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3 Biotech

, 9:2 | Cite as

Keratinolytic activity of Bacillus subtilis LFB-FIOCRUZ 1266 enhanced by whole-cell mutagenesis

  • Daniel Pereira de Paiva
  • Samara Sant’Anna de Oliveira
  • Ana Maria Mazotto
  • Alane Beatriz Vermelho
  • Selma Soares de OliveiraEmail author
Original Article
  • 94 Downloads

Abstract

Discarded feathers represent an important residue from the poultry industry and are a rich source of keratin. Bacillus subtilis LFB-FIOCRUZ 1266, previously isolated from industrial poultry wastes, was used in this work and, through random mutation using ethyl methanesulfonate, ten strains were selected based on the size of their degradation halos. The feather degradation was increased to 115% and all selected mutants showed 1.4- to 2.4-fold increase in keratinolytic activity compared to their wild-type counterparts. The protein concentrations in the culture supernatants increased approximately 2.5 times, as a result of feather degradation. The mutants produced more sulfide than the wild-type bacteria that produced 0.45 µg/ml, while mutant D8 produced 1.45 µg/ml. The best pH for enzyme production and feather degradation was pH 8. Zymography showed differences in the intensity and molecular mass of some bands. The peptidase activity of the enzyme blend was predominantly inhibited by PMSF and EDTA, suggesting the presence of serine peptidases. HPTLC analysis evidenced few differences in band intensities of the amino acid profiles produced by the mutant peptidase activities. The mutants showed an increase in keratinolytic and peptidase activities, demonstrating their biotechnological potential to recycle feather and help to reduce the environmental impact.

Keywords

Keratin Bacillus Biodegradation Peptidase Mutagenesis 

Notes

Acknowledgements

This work was financed in part by the Coordenação de Aperfeiçoamento Pessoal de Nível Superior-Brasil (CAPES), Financecode001, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro—FAPERJ (Daniel Pereira de Paiva: 202.941/2016).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

References

  1. Arikawa Y, Ozawa T, Iwasaki I (1971) An improved photometric method for the determination of sulfite with pararosaniline and formaldehyde. Bull Chem Soc Jpn 41:1454–1456CrossRefGoogle Scholar
  2. Bernal C, Vidal L, Valdivieso E, Coello N (2003) Keratinolytic activity of Kocuria rosea. World J Microbiol Biotechnol 19:255–261CrossRefGoogle Scholar
  3. Bertsch A, Coello N (2005) A biotechnological process for treatment and recycling poultry feathers as a feed ingredient. Bioresour Technol 96:1703–1708.  https://doi.org/10.1016/j.biortech.2004.12.026 CrossRefPubMedGoogle Scholar
  4. Bohacz J (2017) Biodegradation of feather waste keratin by a keratinolytic soil fungus of the genus Chrysosporium and statistical optimization of feather mass loss. World J Microbiol Biotechnol 33:1–16.  https://doi.org/10.1007/s11274-016-2177-2 CrossRefGoogle Scholar
  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 CrossRefPubMedGoogle Scholar
  6. Brenner M, Niederwieser A (1965) Thin-layer chromatography (TLC) of amino acids. In: Hirs CHW (ed) Methods in enzymology. Academic Press, New York, pp 39–59Google Scholar
  7. Bressollier P, Letourneau F, Urdaci M, Verneuil B (1999) Purification and characterization of a keratinolytic serine proteinase from streptomyces albidoflavus. Appl Environ Microbiol 65(6):2570–2576PubMedPubMedCentralGoogle Scholar
  8. Cai C, Lou B, Zheng X (2008) Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. J Zhejiang Univ Sci B 9:60–67.  https://doi.org/10.1631/jzus.B061620 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cedrola SML, Melo ACN, Mazotto AM, Lins U, Zingali RB, Rosado AS, Peixoto RS, Vermelho AB (2012) Keratinases and sulfide from Bacillus subtilis SLC to recyclefeather waste. World J Microbiol Biotechnol 28:1259–1269.  https://doi.org/10.1007/s11274-011-0930-0 CrossRefPubMedGoogle Scholar
  10. Daroit DJ, Corrêa APF, Brandelli A (2009) Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractusmesopotamicus. Int Biodeter Biodegrad 63:358–363.  https://doi.org/10.1016/j.ibiod.2008.11.008 CrossRefGoogle Scholar
  11. Den Abt T, Souffriau B, Foulquié-Moreno MR et al (2016) Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait. Microb Cell 3:159–175.  https://doi.org/10.15698/mic2016.04.491 CrossRefGoogle Scholar
  12. Deng C. Li J, Shin HD, Du G, Chen J, Liu L (2017) Efficient expression of cyclodextrin glycosyltransferase from Geobacillus stearothermophilus in Escherichia coli by promoter engineering and downstream box evolution. J Biotechnol 266:77–83.  https://doi.org/10.1016/j.jbiotec.2017.12.009 CrossRefPubMedGoogle Scholar
  13. Duarte TR, Oliveira SS, Macrae A et al (2011) Increased expression of keratinase and other peptidases by Candida parapsilosis mutants. Braz J Med Biol Res 44:212–216.  https://doi.org/10.1590/S0100-879X2011007500011 CrossRefPubMedGoogle Scholar
  14. El-Gendy MMA (2010) Keratinase production by endophytic Penicillium spp. Morsy1 under solid-state fermentation using rice straw. Appl Biochem Biotechnol 162:780–794.  https://doi.org/10.1007/s12010-009-8802-x CrossRefPubMedGoogle Scholar
  15. Fellahi S, Chibani A, Feuk-Lagerstedt E, Taherzadeh MJ (2016) Identification of two new keratinolytic proteases from a Bacillus pumilus strain using protein analysis and gene sequencing. AMB Express.  https://doi.org/10.1186/s13568-016-0213-0 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Giarma E, Amanetidou E, Toufexi A, Touraki M (2017) Defense systems in developing Artemia franciscana nauplii and their modulation by probiotic bacteria offer protection against a Vibrio anguillarum challenge. Fish Shellfish Immunol 66:163–172CrossRefGoogle Scholar
  17. Grzywnowicz G, Lobarzewski J, Wawrzkiewicz K, Wolski T (1989) Comparative characterization of proteolytic enzymes from Trichophyton gallinae and Trichophyton verrucosum. Med Mycol 27:319–328.  https://doi.org/10.1080/02681218980000431 CrossRefGoogle Scholar
  18. Guo M, Wu F, Hao G, Qi Q, Li R, Li N, Wei L, Chai T (2017) Bacillus subtilis improves immunity and disease resistance in rabbits. Front Immunol 8:354PubMedPubMedCentralGoogle Scholar
  19. Gupta R, Ramnani P (2006) Microbial keratinases and their prospective applications: an overview. Appl Microbiol Biotechnol 70:21–33.  https://doi.org/10.1007/s00253-005-0239-8 CrossRefPubMedGoogle Scholar
  20. Gupta R, Rajput R, Sharma R, Gupta N (2013) Biotechnological applications and prospective market of microbial keratinases. Appl Microbiol Biotechnol 97:9931–9940.  https://doi.org/10.1007/s00253-013-5292-0 CrossRefPubMedGoogle Scholar
  21. He XS, Brüickner R, Doi RH (1991) The protease genes of Bacillus subtilis. Res Microbiol 142:797–803CrossRefGoogle Scholar
  22. Isaac GS, Abu-Tahon MA (2015) Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate. Braz J Microbiol 46:1269–1277.  https://doi.org/10.1590/S1517-838246420140958 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Jahromi ST, Bazkar N (2018) Future direction in marine bacterial agarases for industrial applications. Appl Microbiol Biotechnol 102(16):6847–6863CrossRefGoogle Scholar
  24. Jones BL, Fontanini D, Jarvinen M, Pekkarinen A (1998) Simplified endoproteinase assays using gelatin or azogelatin. Anal Biochem 263(2):214–220CrossRefGoogle Scholar
  25. Kanaki NS, Rajani M (2005) Development and validation of a thin-layer chromatography-densitometric method for the quantitation of alliin from garlic (Allium sativum) and its formulations. J AOAC Int 88:1568–1570PubMedGoogle Scholar
  26. Kim JS, Kluskens LD, de Vos WM, Huber R, van der Oost J (2004) Crystal structure of fervidolysin from Fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin. J Mol Biol 335(3):787–797CrossRefGoogle Scholar
  27. Kublanov IV, Bidjieva SK, Mardanov AV, Bonch-Osmolovskaya EA (2009) Desulfurococcus kamchatkensis sp. nov., a novel hyperthermophilic protein-degrading archaeon isolated from a Kamchatka hot spring. Int J Syst Evol Microbiol 59:1743–1747.  https://doi.org/10.1099/ijs.0.006726-0 CrossRefPubMedGoogle Scholar
  28. 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.  https://doi.org/10.1007/s00253-015-7262-1 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lindegren G, Hwang YL, Oshima Y, Lindegren CC (1965) Genetical mutants induced in ethyl methanesulfonate in Saccharomyces. Can J Genet Cytol 7:491–499.  https://doi.org/10.1139/g65-064 CrossRefPubMedGoogle Scholar
  30. LiZ SuL, Duan X, Wu D, Wu J (2017) Efficient expression of maltohexaose-forming α-amylase from Bacillus stearothermophilus in Brevibacilluschoshinensis SP3 and Its use in maltose production. J Biotechnol 266:77–83Google Scholar
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275.  https://doi.org/10.1016/0304-3894(92)87011-4 CrossRefPubMedGoogle Scholar
  32. Mabrouk MEM (2008) Feather degradation by a new keratinolytic Streptomyces sp. MS-2. World J Microbiol Biotechnol 24:2331–2338.  https://doi.org/10.1007/s11274-008-9748-9 CrossRefGoogle Scholar
  33. Manczinger L, Rozs M, Vágvölgyi C, Kevei F (2003) Isolation and characterization of a new keratinolytic Bacillus licheniformis strain. World J Microbiol Biotechnol 19:35–39.  https://doi.org/10.1023/A:1022576826372 CrossRefGoogle Scholar
  34. Mazotto AM, LageCedrola SM, Lins U et al (2010) Keratinolytic activity of Bacillus subtilis AMR using human hair. Lett Appl Microbiol 50:89–96.  https://doi.org/10.1111/j.1472-765X.2009.02760.x CrossRefPubMedGoogle Scholar
  35. Mazotto AM, Coelho RRR, Cedrola SML et al (2011) Keratinase production by three Bacillus spp. using feather meal and whole feather as substrate in a submerged fermentation. Enzyme Res 2011:1–7.  https://doi.org/10.4061/2011/523780 CrossRefGoogle Scholar
  36. Mazotto AM, Ascheri JLR, de Oliveira Godoy RL et al (2017) Production of feather protein hydrolyzed by B. subtilis AMR and its application in a blend with cornmeal by extrusion. LWT Food Sci Technol 84:701–709.  https://doi.org/10.1016/j.lwt.2017.05.077 CrossRefGoogle Scholar
  37. Mokrejs P, Svoboda P, Hrncirik J et al (2011) Processing poultry feathers into keratin hydrolysate through alkaline-enzymatic hydrolysis. Waste Manag Res 29:260–267.  https://doi.org/10.1177/0734242X10370378 CrossRefPubMedGoogle Scholar
  38. Nam GW, Lee DW, Lee HS, Lee NJ, Kim BC, Choe EA, Hwang JK, Suhartono MT, Pyun Y (2002) Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbiol 178(6):538–547CrossRefGoogle Scholar
  39. Navone L, Speight R (2018) Understanding the dynamics of keratin weakening and hydrolysis by proteases. PLoS One 13(8):e0202608CrossRefGoogle Scholar
  40. Nogueira De Melo AC, Dornelas-Ribeiro M, Paraguai De Souza E et al (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.  https://doi.org/10.1111/j.1567-1364.2007.00269.x CrossRefGoogle Scholar
  41. Okoroma EA, Purchase D, Garelick H et al (2013) Enzymatic formulation capable of degrading scrapie prion under mild digestion conditions. PLoS One 8:1–7.  https://doi.org/10.1371/journal.pone.0068099 CrossRefGoogle Scholar
  42. Onifade AA, Al-Sane NA, Al-Musallam AA, Al-Zarban S (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.  https://doi.org/10.1016/S0960-8524(98)00033-9 CrossRefGoogle Scholar
  43. Prakash P, Jayalakshmi SK, Sreeramulu K (2010) Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2. Appl Microbiol Biotechnol 87:625–633.  https://doi.org/10.1007/s00253-010-2499-1 CrossRefPubMedGoogle Scholar
  44. Rabbani M, Soleymani S, Sadeghi HM, Soleimani N, Moazen F (2014) Inactivation of aprE gene in Bacillus subtilis 168 by homologus recombination. Avicenna J Med Biotechnol 6(3):185–189PubMedPubMedCentralGoogle Scholar
  45. Ramakrishnan J, Balakrishnan H, Raja STK et al (2011) Formulation of economical microbial feed using degraded chicken feathers by a novel Streptomyces sp: mitigation of environmental pollution. Braz J Microbiol 42:825–834.  https://doi.org/10.1590/S1517-83822011000300001 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Ramnani P, Singh R, Gupta R (2005) Keratinolytic potential of Bacillus licheniformis RG1: structural and biochemical mechanism of feather degradation. Can J Microbiol. 51(3):191–196CrossRefGoogle Scholar
  47. Rawlings ND, Barrett AJ, Bateman A (2010) MEROPS: the peptidase database. Nucleic Acids Res 38(suppl_1):D227–D233CrossRefGoogle Scholar
  48. Ribeiro O, Magalhães F, Aguiar TQ et al (2013) Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii. Bioengineered 4:322–331.  https://doi.org/10.4161/bioe.24653 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Sanghvi G, Patel H, Vaishnava D, Ozaa T, Dave G, Kunjadiac P, Shetha N (2016) A novel alkaline keratinase from Bacillus subtilis DP1 with potential utility in cosmetic formulation. Int J Biol Macromol 8:256–262CrossRefGoogle Scholar
  50. Syed DG, Lee JC, Li WJ et al (2009) Production, characterization and application of keratinase from Streptomyces gulbargensis. Bioresour Technol 100:1868–1871.  https://doi.org/10.1016/j.biortech.2008.09.047 CrossRefPubMedGoogle Scholar
  51. Vasileva-Tonkova E, Gousterova A, Neshev G (2009) Ecologically safe method for improved feather wastes biodegradation. Int Biodeterior Biodegrad 63:1008–1012.  https://doi.org/10.1016/j.ibiod.2009.07.003 CrossRefGoogle Scholar
  52. Villa ALV, Aragão MRS, dos Santos EP et al (2013) Feather keratin hydrolysates obtained from microbial keratinases: effect on hair fiber. BMC Biotechnol 13:1–11.  https://doi.org/10.1186/1472-6750-13-15 CrossRefGoogle Scholar
  53. Wang X, Parsons CM (1997) Effect of processing systems on protein quality of feather meals and hog hair meals. Poult Sci 76:491–496CrossRefGoogle Scholar
  54. Wang HY, Liu DM, Liu Y et al (2007) Screening and mutagenesis of a novel Bacillus pumilus strain producing alkaline protease for dehairing. Lett Appl Microbiol 44:1–6.  https://doi.org/10.1111/j.1472-765X.2006.02039.x CrossRefPubMedGoogle Scholar
  55. Wawrzkiewicz K, Łobarzewski J, Wolski T (1987) Intracellular keratinase of Trichophyton gallinae. J Med Vet Mycol 25:261–268CrossRefGoogle Scholar
  56. Yoshioka M, Miwa T, Horii H et al (2007) Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein. J Appl Microbiol 102:509–515.  https://doi.org/10.1111/j.1365-2672.2006.03080.x CrossRefPubMedGoogle Scholar
  57. Zhu BW, Xiong QNF, Sun Y, Yao Z (2018) High-level expression and characterization of a new κ-carrageenase from marine bacterium Pedobacterhainanensis NJ-02. Lett Appl Microbiol 66(5):409–415.  https://doi.org/10.1111/lam.12865 CrossRefPubMedGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2018

Authors and Affiliations

  • Daniel Pereira de Paiva
    • 1
  • Samara Sant’Anna de Oliveira
    • 1
  • Ana Maria Mazotto
    • 2
  • Alane Beatriz Vermelho
    • 2
  • Selma Soares de Oliveira
    • 1
    Email author
  1. 1.Genetic Microorganism Laboratory: Food and Industry Applications, Institute of Microbiology Paulo de GóesFederal University of Rio de JaneiroRio de JaneiroBrazil
  2. 2.Bioinovar-Biotechnology Laboratories: Biocatalysis, Bioproducts and Bioenergy, Institute of Microbiology Paulo de GóesFederal University of Rio de JaneiroRio de JaneiroBrazil

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