Journal of Microbiology and Biotechnology
The Korean Society for Microbiology and Biotechnology publishes the Journal of Microbiology and Biotechnology.

2017 ; Vol.27-12: 2190~2198

AuthorLin Wang, Yuting Qian, Yun Cao, Ying Huang, Zhizhou Chang, Hongying Huang
Place of dutyCircular Agriculture Research Center, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R. China,Nanjing Institute of Agricultural Sciences in Jiangsu Hilly Area, Nanjing 210046, P.R. China
TitleProduction and Characterization of Keratinolytic Proteases by a Chicken Feather-Degrading Thermophilic Strain, Thermoactinomyces sp. YT06
PublicationInfo J. Microbiol. Biotechnol.2017 ; Vol.27-12
AbstractThermoactinomyces sp. strain YT06 was isolated from poultry compost and observed to degrade integral chicken feathers completely at 60°C, resulting in the formation of 3.24 mg/ml of free amino acids from 50 ml of culture containing 10 g/l chicken feathers. Strain YT06 could grow and secrete keratinase using feather as the only carbon and nitrogen sources without other supplement, but complementation of 10 g/l sucrose and 4 g/l NaNO3 increased the production of the keratinolytic enzyme. The maximum protease activity obtained was 110 U/ml and for keratinase was 42 U/ml. The keratinase maintained active status over a broad pH (pH 8-11) and temperature (60-75°C). It was inhibited by serine protease inhibitors and most metal ions; however, it could be stimulated by Mn2+ and the surfactant Tween-20. A reductive agent (β-mercaptoethanol) was observed to cleave the disulfide bond of keratin and improve the access of the enzyme to the keratinaceous substrate. Zymogram analysis showed that strain YT06 primarily secreted keratinase with a molecular mass of approximately 35 kDa. The active band was assessed by MALDI-TOF mass spectrometry and was observed to be completely identical to an alkaline serine protease from Thermoactinomyces sp. Gus2-1. Thermoactinomyces sp. strain YT06 shows great potential as a novel candidate in enzymatic processing of hard-to-degrade proteins into high-value products, such as keratinous wastes.
Full-Text
Key_wordThermophile, keratinase, feather degradation, characterization
References
  1. Lange L, Huang Y, Busk PK. 2016. Microbial decomposition of keratin in nature - a new h ypothesis of industrial relevance. Appl. Microbiol. Biotechnol. 100: 2083-2096.
    Pubmed CrossRef Pubmed Central
  2. Mckittrick J, Chen PY, Bodde SG, Yang W, Novitskaya EE, Meyer MA. 2012. The structure, functions, and mechanical properties of keratin. JOM 64: 449-468.
    CrossRef
  3. Wang L, Cheng G, Ren Y, Dai Z, Zhao ZS, Liu F, et al. 2015. Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease. Appl. Microbiol. Biotechnol. 99: 3949-3959.
    Pubmed CrossRef
  4. Gradisar H, Friedrich J, Krizaj I, Jerala R. 2005. Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl. Environ. Microbiol. 71: 3420-3426.
    Pubmed CrossRef Pubmed Central
  5. Huang Y, Busk PK, Lange L. 2015. Production and characterization of keratinolytic proteases produced by Onygena corvina. Fungal Genom. Biol. 5: 119.
  6. Matsui T, Yamada Y, Mitsuya H, Shigeri Y, Yoshida Y, Saito Y, et al. 2009. Sustainable and practical degradation of intact chicken feathers by cultivating a newly isolated thermophilic Meiothermus ruber H328. Appl. Microbiol. Biotechnol. 82: 941-950.
    Pubmed CrossRef
  7. Riffel A, Daroit DJ, Brandelli A. 2011. Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6. New Biotechnol. 28: 153-157.
    Pubmed CrossRef
  8. 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 6: 1-8.
    Pubmed CrossRef Pubmed Central
  9. Abdel-Naby MA, Ibrahim MH, El-Refai HA. 2016. Catalytic, kinetic and thermodynamic properties of Bacillus pumilus FH9 keratinase conjugated with activated pectin. Int. J. Biol. Macromol. 85: 238-245.
    Pubmed CrossRef
  10. Kunert DJ. 2010. Biochemical mechanism of keratin degradation by the actinomycete Streptomyces fradiae and the fungus Microsporum gypseum: a comparison. J. Basic Microbiol. 29: 597-604.
    CrossRef
  11. Błyskal B. 2009. Fungi utilizing keratinous substrates. Int. Biodeterior. Biodegradation 63: 631-653.
    CrossRef
  12. Gupta R, Sharma R, Beg QK. 2013. Revisiting microbial keratinases: next generation proteases for sustainable biotechnology. Crit. Rev. Biotechnol. 33: 216-228.
    Pubmed CrossRef
  13. Korniłłowicz-Kowalska T, Bohacz J. 2011. Biodegradation of keratin waste: theory and practical aspects. Waste Manag. 31: 1689-1701.
    Pubmed CrossRef
  14. Brandelli A, Daroit DJ, Riffel A. 2010. Biochemical features of microbial keratinases and their production and applications. Appl. Microbiol. Biotechnol. 85: 1735-1750.
    Pubmed CrossRef
  15. Petrova DH, Shishkov SA, Vlahov SS. 2006. Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties. J. Basic Microbiol. 46: 275-285.
    Pubmed CrossRef
  16. 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: 483-492.
    Pubmed CrossRef
  17. Verma A, Singh H, Anwar MS, Kumar S, Ansari MW, Agrawal S. 2016. Production of thermostable organic solvent tolerant keratinolytic protease from Thermoactinomyces sp. RM4: IAA production and plant growth promotion. Front. Microbiol. 7: 1189.
    Pubmed CrossRef Pubmed Central
  18. Sambrook J, Russell DW. 2016. Molecular Cloning: A Laboratory Manual, pp. 895-909. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  19. Fang Z, Zhang J, Liu B, Jiang L, Du G, Chen J. 2014. Cloning, heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1. Process Biochem. 49: 647-654.
    CrossRef
  20. Gatti R, Gioia MG, Andreatta P, Pentassuglia G. 2004. HPLC-fluorescence determination of amino acids in pharmaceuticals after pre-column derivatization with phanquinone. J. Pharm. Biomed. Anal. 35: 339-348.
    CrossRef
  21. Liang X, Bian Y, Tang XF, 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.
    Pubmed CrossRef
  22. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
    Pubmed CrossRef
  23. Jaouadi B, Ellouz-Chaabouni S, Rhimi M, Bejar S. 2008. Biochemical and molecular characterization of a detergentstable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency. Biochimie 90: 1291-1305.
    Pubmed CrossRef
  24. Gupta R , Ramnani P. 2 006. Microbial k eratinases a nd t h eir prospective applications: an overview. Appl. Microbiol. Biotechnol. 70: 21-33.
    Pubmed CrossRef
  25. Kumar R, Balaji S, Uma TS, Mandal AB, Sehgal PK. 2010. Optimization of influential parameters for extracellular keratinase production by Bacillus subtilis (MTCC9102) in solid state fermentation using horn meal - a biowaste management. Appl. Biochem. Biotechnol. 160: 30-39.
    Pubmed CrossRef
  26. Ramnani P, Gupta R. 2004. Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology. Biotechnol. Appl. Biochem. 40: 191-196.
    Pubmed CrossRef
  27. Gioppo NMD, Moreira-Gasparin FG, Costa AM, Alexandrino AM, de Souza CG, Peralta RM. 2009. Influence of the carbon and nitrogen sources on keratinase production by Myrothecium verrucaria in submerged and solid state cultures. J. Ind. Microbiol. Biotechnol. 36: 705-711.
    Pubmed CrossRef
  28. Anbu P, Gopinath S, Hilda A, Lakshmipriya TG. 2007. Optimization of extracellular keratinase production by poultry farm isolate Scopulariopsis brevicaulis. Bioresour. Technol. 98: 1298-1303.
    Pubmed CrossRef
  29. Elbondkly AM. 2010. Keratinolytic activity from new recombinant fusant AYA2000, derived from endophytic Micromonospora strains. Can. J. Microbiol. 56: 748-760.
    Pubmed CrossRef
  30. 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 Pubmed Central
  31. Liang JD, Han YF, Zhang JW, Du W, Liang ZQ, Li ZZ. 2011. Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFRH49-1. J. Appl. Microbiol. 110: 871-880.
    Pubmed CrossRef
  32. Wang B, Yang W, Mckittrick J, Meyers MA. 2016. Keratin:structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration. Prog. Mater. Sci. 76: 229-318.
    CrossRef
  33. Nam GW, Lee DW, Lee HS, Lee NJ, Kim BC, Choe EA, et al. 2003. Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch. Microbiol. 178: 538-547.
    Pubmed CrossRef
  34. Kublanov IV, Tsirul’Nikov KB, Kaliberda EN, Rumsh LD, Haertle T, Bonchosmolovskaia EA. 2009. Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. strain 1004-09 isolated from a hot spring in the Baikal Rift zone. Microbiology 78: 79-88.
    Pubmed CrossRef
  35. Riessen S, Antranikian G. 2001. Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophiles 5: 399-408.
    Pubmed CrossRef
  36. Habbeche A, Saoudi B, Jaouadi B, Haberra S, Kerouaz B, Boudelaa M, et al. 2014. Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 isolated from poultry compost. J. Biosci. Bioeng. 117: 413-421.
    Pubmed CrossRef
  37. 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.
    Pubmed CrossRef



Copyright © 2009 by the Korean Society for Microbiology and Biotechnology.
All right reserved. Mail to jmb@jmb.or.kr
Online ISSN: 1738-8872    Print ISSN: 1017-7825    Powered by INFOrang Co., Ltd