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Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes

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Abstract

A novel feather-degrading bacterium named CA-1 was isolated from the gut of the spider Chilobrachys guangxiensis, which degrades native whole chicken feathers within 20 h. The CA-1 was confirmed to belong to Stenotrophomonas maltophilia based on morphologic and molecular analysis. Maximum feather degradation activity of the bacterium was observed at 37 °C in basal feather medium (NaCl 0.5 g/L, KH2PO4 0.3 g/L, K2HPO4 0.4 g/L, feather powder 10.0 g/L, pH 8.0), which was inhibited when glucose and ammonium nitrate were added in the medium. Furthermore, the purified enzymes under the optimal and suppressive conditions were analyzed respectively by SDS-PAGE and LC–MS/MS. Three enzymes, namely alkaline serine protease (29.1 kDa), ABC transporter permease (27.5 kDa), and alkaline phosphatase (40.8 kDa), were isolated and identified from the supernatant of the optimal culture and were considered to play principal roles. On the other hand, the potential synergic effects of the three proteins in S. maltophilia CA-1 feather degradation system were analyzed theoretically. CA-1 may product outer-membrane vesicles comprised of membranes and periplasmic proteins in the feather medium. The newly identified CA-1 and its synergic enzymes provide a new insight into further understanding the molecular mechanism of feather degradation by microbes. They also have potential application in cost-effectively degrading feathers into feeds and fertilizers through careful optimization and engineering of the three newly identified enzymes.

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References

  1. Riffel A et al (2007) Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6. J Biotechnol 128(3):693–703

    Article  CAS  Google Scholar 

  2. Zaghloul TI, Embaby AM, Elmahdy AR (2011) Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor. Bioresour Technol 102(3):2387–2393

    Article  CAS  Google Scholar 

  3. Paul T, Halder SK, Das A, Bera S, Maity C, Mandal A, Das PS, Mohapatra PKD, Pati BR, Mondal KC (2013) Exploitation of chicken feather waste as a plant growth promoting agent using keratinase producing novel isolate Paenibacillus woosongensis TKB2. Biocatal Agric Biotechnol 2:50–57

    Article  Google Scholar 

  4. Eslahi N, Dadashian F, Nejad NH (2013) An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis. Prep Biochem Biotechnol 43(7):624–648

    Article  CAS  Google Scholar 

  5. Gupta R, Ramnani P (2006) Microbial keratinases and their prospective applications: an overview. Appl Microbiol Biotechnol 70(1):21–33

    Article  CAS  Google Scholar 

  6. Daroit DJ, Brandelli A (2014) A current assessment on the production of bacterial keratinases. Crit Rev Biotechnol 34(4):372–384

    Article  CAS  Google Scholar 

  7. Gupta R, Sharma R, Beg QK (2013) Revisiting microbial keratinases: next generation proteases for sustainable biotechnology. Crit Rev Biotechnol 33(2):216–228

    Article  CAS  Google Scholar 

  8. Vesela M, Friedrich J (2009) Amino acid and soluble protein cocktail from waste keratin hydrolysed by a fungal keratinase of Paecilomyces marquandii. Biotechnol Bioprocess Eng 14(1):84–90

    Article  CAS  Google Scholar 

  9. Poopathi S et al (2014) Purification and characterization of keratinase from feather degrading bacterium useful for mosquito control: a new report. Trop Biomed 31(1):97–109

    CAS  PubMed  Google Scholar 

  10. Sharma R, Gupta R (2012) Coupled action of gamma-glutamyl transpeptidase-glutathione and keratinase effectively degrades feather keratin and surrogate prion protein, Sup 35NM. Bioresour Technol 120:314–317

    Article  CAS  Google Scholar 

  11. Fang Z et al (2017) Keratinolytic protease: a green biocatalyst for leather industry. Appl Microbiol Biotechnol 101(21):7771–7779

    Article  CAS  Google Scholar 

  12. Cheng SW et al (1995) Production and characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-1. Biosci Biotechnol Biochem 59(12):2239–2243

    Article  CAS  Google Scholar 

  13. Bernal C, Diaz I, Coello N (2006) Response surface methodology for the optimization of keratinase production in culture medium containing feathers produced by Kocuria rosea. Can J Microbiol 52(5):445–450

    Article  CAS  Google Scholar 

  14. Lee H et al (2002) Characterization of a keratinolytic metalloprotease from Bacillus sp. SCB-3. Appl Biochem Biotechnol 97(2):123–133

    Article  CAS  Google Scholar 

  15. Thys RC, Brandelli A (2006) Purification and properties of a keratinolytic metalloprotease from Microbacterium sp. J Appl Microbiol 101(6):1259–1268

    Article  CAS  Google Scholar 

  16. 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(2):625–633

    Article  CAS  Google Scholar 

  17. Cao ZJ et al (2009) Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme. J Ind Microbiol Biotechnol 36(2):181–188

    Article  CAS  Google Scholar 

  18. Yamamura S et al (2002) Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp. Biochem Biophys Res Commun 294(5):1138–1143

    Article  CAS  Google Scholar 

  19. Fang Z et al (2013) Biodegradation of wool waste and keratinase production in scale-up fermenter with different strategies by Stenotrophomonas maltophilia BBE11-1. Bioresour Technol 140:286–291

    Article  CAS  Google Scholar 

  20. Fang Z, Zang J, Du G, Chen J (2017) Rational protein engineering approaches to further improve the keratinolytic activity and thermostability of engineered keratinase KerSMD. Biochem Eng J 127:7

    Article  Google Scholar 

  21. 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

    Article  CAS  Google Scholar 

  22. Liu QY et al (2014) Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis. Int Biodeterior Biodegradation 96:26–32

    Article  Google Scholar 

  23. Tamura K et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

    Article  CAS  Google Scholar 

  24. Kaul S, Sumbali G (1997) Keratinolysis by poultry farm soil fungi. Mycopathologia 139(3):137–140

    Article  CAS  Google Scholar 

  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  Google Scholar 

  26. Olsen JV et al (2007) Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4(9):709–712

    Article  CAS  Google Scholar 

  27. Neilson KA et al (2011) Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics 11(4):535–553

    Article  CAS  Google Scholar 

  28. Ribitsch D et al (2012) Extracellular serine proteases from Stenotrophomonas maltophilia: screening, isolation and heterologous expression in E. coli. J Biotechnol 157(1):140–147

    Article  CAS  Google Scholar 

  29. Bhange K, Chaturvedi V, Bhatt R (2016) Feather degradation potential of Stenotrophomonas maltophilia KB13 and feather protein hydrolysate (FPH) mediated reduction of hexavalent chromium. 3 Biotech 6(1):42

    Article  Google Scholar 

  30. Jankiewicz U, Larkowska E, Brzezinska MS (2016) Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4. J Biosci Bioeng 121(6):614–618

    Article  CAS  Google Scholar 

  31. Gouveia C et al (2008) Study on the bacterial midgut microbiota associated to different Brazilian populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae). Neotrop Entomol 37(5):597–601

    Article  Google Scholar 

  32. Hughes GL et al., Genome sequence of Stenotrophomonas maltophilia strain SmAs1, isolated from the Asian malaria mosquito Anopheles stephensi. Genome Announc (2016) 4(2)

  33. Indiragandhi P et al (2007) Cultivable bacteria associated with larval gut of prothiofos-resistant, prothiofos-susceptible and field-caught populations of diamondback moth, Plutella xylostella and their potential for, antagonism towards entomopathogenic fungi and host insect nutrition. J Appl Microbiol 103(6):2664–2675

    Article  CAS  Google Scholar 

  34. Morales-Jimenez J et al (2009) Bacterial community and nitrogen fixation in the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae). Microb Ecol 58(4):879–891

    Article  CAS  Google Scholar 

  35. Wang Z et al (2016) The optimization of fermentation conditions and enzyme properties of Stenotrophomonas maltophilia for protease production. Biotechnol Appl Biochem 63(2):292–299

    Article  CAS  Google Scholar 

  36. Blow F, Vontas J, Darby AC (2016) Draft genome sequence of Stenotrophomonas maltophilia SBo1 isolated from Bactrocera oleae. Genome Announc 4(5)

  37. Fang Z, Zhang J, Liu B, Du G, Chen J (2013) Biochemical characterization of three keratinolytic enzymes from Stenotrophomonas maltophilia BBE11-1 for biodegrading keratin wastes. Int Biodeterior Biodegradation 82:166–172

    Article  CAS  Google Scholar 

  38. Parrado J et al (2014) Proteomic analysis of enzyme production by Bacillus licheniformis using different feather wastes as the sole fermentation media. Enzyme Microb Technol 57:1–7

    Article  CAS  Google Scholar 

  39. Riessen S, Antranikian G (2001) Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophiles 5(6):399–408

    Article  CAS  Google Scholar 

  40. Maranhao FC et al (2009) Membrane transporter proteins are involved in Trichophyton rubrum pathogenesis. J Med Microbiol 58(Pt 2):163–168

    Article  CAS  Google Scholar 

  41. Jan AT (2017) Outer membrane vesicles (OMVs) of gram-negative bacteria: a perspective update. Front Microbiol 8:1053

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Educational Department Foundation of Hunan Province (Grant No. 16A098), Key R & D project of Hunan Provincial Science and Technology Department (Grant No. 2017NK2311) and National Natural Science Foundation of China (Grant No. 31772865). We thank Dr. Fangjia Lu in Purdue University for the professional edit.

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Authors and Affiliations

  1. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, People’s Republic of China

    Fang Qu, Qingwang Chen, Yiying Ding, Zihao Liu, Yan Zhao, Xuewen Zhang, Zikui Liu & Jinjun Chen

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  1. Fang Qu
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  2. Qingwang Chen
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  3. Yiying Ding
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  4. Zihao Liu
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  5. Yan Zhao
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  6. Xuewen Zhang
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  7. Zikui Liu
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  8. Jinjun Chen
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Correspondence to Jinjun Chen.

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Ethical approval

All experimental protocols used in the study were carried out in accordance with the guidelines and regulations of College of Bioscience and Biotechnology, Hunan Agricultural University, China. The feathers were collected from a local poultry-processing store. Here, the poultry were slaughtered, dissected and after carcass evaluation, meat was intended for consumption. Therefore, our research does not require the approval of Animal Experimentation committee.

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Qu, F., Chen, Q., Ding, Y. et al. Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes. Mol Biol Rep 45, 1681–1689 (2018). https://doi.org/10.1007/s11033-018-4311-8

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  • DOI: https://doi.org/10.1007/s11033-018-4311-8

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