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.
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Arikawa Y, Ozawa T, Iwasaki I (1971) An improved photometric method for the determination of sulfite with pararosaniline and formaldehyde. Bull Chem Soc Jpn 41:1454–1456
Bernal C, Vidal L, Valdivieso E, Coello N (2003) Keratinolytic activity of Kocuria rosea. World J Microbiol Biotechnol 19:255–261
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
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
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
Brenner M, Niederwieser A (1965) Thin-layer chromatography (TLC) of amino acids. In: Hirs CHW (ed) Methods in enzymology. Academic Press, New York, pp 39–59
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):2570–2576
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
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
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
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
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
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
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
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
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 66:163–172
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
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 8:354
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
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
He XS, Brüickner R, Doi RH (1991) The protease genes of Bacillus subtilis. Res Microbiol 142:797–803
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
Jahromi ST, Bazkar N (2018) Future direction in marine bacterial agarases for industrial applications. Appl Microbiol Biotechnol 102(16):6847–6863
Jones BL, Fontanini D, Jarvinen M, Pekkarinen A (1998) Simplified endoproteinase assays using gelatin or azogelatin. Anal Biochem 263(2):214–220
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 88:1568–1570
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):787–797
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
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
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
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 266:77–83
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
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
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
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
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
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
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
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):538–547
Navone L, Speight R (2018) Understanding the dynamics of keratin weakening and hydrolysis by proteases. PLoS One 13(8):e0202608
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
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
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
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
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):185–189
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
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):191–196
Rawlings ND, Barrett AJ, Bateman A (2010) MEROPS: the peptidase database. Nucleic Acids Res 38(suppl_1):D227–D233
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
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 8:256–262
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
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
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
Wang X, Parsons CM (1997) Effect of processing systems on protein quality of feather meals and hog hair meals. Poult Sci 76:491–496
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
Wawrzkiewicz K, Łobarzewski J, Wolski T (1987) Intracellular keratinase of Trichophyton gallinae. J Med Vet Mycol 25:261–268
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
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
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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).
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de Paiva, D.P., de Oliveira, S.S., Mazotto, A.M. et al. Keratinolytic activity of Bacillus subtilis LFB-FIOCRUZ 1266 enhanced by whole-cell mutagenesis. 3 Biotech 9, 2 (2019). https://doi.org/10.1007/s13205-018-1527-1
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DOI: https://doi.org/10.1007/s13205-018-1527-1