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Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium

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Summary

Isolation and identification of a thermotolerant feather-degrading bacterial strain from Thai soil as well as purification and properties of its keratinase were investigated. The thermotolerant bacterium was identified as Bacillus licheniformis. The keratinase was purified to homogeneity by three-step chromatography. The purified enzyme exhibited a high specific activity (218 U mg−1) with 86-fold purification and 25% yield. The enzyme was monomeric and had a molecular mass of 35 kDa. The optimum pH and temperature for the enzyme were 8.5 and 60 °C, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA and iodoacetamide, but was stimulated by metal ions. It hydrolysed soluble proteins with a relative activity of 4–100% and insoluble proteins, including keratins, with a relative activity of 3–35%. Therefore, the enzyme could improve the nutritional value of meat- and poultry-processing wastes containing keratins, collagen and gelatin.

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References

  • K. Altalo B.A. Gashe (1993) ArticleTitleProtease production by a thermophilic Bacillus species (P-001A) which degrade various kinds of fibrous protein Biotechnology Letters 15 1151–1156

    Google Scholar 

  • P. Andrews (1964) ArticleTitleEstimation of the molecular weights of proteins by Sephadex gel-filtration Biochemical Journal 91 222–233 Occurrence Handle1:CAS:528:DyaF2cXns1Crug%3D%3D

    CAS  Google Scholar 

  • J. Bajorath W. Hinrichs W. Saenger (1988) ArticleTitleThe enzymatic activity of proteinase K is controlled by calcium European Journal of Biochemistry 176 441–447 Occurrence Handle10.1111/j.1432-1033.1988.tb14301.x Occurrence Handle1:CAS:528:DyaL1cXmtVOgtL4%3D

    Article  CAS  Google Scholar 

  • B. Bockle B. Galunsky R. Muller (1995) ArticleTitleCharacterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530 Applied and Environmental Microbiology 61 3705–3710 Occurrence Handle1:STN:280:DyaK28%2FptVKhtg%3D%3D

    CAS  Google Scholar 

  • P. Bressollier F. Letourneau M. Urdaci B. Verneuil (1999) ArticleTitlePurification and characterization of a keratinolytic serine protease from Streptomyces albidoflavus Applied and Environmental Microbiology 65 2570–2576 Occurrence Handle1:CAS:528:DyaK1MXjs12gtrg%3D

    CAS  Google Scholar 

  • S.W. Cheng H.M. Hu S.W. Shen H. Takagi M. Asono Y.C. Tsai (1995) ArticleTitleProduction and characterization of keratinase of a feather degrading Bacillus licheniformis PWD–1 Bioscience Biotechnology and Biochemistry 59 2239–2243 Occurrence Handle1:CAS:528:DyaK28XitFantg%3D%3D Occurrence Handle10.1271/bbb.59.2239

    Article  CAS  Google Scholar 

  • R.R. Chitte V.K. Nalawade S. Dey (1999) ArticleTitleKeratinolytic activity from the broth of a feather-degrading thermophilic Streptomyces thermoviolaceus strain SD8 Letters in Applied Microbiology 28 131–136 Occurrence Handle10.1046/j.1365-2672.1999.00484.x Occurrence Handle1:CAS:528:DyaK1MXhs12nsrg%3D

    Article  CAS  Google Scholar 

  • I.N.S. Dozie C.N. Okeke N.C. Unaeze (1994) ArticleTitleA thermostable, alkaline-active, keratinolytic proteinase from Chrysosporium keratinophilum World Journal of Microbiology and Biotechnology 10 563–567 Occurrence Handle10.1007/BF00367668 Occurrence Handle1:CAS:528:DyaK2MXitVWktLk%3D

    Article  CAS  Google Scholar 

  • A.B. Friedrich G. Antranikian (1996) ArticleTitleKeratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales Applied and Environmental Microbiology 62 2875–2882 Occurrence Handle1:CAS:528:DyaK28Xkslegu7Y%3D

    CAS  Google Scholar 

  • J. Friedrich H. Gradisar D. Mandin J.P. Chaumont (1999) ArticleTitleScreening fungi for synthesis of keratinolytic enzymes Letters in Applied Microbiology 28 127–130 Occurrence Handle10.1046/j.1365-2672.1999.00485.x Occurrence Handle1:CAS:528:DyaK1MXhs12nsrs%3D

    Article  CAS  Google Scholar 

  • A. Gessesse R. Hatti-Kaul B.A. Gashe B. Mattiasson (2003) ArticleTitleNovel alkaline proteases from alkaliphilic bacteria grown on chicken feather Enzyme and Microbial Technology 32 519–524 Occurrence Handle10.1016/S0141-0229(02)00324-1 Occurrence Handle1:CAS:528:DC%2BD3sXitFals7c%3D

    Article  CAS  Google Scholar 

  • H. Gradisar S. Kern J. Friedrich (2000) ArticleTitleKeratinase of Doratomyces microsporus Applied Microbiology and Biotechnology 53 196–200 Occurrence Handle1:CAS:528:DC%2BD3cXhsVOltrg%3D

    CAS  Google Scholar 

  • B.D. Hames (1998) Gel Electrophoresis of Proteins: A Practical Approach EditionNumber3 Oxford University Press New York

    Google Scholar 

  • C. Heussen E.B. Dowdle (1980) ArticleTitleElectrophoresis analysis of plasminogen activators in polyacrylamine gels containing sodium dodecyl sulphate and copolymerized substrates Analytical Biochemistry 102 196–202 Occurrence Handle10.1016/0003-2697(80)90338-3 Occurrence Handle1:CAS:528:DyaL3cXovFOmsg%3D%3D

    Article  CAS  Google Scholar 

  • M.A. Innis D.H. Gelfand (1990) Optimization of PCRs M.A. Innis D.H. Gelfand J.J. Sninsky T.J. White (Eds) PCR Protocols: A Guide to Methods and Application San Diego Academic Press 3–12

    Google Scholar 

  • J.M. Kim W.J. Lim H.J. Suh (2001) ArticleTitleFeather-degrading Bacillus species from poultry waste Process Biochemistry 37 287–291 Occurrence Handle10.1016/S0032-9592(01)00206-0 Occurrence Handle1:CAS:528:DC%2BD3MXmsFWqsbk%3D

    Article  CAS  Google Scholar 

  • C.G. Kumar M.P. Tiwari K.D. Jany (1999) ArticleTitleNovel alkaline serine protease from alkalophilic Bacillus spp.: purification and some properties Process Biochemistry 34 441–449 Occurrence Handle10.1016/S0032-9592(98)00110-1 Occurrence Handle1:CAS:528:DyaK1MXlt1Omtbg%3D

    Article  CAS  Google Scholar 

  • U.K. Laemmli (1970) ArticleTitleCleavage of structure protein during the assembly of the head of bacteriophage T4 Nature 227 680–685 Occurrence Handle10.1038/227680a0 Occurrence Handle1:CAS:528:DC%2BD3MXlsFags7s%3D

    Article  CAS  Google Scholar 

  • S. Lal R. Rajak C. Hasijia (1999) ArticleTitleIn vitro degradation of keratin by two species of Bacillus Journal of General and Applied Microbiology 45 283–287 Occurrence Handle1:CAS:528:DC%2BD3cXisVeksbs%3D

    CAS  Google Scholar 

  • D.J. Lane B. Pace G.J. Olsen D.A. Stahl M.L. Sogin N.R. Pace (1985) ArticleTitleRapid determination of 16S ribosomal RNA sequences for phylogenic analyses Proceedings of the National Academy of Sciences, USA 82 6955–6959 Occurrence Handle1:CAS:528:DyaL28XjtFalug%3D%3D

    CAS  Google Scholar 

  • C.G. Lee P.R. Ferket J.C.H. Shih (1991) ArticleTitleImprovement of feather digestibility by bacterial keratinase as a feed additive Federation of American Societies of Experimental Biology Journal 5 A596

    Google Scholar 

  • H. Lee D.B. Suh J.H. Hwang H.J. Suh (2002) ArticleTitleCharacterization of a keratinolytic metalloprotease from Bacillus sp. SCB-3 Applied Biochemistry and Biotechnology 97 123–133 Occurrence Handle10.1385/ABAB:97:2:123 Occurrence Handle1:CAS:528:DC%2BD38XivFyktLs%3D

    Article  CAS  Google Scholar 

  • F. Letourneau V. Soussolte P. Bressollier P. Branland B. Verneuil (1998) ArticleTitleKeratinolytic activity of Streptomyces sp.S.K1-02: a new isolated strain Letters in Applied Microbiology 26 77–88 Occurrence Handle10.1046/j.1472-765X.1998.00281.x Occurrence Handle1:CAS:528:DyaK1cXhtlOqsL0%3D

    Article  CAS  Google Scholar 

  • X. Lin G.D. Inglis L.J. Yanke K.J. Cheng (1999) ArticleTitleSelection and characterization of feather-degrading bacteria from canola meal compost Journal of Industrial Microbiology and Biotechnology 23 149–153 Occurrence Handle10.1038/sj.jim.2900706 Occurrence Handle1:CAS:528:DyaK1MXmtlOntr4%3D

    Article  CAS  Google Scholar 

  • X. Lin C.G. Lee E.S. Casale J.C.H. Shih (1992) ArticleTitlePurification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain Applied and Environmental Microbiology 58 3271–3275 Occurrence Handle1:CAS:528:DyaK3sXnsFynsA%3D%3D

    CAS  Google Scholar 

  • P.L. Manachini M.G. Fortina C. Parini (1988) ArticleTitleThermostable alkaline protease produced by Bacillus thermoruber – a new sp. of Bacillus Applied Microbiology and Biotechnology 28 409–413 Occurrence Handle10.1007/BF00268205 Occurrence Handle1:CAS:528:DyaL1cXkvVymtrc%3D

    Article  CAS  Google Scholar 

  • M. Okamoto Y. Yonejima Y. Tsujimoto Y. Suzuki K. Watanabe (2001) ArticleTitleA thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1 Applied Microbiology and Biotechnology 57 103–108 Occurrence Handle1:CAS:528:DC%2BD3MXotVSnu7g%3D

    CAS  Google Scholar 

  • A.A. Onifade N.A. Al-Sane A.A. Al Musallam S. Al-Zarban (1998) ArticleTitleA review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources Bioresource Technology 66 1–11 Occurrence Handle10.1016/S0960-8524(98)00033-9 Occurrence Handle1:CAS:528:DyaK1cXkvVWms7o%3D

    Article  CAS  Google Scholar 

  • D. Pissuwan W. Suntornsuk (2001) ArticleTitleProduction of keratinase by Bacillus sp. FK 28 isolated in Thailand Kasetsart Journal (Natural Science) 35 171–178 Occurrence Handle1:CAS:528:DC%2BD3sXhsFymtLw%3D

    CAS  Google Scholar 

  • S. Riessen G. Antranikian (2001) ArticleTitleIsolation of Thermoanaerobacter keratinophilus, a novel thermophilic, anaerobic bacterium with keratinolytic activity Extremophiles 5 399–408 Occurrence Handle10.1007/s007920100209 Occurrence Handle1:CAS:528:DC%2BD38XksFKksg%3D%3D

    Article  CAS  Google Scholar 

  • H. Rosen (1957) ArticleTitleA modified ninhydrin colorimetic analysis for amino acids Archives of Biochemistry and Biophysics 67 10–15 Occurrence Handle1:CAS:528:DyaG2sXks1ersA%3D%3D

    CAS  Google Scholar 

  • M. Rozs L. Manczinger C. Vagvolgyi F. Kevei (2001) ArticleTitleSecretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis FEMS Microbiology Letters 205 221–224 Occurrence Handle10.1016/S0378-1097(01)00462-1 Occurrence Handle1:CAS:528:DC%2BD3MXpt1alsrg%3D

    Article  CAS  Google Scholar 

  • J. Sambrook E.F. Fritsch T. Maniatis (1989) Molecular Cloning: A Laboratory Manual EditionNumber2 Cold Spring Harbor Laboratory Cold Spring Harbor

    Google Scholar 

  • S. Sangali A. Brandelli (2000a) ArticleTitleFeather keratin hydrolysis by a Vibrio sp. strain kr2 Journal of Applied Microbiology 89 735–743 Occurrence Handle10.1046/j.1365-2672.2000.01173.x Occurrence Handle1:CAS:528:DC%2BD3cXos1Oqt7k%3D

    Article  CAS  Google Scholar 

  • S. Sangali A. Brandelli (2000b) ArticleTitleIsolation and characterization of a novel feather-degrading bacterial strain Applied Biochemistry and Biotechnology 87 17–24 Occurrence Handle10.1385/ABAB:87:1:17 Occurrence Handle1:CAS:528:DC%2BD3cXjvFels70%3D

    Article  CAS  Google Scholar 

  • J. Singh R.M. Vohra D.K. Sahoo (2001) ArticleTitlePurification and characterization of two extracelluar alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus Journal of Industrial Microbiology and Biotechnology 26 387–393 Occurrence Handle10.1038/sj.jim.7000151 Occurrence Handle1:CAS:528:DC%2BD3MXms1Khsrk%3D

    Article  CAS  Google Scholar 

  • P.H.A. Sneath N.S. Mair M.E. Sharpe J.G. Holt (1986) Bergey’s Manual of Systematic Bacteriology NumberInSeriesVol. 2 Williams and Wilkins Baltimore

    Google Scholar 

  • H. Takami N. Satoshi R. Aono K. Horikoshi (1992) ArticleTitleDegradation of human hair by a thermostable alkaline protease from alkaliphilic Bacillus sp. no. AH-101 Bioscience Biotechnology and Biochemistry 56 1667–1669 Occurrence Handle1:CAS:528:DyaK38XmsFOnsrk%3D

    CAS  Google Scholar 

  • N.H. Towatana A. Painupong P. Suntinanalert (1999) ArticleTitlePurification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719 Journal of Bioscience and Bioengineering 87 581–587

    Google Scholar 

  • K. Wawrzkiewicz T. Wolski J. Lobarzewski (1991) ArticleTitleScreening the keratinolytic activity of dermatophytes in vitro Mycopathologia 114 1–8 Occurrence Handle10.1007/BF00436684 Occurrence Handle1:CAS:528:DyaK38Xjt1Cr

    Article  CAS  Google Scholar 

  • C.M. Williams C.G. Lee J.D. Garlich J.C.H. Shih (1991) ArticleTitleEvaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein Poultry Science 70 85–94 Occurrence Handle1:CAS:528:DyaK3MXisVKht74%3D

    CAS  Google Scholar 

  • C.M. Williams C.S. Richter J.M. MacKenzie J.C.H. Shih (1990) ArticleTitleIsolation, identification and characterization of a feather-degrading bacterium Applied and Environmental Microbiology 56 1509–1515 Occurrence Handle1:CAS:528:DyaK3cXksFGltrg%3D

    CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, 10140, Thailand

    Worapot Suntornsuk, Junthip Tongjun & Poranee Onnim

  2. Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, 606-8585, Japan

    Hiroshi Oyama & Kohei Oda

  3. Department of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok, 10140, Thailand

    Kanok Ratanakanokchai

  4. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Thanit Kusamran

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  1. Worapot Suntornsuk
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  2. Junthip Tongjun
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  3. Poranee Onnim
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  4. Hiroshi Oyama
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  5. Kanok Ratanakanokchai
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  6. Thanit Kusamran
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  7. Kohei Oda
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Correspondence to Worapot Suntornsuk.

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Suntornsuk, W., Tongjun, J., Onnim, P. et al. Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium. World J Microbiol Biotechnol 21, 1111–1117 (2005). https://doi.org/10.1007/s11274-005-0078-x

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  • DOI: https://doi.org/10.1007/s11274-005-0078-x

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