRefGoogle Scholar
  42. Saeki K, Ozaki K, Kobayashi T, Ito S (2007) Detergent Alkaline Proteases: enzymatic properties, genes, and crystal structures. J Biosci Bioeng 103:501–508. doi: 10.1263/jbb.103.501 CrossRefGoogle Scholar
  43. Son H, Park H, Kim H, Lee C (2008) Nutritional regulation of keratinolytic activity in Bacillus pumilis. Biotechnol Lett 30:461–465. doi: 10.1007/s10529-007-9567-3 CrossRefGoogle Scholar
  44. Sousa F, Jus S, Erbel A, Kokol V, Cavaco-Paulo A, Gubitz GM (2007) A novel metalloprotease from Bacillus cereus for protein fiber processing. Enzyme Microb Technol 40:1772–1781. doi: 10.1016/j.enzmictec.2006.12.017 CrossRefGoogle Scholar
  45. Suh HJ, Lee HK (2001) Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1. Protein Chem 20:165–169. doi: 10.1023/A:1011075707553 CrossRefGoogle Scholar
  46. Suzuki Y, Tsujimoto Y, Matsui H, Watanabe K (2006) Decomposition of extremely hard-to-degrade animal proteins by thermophilic bacteria. J Biosci Bioeng 102:73–81. doi: 10.1263/jbb.102.73 CrossRefGoogle Scholar
  47. Tang X, Lakay FM, Shen W, Shao W, Fang H, Prior BA, Wang Z, Zhuge J (2004) Purification and characterization of an alkaline protease used in tannery industry from Bacillus licheniformis. Biotechol Lett 26:1421–1424. doi: 10.1023/B:BILE.0000045642.19299.3f CrossRefGoogle Scholar
  48. Tari C, Genckal H, Tokatli F (2006) Optimization of a growth medium using a statistical approach for the production of an alkaline protease from newly isolated Bacillus sp. L21. Proc Biochem 41:659–665. doi: 10.1016/j.procbio.2005.08.012 CrossRefGoogle Scholar
  49. Thys RCS, Brandelli A (2006) Purification and properties of a keratinolytic metalloprotease from Microbacterium sp. J Appl Microbiol 101:1259–1268. doi: 10.1111/j.1365-2672.2006.03050.x CrossRefGoogle Scholar
  50. Vasconcelos FJM, Rabinovitch L (1994) A new formula for an alternative culture medium, without antibiotics, for isolation and presumptive quantification of Bacillus cereus food. J Food Microbiol 58:235–238Google Scholar
  51. Vermelho AB, Mazotto AM, Nogueira de Melo AC, Vieira FHC, Duarte TR, Macrae A, Nishikawa MM, Bon EPS (2009) Identification of a Candida parapsilosis strain producing extracellular serine peptidase with keratinolytic activity. Mycopathol. doi: 10.1007/s11046-009-9231-7
  52. Wawrzkiewicz K, Wolski T, Lobarewski J (1991) Screening the keratinolytic activity of dermatophytes in vitro. Mycopathologia 114:1–8. doi: 10.1007/BF00436684 CrossRefGoogle Scholar
  53. Williams CM, Richter CS, Mackeinze JM Jr, Shih JCH (1990) Isolation, identification, and characterization of a feather-degrading bacterium. Appl Environ Microbiol 56:1509–1515Google Scholar
  54. Yoshioka M, Miwa T, Horii H, Takata M, Nishizawa K, Watanabe M, Shinagawa M, Murayama Y (2007) Characterization of a proteolytic enzyme derived from Bacillus strain that effectively degrades prion protein. J Appl Microbiol 102:509–515. doi: 10.1111/j.1365-2672.2006.03080.x CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Ana Maria Mazotto
    • 1
  • Ana Cristina N. de Melo
    • 1
  • Andrew Macrae
    • 1
  • Alexandre Soares Rosado
    • 1
  • Raquel Peixoto
    • 1
  • Sabrina M. L. Cedrola
    • 1
  • Sônia Couri
    • 2
  • Russolina B. Zingali
    • 3
  • Ana Lúcia V. Villa
    • 4
  • Leon Rabinovitch
    • 5
  • Jeane Q. Chaves
    • 5
  • Alane B. Vermelho
    • 1
    Email author
  1. 1.Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Bloco I, Centro de Ciências da Saúde (CCS)Universidade Federal do Rio de Janeiro (UFRJ), Cidade UniversitáriaIlha do Fundão, Rio de JaneiroBrazil
  2. 2.Instituto Federal de Educação Ciência e Tecnologia do Rio de janeiroMaracanã, Rio de JaneiroBrazil
  3. 3.Departamento de Bioquímica, Instituto de Ciências Biomédicas, Bloco H, Centro de Ciências da Saúde (CCS)Universidade Federal do Rio de Janeiro (UFRJ), Cidade UniversitáriaIlha do Fundão, Rio de JaneiroBrazil
  4. 4.Universidade federal do Rio de Janeiro, Campus MacaéGranja do Cavaleiros, MacaéBrazil
  5. 5.Departamento de BacteriologiaInstituto Oswaldo CruzRio de JaneiroBrazil

Personalised recommendations