Skip to main content
SpringerLink
Log in

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50–300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and β-keratins at 60–80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

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

Access this article

Log in via an institution

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

Similar content being viewed by others

References

  • Ahluwalia SS, Goyal D (2007) Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresour Technol 98:2243–2257

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Blumentals II, Robinson AS, Kelly RM (1990) Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl Environ Microbiol 56:1992–1998

    PubMed Central  CAS  PubMed  Google Scholar 

  • Bockle B, Muller R (1997) Reduction of disulfide bonds by Streptomyces pactum during growth on chicken feathers. Appl Environ Microbiol 63:790–792

    PubMed Central  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brandelli A, Daniel DJ, Riffel A (2010) Biochemical features of microbial keratinases and their production and applications. Appl Microbiol Biotechnol 85:1735–1750

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cheng G, Zhao P, Tang XF, Tang B (2009) Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF Microbiol 155:3661–3672

    Article  CAS  Google Scholar 

  • Clamp AR, Jayson GC (2005) The clinical potential of antiangiogenic fragments of extracellular matrix proteins. Br J Cancer 93:967–972

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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 Biodegrad 63:358–363

    Article  CAS  Google Scholar 

  • Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H (1974) Proteinase K from Tritirachium album Limber. Eur J Biochem 47:91–97

    Article  CAS  PubMed  Google Scholar 

  • 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

    PubMed Central  CAS  PubMed  Google Scholar 

  • Gousterova A, Braikova D, Goshev I, Christov P, Tishinov K, Vasileva-Tonkova E, Haertle T, Nedkov P (2005) Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis. Lett Appl Microbiol 40:335–340

    Article  CAS  PubMed  Google Scholar 

  • Gu ZH, Xie XL, Liu XD, Feng GD, Zhu HH, Yao Q (2013) Isolation and characterization of a feather-degrading bacterium. Microbiol China 40(5):792–801

    Google Scholar 

  • Gupta R, Mohapatra H (2003) Microbial biomass: an economical alternative for removal from waste water. Indian J Exp Biol 41:945–966

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Hadas A, Kautsky L (1994) Feather meal, a semi-slow-release nitrogen fertilizer for organic farming. Fertil Res 38:165–170

    Article  Google Scholar 

  • Ignatova Z, Gousterova A, Spassov G, Nedkov P (1999) Isolation and partial characterisation of extracellular keratinase from a wool degrading thermophilic actinomycete strain Thermoactinomyces candidus. Can J Microbiol 45:217–222

    Article  CAS  PubMed  Google Scholar 

  • Kataoka M, Yamaoka A, Kawasaki K, Shigeri Y, Watanabe K (2014) Extraordinary denaturant tolerance of keratinolytic protease complex assemblies produced by Meiothermus ruber H328. Appl Microbiol Biotechnol 98(7):2973–2980

    Article  CAS  PubMed  Google Scholar 

  • Khardenavis AA, Kapley A, Purohit HJ (2009) Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383. Waste Manag 29:1409–1415

    Article  CAS  PubMed  Google Scholar 

  • King J, Laemmli UK (1971) Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol 62:465–477

    Article  CAS  PubMed  Google Scholar 

  • Kluskens LD, Voorhorst WG, Siezen RJ, Schwerdtfeger RM, Antranikian G, van der Oost J, de Vos WM (2002) Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans. Extremophiles 6:185–194

    Article  CAS  PubMed  Google Scholar 

  • Kublanov IV, Tsiroulnikov KB, Kaliberda EM, Rumsh LD, Haertlé T, Bonch-Osmolovskaya EA (2009) Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. strain 1004–09 isolated from a hot spring in the Baikal rift zone. Microbiology (Russia) 78:67–75

    CAS  Google Scholar 

  • Kunert J (1989) Biochemical mechanism of keratin degradation by the actinomycete Streptomyces fradiae and the fungus Microsporum gypseum: a comparison. J Basic Microbiol 29:597–604

    Article  CAS  Google Scholar 

  • Langeveld JP, Wang JJ, Van de Wiel DF, Shih GC, Garssen GJ, Bossers A, Shih JC (2003) Enzymatic degradation of prion protein in brain stem from infected cattle and sheep. J Infect Dis 188:1782–1789

    Article  CAS  PubMed  Google Scholar 

  • Lee JK, Kim YO, Kim HK, Park YS, Oh TK (1996) Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. Biosci Biotechnol Biochem 60:840–846

    Article  CAS  PubMed  Google Scholar 

  • Liang X, Bian Y, Tang X-F, Xiao G, Tang B (2010) Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions. Appl Microbiol Biotechnol 87:999–1006

    Article  CAS  PubMed  Google Scholar 

  • Lin X, Lee CG, Casale ES, Shih JC (1992) Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain. Appl Environ Microbiol 58:3271–3275

    PubMed Central  CAS  PubMed  Google Scholar 

  • Lin X, Kelemen DW, Miller ES, Shih JC (1995) Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1. Appl Environ Microbiol 61:1469–1474

  • Lin X, Shih J, Swaisgood HE (1996) Hydrolysis of feather keratin by immobilized keratinase. Appl Environ Microbiol 62:4273–4275

    PubMed Central  CAS  PubMed  Google Scholar 

  • Lin X, Wong SL, Miller ES, Shih JC (1997) Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis. J Ind Microbiol Biotechnol 19:134–138

    Article  CAS  PubMed  Google Scholar 

  • Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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 50:89–96

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Nam GW, Lee DW, 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Petrova DH, Shishkov SA, Vlahov SS (2006) Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties. J Basic Microbiol 46(4):275–285

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Ramnani P, Gupta R (2007) Keratinases vis-à-vis conventional proteases and feather degradation. World J Microbiol Biotechnol 23:1537–1540

    Article  CAS  Google Scholar 

  • Ramnani P, Singh R, Gupta R (2005) Keratinolytic potential of Bacillus licheniformis RG1: structural and biochemical mechanism of feather degradation. Can J Microbiol 51:191–196

    Article  CAS  PubMed  Google Scholar 

  • Riessen S, Antranikian G (2001) Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophiles 5:399–408

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Siezen RJ, Leunissen JA (1997) Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6:501–523

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Tang B, Nirasawa S, Kitaoka M, Hayashi K (2002) In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64. Biochem Biophys Acta 1596:16–27

    CAS  PubMed  Google Scholar 

  • Teplyakov AV, Kuranova IP, Harutyunyan EH, Vainshtein BK, Frommel C, Hohne WE, Wilson KS (1990) Crystal structure of thermitase at 1.4 Å resolution. J Mol Biol 214:261–279

    Article  CAS  PubMed  Google Scholar 

  • Teresa KK, Justyna B (2011) Biodegradation of keratin waste: theory and practical aspects. Waste Manag 31:1689–1701

    Article  Google Scholar 

  • Tsiroulnikov K, Rezai H, Bonch-Osmolovskaya E, Nedkov P, Gousterova A, Cueff V, Godfroy A, Barbier G, Métro F, Chobert JM, Clayette P, Dormont D, Grosclaude J, Haertlé T (2004) Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and Streptomyces subspecies. J Agric Food Chem 52:6353–6360

    Article  CAS  PubMed  Google Scholar 

  • Tsuchiya K, Nakamura Y, Sakashita H, Kimura T (1992) Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682. Biosci Biotechnol Biochem 56:246–250

    Article  CAS  PubMed  Google Scholar 

  • Vermelho AB, Mazotto AM, de Melo ACN, Vieira FHC, Duarte TR, Macrae A, Nishikawa MM, da Silva Bon EP (2009) Identification of a Candida parapsilosis strain producing extracellular serine peptidase with keratinolytic activity. Mycopathologia 169:57–65

    Article  Google Scholar 

  • Vignardet C, Guillaume YC, Michel L, Friedrich J, Millet J (2001) Comparison of two hard keratinous substrates submitted to the action of a keratinase using an experimental design. Int J Pharm 224:115–122

    Article  CAS  PubMed  Google Scholar 

  • Wawrzkiewicz K, Wolski T, Lobarewski J (1991) Screening the keratinolytic activity of dermatophytes in vitro. Mycopathologia 114:1–8

    Article  CAS  PubMed  Google Scholar 

  • Williams CM, Richter CS, Mackenzie JM, Shih JC (1990) Isolation, identification, and characterization of a feather-degrading bacterium. Appl Environ Microbiol 56:1509–1515

    PubMed Central  CAS  PubMed  Google Scholar 

  • Yamamura S, Morita Y, Hasan Q, Yokoyama K, Tamiya E (2002) Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp. Biochem Biophys Res Commun 294:1138–1143

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Zabolotskaya MV, Demidyuk IV, Akimkina TV, Kostrov SV (2004) A novel neutral protease from Thermoactinomyces species 27a: sequencing of the gene, purification, and characterization of the enzyme. Protein J 23(7):483–492

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the National Natural Science Foundation of China (30970053 and 31270099) and the National Innovation Experiment Program for University Students, China (081048619).

Author information

Authors and Affiliations

  1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China

    Liyuan Wang, Guyue Cheng, Yuxia Ren, Zheng Dai, Zhong-Shu Zhao, Feng Liu, Shiyong Li, Yahan Wei, Jing Xiong, Xiao-Feng Tang & Bing Tang

  2. Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, 430072, China

    Xiao-Feng Tang & Bing Tang

  3. Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China

    Liyuan Wang

Authors
  1. Liyuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guyue Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuxia Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zheng Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhong-Shu Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Feng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shiyong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yahan Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jing Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xiao-Feng Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Bing Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bing Tang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 237 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, L., Cheng, G., Ren, Y. et al. Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease. Appl Microbiol Biotechnol 99, 3949–3959 (2015). https://doi.org/10.1007/s00253-014-6207-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-6207-4

Keywords

Search

Navigation