Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity

  • Li, Xiaoguang (Key Laboratory of Leather Chemistry and Engineering, Ministry of Education and College of Biomass Science and Engineering, Sichuan University) ;
  • Zhang, Qian (Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education and College of Life Sciences, Sichuan University) ;
  • Gan, Longzhan (Key Laboratory of Leather Chemistry and Engineering, Ministry of Education and College of Biomass Science and Engineering, Sichuan University) ;
  • Jiang, Guangyang (Key Laboratory of Leather Chemistry and Engineering, Ministry of Education and College of Biomass Science and Engineering, Sichuan University) ;
  • Tian, Yongqiang (Key Laboratory of Leather Chemistry and Engineering, Ministry of Education and College of Biomass Science and Engineering, Sichuan University) ;
  • Shi, Bi (Key Laboratory of Leather Chemistry and Engineering, Ministry of Education and College of Biomass Science and Engineering, Sichuan University)
  • Received : 2021.08.30
  • Accepted : 2021.11.18
  • Published : 2022.01.28
Download PDF
⟨ Previous Next ⟩

Abstract

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80℃) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70℃ and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

Keywords

Acknowledgement

This work was supported by the National Key Research and Development Program of China (2017YFB0308401).

References

  1. Kandasamy N, Velmurugan P, Sundarvel A, Rao JR, Bangaru C, Palanisamy T. 2012. Eco-benign enzymatic dehairing of goatskins utilizing a protease from a Pseudomonas fluorescens species isolated from fish visceral waste. J. Clean. Prod. 25: 27-33. https://doi.org/10.1016/j.jclepro.2011.12.007
  2. Ockerman HW, Basu L. 2014. BY-PRODUCTS | Hides and Skins. In Dikeman M, Devine C (eds.), pp. 112-124. Encyclopedia of Meat Sciences (Second Ed.) Academic Press, Oxford.
  3. Kanagaraj J, Senthilvelan T, Panda RC, Kavitha S. 2015. Eco-friendly waste management strategies for greener environment towards sustainable development in leather industry: A comprehensive review. J. Clean. Prod. 89: 1-17. https://doi.org/10.1016/j.jclepro.2014.11.013
  4. Thanikaivelan P, Rao JR, Nair BU, Ramasami T. 2004. Progress and recent trends in biotechnological methods for leather processing. Trends Biotechnol. 22: 181-188. https://doi.org/10.1016/j.tibtech.2004.02.008
  5. Paul T, Jana A, Mandal AK, Mandal A, Das Mohpatra PK, Mondal KC. 2016. Bacterial keratinolytic protease, imminent starter for nextgen leather and detergent industries. Sustain. Chem. Pharm. 3: 8-22. https://doi.org/10.1016/j.scp.2016.01.001
  6. Ghafoori H, Askari M, Sarikhan S. 2016. Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis (X5B). Extremophiles 20: 115-123. https://doi.org/10.1007/s00792-015-0804-8
  7. Barzkar N. 2020. Marine microbial alkaline protease: an efficient and essential tool for various industrial applications. Int. J. Biol. Macromol. 161: 1216-1229. https://doi.org/10.1016/j.ijbiomac.2020.06.072
  8. 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. https://doi.org/10.1128/AEM.71.7.3420-3426.2005
  9. Dettmer A, Cavalli E, Ayub MAZ, Gutterres M. 2013. Environmentally friendly hide unhairing: enzymatic hide processing for the replacement of sodium sulfide and delimig. J. Clean. Prod. 47: 11-18. https://doi.org/10.1016/j.jclepro.2012.04.024
  10. Sun F, Sun Q, Zhang H, Kong B, Xia X. 2019. Purification and biochemical characteristics of the microbial extracellular protease from Lactobacillus curvatus isolated from Harbin dry sausages. Int. J. Biol. Macromol. 133: 987-997. https://doi.org/10.1016/j.ijbiomac.2019.04.169
  11. Sharma AK, Kikani BA, Singh SP. 2020. Biochemical, thermodynamic and structural characteristics of a biotechnologically compatible alkaline protease from a haloalkaliphilic, Nocardiopsis dassonvillei OK-18. Int. J. Biol. Macromol.153: 680-696. https://doi.org/10.1016/j.ijbiomac.2020.03.006
  12. Ben Elhoul M, Zarai Jaouadi N, Rekik H, Bejar W, Boulkour Touioui S, Hmidi M, et al. 2015. A novel detergent-stable solvent-tolerant serine thiol alkaline protease from Streptomyces koyangensis TN650. Int. J. Biol. Macromol. 79: 871-882. https://doi.org/10.1016/j.ijbiomac.2015.06.006
  13. Brandelli A. 2007. Bacterial keratinases: useful enzymes for bioprocessing agroindustrial wastes and beyond. Food Bioprocess Tech. 1: 105-116. https://doi.org/10.1007/s11947-007-0025-y
  14. Zarai Jaouadi N, Rekik H, Badis A, Trabelsi S, Belhoul M, Yahiaoui AB, et al. 2013. Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities. PLoS One 8: e76722. https://doi.org/10.1371/journal.pone.0076722
  15. Kostyleva EV, Sereda AS, Velikoretskaya IA, Nefedova LI, Sharikov AY, Tsurikova NV, et al. 2016. A new Bacillus licheniformis mutant strain producing serine protease efficient for hydrolysis of soy meal proteins. Microbiology 85: 462-470. https://doi.org/10.1134/s0026261716040123
  16. Jagadeesan Y, Meenakshisundaram S, Saravanan V, Balaiah A. 2020. Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management. Int. J. Biol. Macromol. 163: 135-146. https://doi.org/10.1016/j.ijbiomac.2020.06.219
  17. Haddar A, Agrebi R, Bougatef A, Hmidet N, Sellami-Kamoun A, Nasri M. 2009. Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive. Bioresour. Technol. 100: 3366-3373. https://doi.org/10.1016/j.biortech.2009.01.061
  18. 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. https://doi.org/10.1007/s00253-008-1355-z
  19. Zhang RX, Gong JS, Su C, Zhang DD, Tian H, Dou WF, et al. 2016. Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis CGMCC 10798. Int. J. Biol. Macromol. 93: 843-851. https://doi.org/10.1016/j.ijbiomac.2016.09.063
  20. Parte AC, Sarda Carbasse J, Meier-Kolthoff JP, Reimer LC, Goker M. 2020. List of prokaryotic names with standing in nomenclature (LPSN) moves to the DSMZ. Int. J. Syst. Evol. Microbiol. 70: 5607-5612. https://doi.org/10.1099/ijsem.0.004332
  21. Li X, Zhang S, Gan L, Cai C, Tian Y, Shi B. 2020. Ornithinibacillus caprae sp. nov, a moderate halophile isolated from the hides of a white goat. Arch. Microbiol. 202: 1469-1476. https://doi.org/10.1007/s00203-020-01855-6
  22. Meier-Kolthoff JP, Goker M. 2019. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 10: 2182. https://doi.org/10.1038/s41467-019-10210-3
  23. Li W, Jaroszewski L, Godzik A. 2002. Tolerating some redundancy significantly speeds up clustering of large protein databases. Bioinformatics 18: 77-82. https://doi.org/10.1093/bioinformatics/18.1.77
  24. State dministration for Quality Supervision and Inspection and Quarantine of the People's Republic of China: The National Standardization Administration Commission GB/T 23527-2009. 2009. Proteinase preparations.
  25. Laemmli UK. 1970. Cleavage of structure protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  26. Garciacarreno FL, Dimes LE, Haard NF. 1993. Substrate gel-electrophoresis for composition and molecular-weight of proteinases of proteinaceous proteinase-inhibitors. Anal. Biochem. 214: 65-69. https://doi.org/10.1006/abio.1993.1457
  27. Li Y, Wu C, Zhou M, Wang ET, Zhang Z, Liu W, Ning J, Xie Z. 2017. Diversity of cultivable protease-producing bacteria in Laizhou Bay sediments, Bohai Sea, China. Front. Microbiol. 8: 405.
  28. Pandey S, Rakholiya KD, Raval VH, Singh SP. 2012. Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature. J. Biosci. Bioeng. 114: 251-256. https://doi.org/10.1016/j.jbiosc.2012.03.003
  29. Thanapun T. 2013. Screening and characterization of protease-producing Virgibacillus, Halobacillus and Oceanobacillus strains from Thai fermented fish. J. Appl. Pharm. Sci. 3: 025-030.
  30. Rohban R, Amoozegar MA, Ventosa A. 2009. Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. J. Ind. Microbiol. Biotechnol. 36: 333-340. https://doi.org/10.1007/s10295-008-0500-0
  31. Seghal Kiran G, Nishanth Lipton A, Kennedy J, Dobson AD, Selvin J. 2014. A halotolerant thermostable lipase from the marine bacterium Oceanobacillus sp. PUMB02 with an ability to disrupt bacterial biofilms. Bioengineered 5: 305-318. https://doi.org/10.4161/bioe.29898
  32. Deng A, Wu J, Zhang Y, Zhang G, Wen T. 2010. Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour. Technol. 101: 7111-7117.
  33. Ibrahim ASS, Al-Salamah AA, El-Badawi YB, El-Tayeb MA, Antranikian G. 2015. Detergent-, solvent- and salt-compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization. Extremophiles 19: 961-971. https://doi.org/10.1007/s00792-015-0771-0
  34. Darwesh OM, Ali SS, Matter IA, Elsamahy T, Mahmoud YA. 2020. Enzymes immobilization onto magnetic nanoparticles to improve industrial and environmental applications. Methods Enzymol. 630: 481-502. https://doi.org/10.1016/bs.mie.2019.11.006
  35. Gong JS, Wang Y, Zhang DD, Zhang RX, Su C, Li H, et al. 2015. Biochemical characterization of an extreme alkaline and surfactant-stable keratinase derived from a newly isolated actinomycete Streptomyces aureofaciens K13. Rsc. Adv. 5: 24691-24699. https://doi.org/10.1039/C4RA16423G
  36. Tork SE, Shahein YE, El-Hakim AE, Abdel-Aty AM, Aly MM. 2013. Production and characterization of thermostable metallo-keratinase from newly isolated Bacillus subtilis NRC 3. Int. J. Biol. Macromol. 55: 169-175. https://doi.org/10.1016/j.ijbiomac.2013.01.002
  37. El-Khonezya MI, Elgammalb EW, Ahmedb EF, Abd-Elaziz AM. 2021. Detergent stable thiol-dependant alkaline protease produced from the endophytic fungus Aspergillus ochraceus BT21: purification and kinetics. Biocatal. Agric. Biotechnol. 35: 102046. https://doi.org/10.1016/j.bcab.2021.102046
  38. Patil U, Chaudhari A. 2009. Purification and characterization of solvent-tolerant, thermostable, alkaline metalloprotease from alkalophilic Pseudomonas aeruginosa MTCC 7926. J. Chem. Technol. Biotechnol. 84: 1255-1262. https://doi.org/10.1002/jctb.2169
  39. Maruthiah T, Somanath B, Immanuel G, Palavesam A. 2015. Deproteinization potential and antioxidant property of haloalkalophilic organic solvent tolerant protease from marine Bacillus sp. APCMST-RS3 using marine shell wastes. Biotechnol. Rep. 8: 124-132. https://doi.org/10.1016/j.btre.2015.10.009
  40. Suwannaphan S, Fufeungsombut E, Promboon A, Chim-anage P. 2017. A serine protease from newly isolated Bacillus sp. for efficient silk degumming, sericin degrading and colour bleaching activities. Int. Biodeter. Biodegr. 117: 141-149. https://doi.org/10.1016/j.ibiod.2016.12.009
  41. Gegeckas A, Simkute A, Gudiukaite R, Citavicius DJ. 2018. Characterization and application of keratinolytic paptidases from Bacillus spp.. Int. J. Biol. Macromol. 113: 1206-1213. https://doi.org/10.1016/j.ijbiomac.2018.03.046
  42. Ben Elhoul M, Zarai Jaouadi N, Rekik H, Omrane Benmrad M, Mechri S, Moujehed E, et al. 2016. Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50. Int. J. Biol. Macromol. 92: 299-315. https://doi.org/10.1016/j.ijbiomac.2016.07.009
  43. Jaouadi B, Abdelmalek B, Fodil D, Ferradji FZ, Rekik H, Zarai N, et al. 2010. Purification and characterization of a thermostable keratinolytic serine alkaline proteinase from Streptomyces sp. strain AB1 with high stability in organic solvents. Bioresour. Technol. 101: 8361-8369. https://doi.org/10.1016/j.biortech.2010.05.066
  44. Jellouli K, Bougatef A, Manni L, Agrebi R, Siala R, Younes I, et al. 2009. Molecular and biochemical characterization of an extracellular serine-protease from Vibrio metschnikovii J1. J. Ind. Microbiol. Biotechnol. 36: 939-948. https://doi.org/10.1007/s10295-009-0572-5
  45. Sujitha P, Kavitha S, Shakilanishi S, Babu NKC, Shanthi C. 2018. Enzymatic dehairing: a comprehensive review on the mechanistic aspects with emphasis on enzyme specificity. Int. J. Biol. Macromol. 118: 168-179. https://doi.org/10.1016/j.ijbiomac.2018.06.081
  46. Yates JR. 1972. Studies in depilation. Part X. The mechanism of the enzyme depilation process. J. Soc. Leather Trades Chem. 56: 158-177.
  47. Bouacem K, Bouanane-Darenfed A, Zarai Jaouadi N, Joseph M, Hacene H, Ollivier B, et al. 2016. Novel serine keratinase from Caldicoprobacter algeriensis exhibiting outstanding hide dehairing abilities. Int. J. Biol. Macromol. 86: 321-328. https://doi.org/10.1016/j.ijbiomac.2016.01.074