Abstract
The goal of this study was to identify and biochemically characterize a novel hyperthermostable keratinase from microorganisms for feather waste degradation. Here, a hyperthermophilic Geoglobus acetivorans keratinase (GacK) gene was chosen based on a search of a sequence database. The selected GacK gene was synthesized, cloned, and successfully expressed without a signal peptide in the E. coli system. A monomer of approximately 58 kDa was obtained in a soluble form and purified. The recombinant GacK displayed the highest activity at an optimum temperature of 100 °C and a pH of 10. The hyperthermostable GacK enzymatic performance remained high even after incubation in nonionic surfactants and the chelating agent EDTA. The residual and keratinolytic activities of GacK, as determined with azocasein and keratin azure used as substrates, remained significantly greater than 80% at 130 °C for 7 h. The kinetic parameters Km and Vmax for azure keratin were 0.41 mg/ml and 875.14 unit/mg, respectively, while those for azocasein were 1.51 mg/ml and 505.32 unit/mg, respectively. The results suggest that the enzyme is among the most hyperthermostable keratinases. Because of its enzymatic characteristics to degrade keratin azure at high temperatures, GacK may potentially be utilized in future industrial applications.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- GacK:
-
Geoglobus acetivorans Keratinase
- FpeK:
-
Fervidobacterium pennivorans Keratinase
- BalK:
-
Bacillus licheniformis Keratinase
- StmK :
-
Stenotrophomonas maltophilia Keratinase
- BasK :
-
Bacillus subtilis Keratinase
- KerC:
-
KerC Bacillus subtilis keratinase
- BjbK:
-
Bacillus Sp. JB99 keratinase
- IPTG:
-
Isopropylthiogalactoside
- NaCl:
-
Sodium chloride
- SDS-PAGE:
-
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- Asp:
-
Aspartate
- His:
-
Histidine
- Ser:
-
Serine
- KDa:
-
Kilodalton
References
Agrahari S, Wadhwa N (2010) Degradation of chicken feather a poultry waste product by keratinolytic bacteria isolated from dumping site at ghazipur poultry process. Plant Int J S 9:482–489. https://doi.org/10.3923/ijps.2010.482.489
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836-80360-2
Bohacz J (2017) Biodegradation of feather waste keratin by a keratinolytic soil fungus of the genus Chrysosporiumand statistical optimization of feather mass loss. World J Microbio Biotechnol. 33:13–29. https://doi.org/10.1007/s11274-016-2177-2
Bradford MM (1976) A Rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding (PDF). Anal Bioc 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Christopher TS, James AC, Diep MN, Chang-Hao W, Benjamin MZ, James RC, Jonathan MC, Jonathan KO, Gina LL, Gerrit JS, Michael WA, Robert MK (2018) Biotechnology of extremely thermophilic archaea. FEMS Microbiol Rev 42:543–578. https://doi.org/10.1093/femsre/fuy012
Confidence intervals of proportions were calculated using the GraphPad QuickCalcs Web site: https://www.graphpad.com/quickcalcs/ConfInterval1.cfm. Accessed Nov 2015
Cuecas A, Kanoksilapatham W, Gonzalez J (2017) Evidence of horizontal gene transfer by transposase gene analyses in Fervidobacteriumspecies. PLoS ONE 12:e0173961. https://doi.org/10.1371/journal.pone.0173961
Dai Z, Fu H, Zhang Y, Zeng J, Tang B, Tang XF (2012) Insights into the maturation of hyperthermophilic pyrolysin and the roles of its N-terminal propeptide and long C-terminal extension. Appl Environ. 78:4233–4241. https://doi.org/10.1128/AEM.00548-12
Dozie INS, Okeke CN, Unaeze NC (1994) A thermostable, alkaline-active, keratinolytic proteinase from Chrysosporium Keratinophilum. World J Microbio Biotechnol. 5:563–567. https://doi.org/10.1007/BF00367668
El-Ayouty YM, El-Said A, Salama AM (2012) Purification and characterization of a keratinase from the feather-degrading cultures of Aspergillus flavipes. Afr J Biotechnol 11(9):2313–2319
Emeka A, Okoromaa H, Garelicka O, Abiolab O, Diane P (2012) Identification and characterisation of a Bacillus licheniformis strain with profound keratinase activity for degradation of melanised feather. Inter biodeterior Biodegrad 74:54–60. https://doi.org/10.1016/j.ibiod.2012.07.013
Govarthanan M, Selvankumar T, Arunprakash S (2011) Production of keratinolytic enzyme by a newly isolated feather degrading Bacillus sp from chick feather waste. Int J Pharma Bio Sci 2:259–265. https://doi.org/10.1005/2011/295
He L, Wu YQ, Zhang XH (2008) Medium factor optimization and fermentation kinetics for phenazine-1-carboxylic acid production by Pseudomonas sp. M18G. Biotechnol Bioeng 100:250–259. https://doi.org/10.1002/bit.21767
Hirata A, Hori Y, Koga Y, Okada J, Sakudo A, Ikuta K, Kanaya S, Takano K (2013) Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein. BMC Biotech 13:19–38. https://doi.org/10.1186/1472-6750-13-19
Kim JS, Kluskens LD, Vos WM, Huber R, Oost J (2004) Crystal structure of fervidolysin from Fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin. J. Mol. Biol. 335:787–797. https://doi.org/10.1016/j.jmb.2003.11.006
Kluskens LD, Voorhorst WG, Siezen RJ, Schwerdtfeger RM, Antranikian G, Oost J, Vos WM (2002) Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans. Extremophiles 6:185–194. https://doi.org/10.1007/s007920100239
Liu B, Zhang J, Li B, Liao X, Du G, Chen J (2013) Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilisWB600 expression system and its application in wool fiber processing. World J Microbiol Biotechnol. 29:825–832. https://doi.org/10.1007/s11274-012-1237-5
Liu Q, Zhang T, Song N (2014) Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis. Int Biodeterior Biodegrad. 96:26–32. https://doi.org/10.1016/j.ibiod.2014.08.008
Mardanov AV, Galina BS, Alexander IS, Alexey VB, Sergey NG, Ilya VK, Elizaveta AB, Konstantin GS, Nikolai VR (2015) The Geoglobus Acetivorans genome: Fe(III) reduction, acetate utilization, autotrophic growth, and degradation of aromatic compounds in a hyperthermophilic archaeon. Appl Environ Microbiol 11:2705–2714. https://doi.org/10.1128/AEM.02705-14
Mukherjee AK, Adhikari H, Rai SK (2008) Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using imperata cylindrical grass and potato peel as a low-cost medium: characterization application of enzyme in detergent formulation. Biochem Eng J 39:353–361. https://doi.org/10.1016/j.bej.2007.09.017
Muthusamy G, Thangasamy S, Kandasamy S, Chinnappan S, Vincent A, Seralathan KK (2014) Response surface methodology based optimization of keratinase production from alkali-treated feather waste and horn waste using Bacillus sp. MG-MASC-BT. J Ind Eng Chem. 27:25–30. https://doi.org/10.1016/j.jiec.2014.12.022
Nam GW, Dong WL, Han SL, Nam JL, Byoung CK, Eun AC, Jae KH, Maggy TS, Yu RP (2002) Native-feather degradation by Fervidobacterium Islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbio. 178:538–547. https://doi.org/10.1007/s00203-002-0489-0
Nielsen H (2017) Predicting secretory proteins with SignalP. Methods Mol Biol 1611:59–73. https://doi.org/10.1007/978-1-4939-7015-5_6
Ozaltin K, Postnikov PS, Trusova ME, Sedlarik V, Di Martino A (2019) Polysaccharides based microspheres for multiple encapsulations and simultaneous release of proteases. J Biol Macromol 132:24–31
Park MH, Walpola BC, Ho-Yoon M (2013) Purification and characterization of protease enzyme from Burkholderia stabilis. Afr J Biotech. 12:1408–1418. https://doi.org/10.5897/AJB12.1574
Rodriguez-Siordia I, Rojo-Arreola L, Navarrete DTM, García-Carreño F (2017) American lobster Cathepsin D, an aspartic peptidase resistant to proteolysis and active in organic solvents, nonionic detergents and salts. Int J Biol Macromol 107:1501–1509. https://doi.org/10.1016/j.ijbiomac.2017.10.007
Sako Y, Croocker PC, Ishida Y (1997) An extremely heat-stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1. FEBS Lett. 415:329–334. https://doi.org/10.1016/s0014-5793(97)01153-8
Salminen EA, Jukka AR (2002) Semi-continuous anaerobic digestion of solid poultry slaughterhouse waste: effect of hydraulic retention time and loading. Water Res. 36:3175–3182. https://doi.org/10.1016/S0043-1354(02)00010-6
Salminen E, Rintala J (2002) Anaerobic digestion of organic solid poultry slaughterhouse waste—a review. Biores Tech 83:13–26. https://doi.org/10.1016/S0960-8524(01)001997
Shankar S, Rao MR, Lax S (2011) Purification and characterization of an alkaline protease by a new strain of Beauveria sp. Proc Biochem 47:579–585. https://doi.org/10.1016/j.procbio.2010.10.013
Sittipol D, Saelao P, Lohnoob T, Lerksuthiratb T, Kumsang Y, Wanta W, Khunraea P, Rattanarojpong T, Jongruja N (2019) Cloning, expression, purification and characterization of a thermo- and surfactant-stable protease from Thermomonospora curvata. Biocatal Agric Biotechnol 19:118–143. https://doi.org/10.1016/j.bcab.2019.101111
Smith BJ (1984) SDS polyacrylamide gel electrophoresis of proteins. Nucleic Acids Res 1:41–56. https://doi.org/10.1385/0-89603-062-8:41
Suh HJ, Lee HK (2001) Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1. J Protein Chem 20:165–169. https://doi.org/10.1023/A:1011075707553
Takami H, Nogi Y, Horikoshi K (1999) Reidentification of the keratinase-producing facultativel alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans. Extremophiles 3:293–296. https://doi.org/10.1007/s007920050130
Tanaka S, Saito K, Chon H, Matsumura H, Koga Y, Takano K, Kanaya S (2006) Crystallization and preliminary X-ray diffraction study of an active-site mutant of pro-Tk-subtilisin from a hyperthermophilic archaeon. Acta Crystallogr Sect F: Struct Biol Cryst Commun 62:902–905. https://doi.org/10.1107/S1744309106030454
Vidmar B, Vodovnik M (2018) Microbial keratinases: enzymes with promising biotechnological applications. Food Technol Biotechnol 56:312–328. https://doi.org/10.17113/ftb.56.03.18.5658
Voorhorst WG, Eggen RI, Geerling AC, Platteeuw C, Siezen RJ, Vos WM (1996) Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 271:20426–20431. https://doi.org/10.1074/jbc.271.34.20426
Wang X, Parsons CM (1987) Effect of processing systems on protein quality of feather meals and hog hair meals. Poul Sci 76:491–496. https://doi.org/10.1093/ps/76.3.491
Wang P, Wang Q, Cui L (2011) The combined use of cutinase, keratinase and protease treatments for wool bio-antifelting. Fib Polym 12:760–766. https://doi.org/10.1007/s12221-011-0760-6
Waterhouse A, Bertoni A, Bienert M, Studer S, Tauriello G, Gumienny G, Heer De -Beer RFT, Rempfer TAP, Bordoli C, Lepore L, Schwede R (2018) SWISS-MODEL: homology modeling of protein structures and complexes. Nucleic Acids Res. 46:296–303. https://doi.org/10.1093/nar/gky427
Wolf K, Gilbert PA (1992). EDTA—ethylenediaminetetraacetic acid. In: de Oude NT (eds) Detergents. Anthropogenic Compounds, V3. https://doi.org/https://doi.org/10.1007/978-3-540-471080_7
Zhan D, Bai A, Yu L, Han W, Feng Y (2014) Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120. PLoS ONE 9:0103902–0103909. https://doi.org/10.1371/journal.pone.0103902
Acknowledgements
Accession numbers: G. acetivorans (FJ216404.1), GacK (accession number for the gene sequence of strain SBH6 is FJ216404 version FJ216404.1), the accession numbers were deposited in GenBank with accession numbers AIY90051.1.
Funding
This research was funded by the Thailand Science Research and Innovation and Office of the Higher Education Commission (contract number MRG6180189). This work was supported by a research fund from the Faculty of Science, King Mongkut’s University of Technology Thonburi, KMUTT Research Fund, and Petchra Pra Jom Klao Ph.D. scholarship.
Author information
Authors and Affiliations
Contributions
DS: performed the experiments and original draft of the manuscript. SR: assisted in performing the experiments. YSA: proofread the manuscript and provided technical writing assistance. TLo: provision of experimental materials and discussion. TLe: technical writing assistance and methodology. YK: data curation and discussion. WY: Provision of experimental materials and discussion. PK: data curation and conceptualization. TR: data curation and conceptualization. KP: funding and Supervision. NJ: funding acquisition, methodology, data curation and supervision.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest in the publication.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Rights and permissions
About this article
Cite this article
Sittipol, D., Rodpan, S., Ajingi, Y.S. et al. Identification, overexpression, purification, and biochemical characterization of a novel hyperthermostable keratinase from Geoglobus acetivorans. 3 Biotech 11, 2 (2021). https://doi.org/10.1007/s13205-020-02538-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-020-02538-1