DOI QR코드

DOI QR Code

Molecular Cloning, Characterization, and Application of Organic Solvent-Stable and Detergent-Compatible Thermostable Alkaline Protease from Geobacillus thermoglucosidasius SKF4

  • Suleiman D Allison (Department of Food Science and Technology, Faculty of Agriculture and Agricultural Technology, Moddibo Adama University) ;
  • Nur AdeelaYasid (Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra) ;
  • Fairolniza Mohd Shariff (Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia) ;
  • Nor'Aini Abdul Rahman (Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra)
  • Received : 2023.06.28
  • Accepted : 2023.10.30
  • Published : 2024.02.28

Abstract

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca2+ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

Keywords

References

  1. Gupta R, Beg QK, Lorenz P. 2002. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59: 15-32. https://doi.org/10.1007/s00253-002-0975-y
  2. Hartley BS. 1960. Proteolytic enzymes. Annu. Rev. Biochem. 29: 45-72. https://doi.org/10.1146/annurev.bi.29.070160.000401
  3. Sharma M, Gat Y, Arya S, Kumar V, Panghal A, Kumar A. 2019. A review on microbial alkaline protease: an essential tool for various industrial approaches. Ind. Biotechnol. 15: 69-78. https://doi.org/10.1089/ind.2018.0032
  4. Shimogaki H, Takeuchi K, Nishino T, Ohdera M, Kudo, T, Ohba, K, et al. 1991. Purification and properties of a novel surface-active agent-and alkaline-resistant protease from Bacillus sp. Y. Agric. Biol. Chem. 55: 2251-2258. https://doi.org/10.1271/bbb1961.55.2251
  5. Bekler FM, Guven, K. 2015. Production and purification of novel thermostable alkaline protease from Anoxybacillus sp. KP1. Cell. Mol. Biol. 61: 113-120.
  6. Banik RM, Prakash, M. 2004. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res. 159: 135-140. https://doi.org/10.1016/j.micres.2004.01.002
  7. Naveed M, Nadeem F, Mehmood T, Bilal M, Anwar Z, Amjad F. 2021. Protease - a versatile and ecofriendly biocatalyst with multi-industrial applications: an updated review. Catal. Lett. 151: 307-323. https://doi.org/10.1007/s10562-020-03316-7
  8. Esakkiraj P, Meleppat B, Lakra AK, Ayyanna R, Aru V. 2016. Cloning, expression, characterization, and application of protease produced by Bacillus cereus PMW8. Royal Soc. Chem. Adv. 6: 8611-38616.
  9. Fu Z, Ab Hamid SB, Razak CNA, Basri M, Salleh AB, Abd Rahman RNZ. 2003. Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease. Prot. Expr.Purif. 28: 63-68. https://doi.org/10.1016/S1046-5928(02)00637-X
  10. Hawumba JF, Theron J, Volker S, Brozel VS. 2002.Thermophilic protease-producing Geobacillus from Buranga hot springs in western Uganda. Curr. Microbiol. 45: 144-150. https://doi.org/10.1007/s00284-001-0116-3
  11. Thebti W, Riahi Y, Belhadj O. 2016. Purification and characterization of a new thermostable, haloalkaline, solvent stable, and detergent compatible serine protease from Geobacillus toebii strain LBT 77. BioMed. Res. Int. 2016: 9178962-9178962.
  12. Arbige MV, Shetty JK, Chotani GK. 2019. Industrial enzymology: the next chapter. Trend. Biotechnol. 37: 1355-1366. https://doi.org/10.1016/j.tibtech.2019.09.010
  13. Haddar A, Bougatef A, Agrebi R. Sellami-Kamoun A, Nasri M. 2009. A novel surfactanstable alkaline serine-protease from a newly isolated Bacillus mojavensis A21. purification and characterization. Pro. Biochem. 44: 29-35. https://doi.org/10.1016/j.procbio.2008.09.003
  14. Jain D, Pancha I, Mishra SK, Shrivastav A, Mishra S. 2012. Purification and characterization of haloalkaline thermoactive, solvent stable and SDS-induced protease from Bacillus sp.: a potential additive for laundry detergents. Bioresour. Technol. 115: 228-236. https://doi.org/10.1016/j.biortech.2011.10.081
  15. Long Liu, Haiquan Yang, Hyun-dong Shin, Rachel R Chen, Jianghua Li, Guocheng Du, et al. 2013. How to achieve high-level expression of microbial enzymes strategies and perspectives. Bioengineered 4: 212-223. https://doi.org/10.4161/bioe.24761
  16. Galante YM, Formantici C. 2003.Enzyme applications in detergency and in manufacturing industries. Curr. Org. Chem. 7: 1399-1422. https://doi.org/10.2174/1385272033486468
  17. Rigoldi F, Donini S, Redaelli A, Parisini E, Gautier A. 2018. Review: engineering of thermostable enzymes for industrial applications. APL Bioeng. 2: 011501.
  18. Rao MB, Taksale AM, Ghatge MS, Deshpande, VV. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62: 597-635. https://doi.org/10.1128/MMBR.62.3.597-635.1998
  19. Zeigler DR.2013. The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet? Microbiology 160: 1-11. https://doi.org/10.1099/mic.0.071696-0
  20. Hussein, AH, Lisowska BK, Leak DJ. 2015. The genus Geobacillus and their biotechnological potential. Adv. Appl. Microbiol. 92: 1-48. https://doi.org/10.1016/bs.aambs.2015.03.001
  21. Suzuki H. 2018. Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species. Appl. Microbiol. Biotechnol. 102: 10425-10437. https://doi.org/10.1007/s00253-018-9422-6
  22. Sonnleitner B, Fiechter A. 1983. Advantages of using thermophiles in biotechnological processes: expectations and reality. Trend. Biotechnol. 1: 74-80. https://doi.org/10.1016/0167-7799(83)90056-2
  23. Maciver B, McHale RH, Saul DJ, Bergquist PL. 1994. Cloning and sequencing of a serine protease gene from a thermophilic Bacillus species and its expression in Escherichia coli. Appl. Environ. Microbiol. 60: 3981-3988. https://doi.org/10.1128/aem.60.11.3981-3988.1994
  24. Sheng L, Kovacs K, Winzer K, Zhang Y, Minton NP. 2017. Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955. Biotechnol. Biofuels 10: 5.
  25. Zhu D, Adebisi WA, Ahmad F, Sethupathy S, Danso B, Sun J. 2020. Recent development of extremophilic bacteria and their application in biorefinery. Front. Bioeng. Biotechnol. 8: 483.
  26. Aanniz T, Ouadghiri M, Melloul M, Swings J, Elfahime E, Ibijbijen, J, et al. 2015. Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils. Braz. J. Microbiol. 46: 443-453. https://doi.org/10.1590/S1517-838246220140219
  27. Sharma KM, Kumar R, Panwar S, Kumar A. 2017. Microbial alkaline proteases: optimization of production parameters and their properties. J. Gen. Eng. Biotechnol. 15: 115-126. https://doi.org/10.1016/j.jgeb.2017.02.001
  28. Vijayaraghavan P, Lazarus S, Vincent SGP.2014. De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: biosynthesis and properties. Saudi J. Biol. Sc. 21: 27-34. https://doi.org/10.1016/j.sjbs.2013.04.010
  29. Arya PS, Yagnik SM, Rajput KN, Panchal RR, Rava V. H.2021. Understanding the basis of occurrence, biosynthesis, and implications of thermostable alkaline proteases. Appl. Biochem. Biotechnol. 193: 4113-4150. https://doi.org/10.1007/s12010-021-03701-x
  30. Singh S, Bajaj BK. 2017. Agroindustrial/forestry residues as substrates for production of thermoactive alkaline protease from Bacillus licheniformis K-3 having multifaceted hydrolytic potential. Waste Biomass Valorization 8: 453-462. https://doi.org/10.1007/s12649-016-9577-2
  31. Suberu Y, Akande I, Samuel T, Lawal A, Olaniran A. 2019. Cloning, expression,purification and characterisation of serine alkaline protease from Bacillus subtilis RD7. Biocatal. Agric. Biotechnol. 20: 101264.
  32. Subba RC, Sathish T, Ravichandra P, Prakasham RS. 2009. Characterization of thermo-and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco -friendly applications. Proc. Biochem. 44: 262-268. https://doi.org/10.1016/j.procbio.2008.10.022
  33. Straub CT, Counts, JA, Nguyen, DM, Wu CH, Zeldes BM, Crosby, JR. 2018. Biotechnology of extremely thermophilic archaea. FEMS Microbiol. Rev. 42: 543-578. https://doi.org/10.1093/femsre/fuy012
  34. Suleiman AD, Rahman, NA,Yosuf AD, Sarrif, FM , Yasd NA.2020. Effect of cultural conditions on protease production by a thermophilic Geobacillus thermoglucosidasius SKF4 isolated from Sungai Klah hot spring park, Malaysia. Molecules 25: 2609.
  35. Sambrook J, Fritsch EF, Maniatis T. 1989. The identification of recombinant clones. Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, New York, NY, 324-328.
  36. Yildirim V, Baltaci MO, Ozgencli I, Sisecioglu M, Adiguzel A, Adiguzel G. 2017. Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents. J. Enz. Inhibit. Med. Chem. 32: 468-477. https://doi.org/10.1080/14756366.2016.1261131
  37. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  38. MacDonald CE, Chen LL. 1965. Lowry modification of the Folin reagent for determination of proteinase activity. Anal. Biochem. 10: 175-177. https://doi.org/10.1016/0003-2697(65)90255-1
  39. Folin O, Marenzi AD.1929. Tyrosine and tryptophane determinations in one-tenth gram of protein. J. Biol. Chem. 83: 89-102. https://doi.org/10.1016/S0021-9258(20)70840-9
  40. Anson ML. 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol. 22: 79-89. https://doi.org/10.1085/jgp.22.1.79
  41. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  42. Rekik H, Jaouadi NZ, Gargouri F, Bejar W, Frikha F, Jmal N, et al. 2019. Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12. Int. J. Biol. Macromol. 121: 1227-1239. https://doi.org/10.1016/j.ijbiomac.2018.10.139
  43. Vincent SS, John SB. 2009. Buffers: principles and practice. In Methods in Enzymology eds. Richard RB, Murray PD, pp. 43-56. Massachusetts: Elsevier Science and Technology Books.
  44. Benmrad MO, Moujehed E, Elhoul MB, Jaouadi NZ, Mechri S, Rekik H, et al. 2016. A novel organic solvent-and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101. Inter. J. Biol. Macromol. 91: 961-972 https://doi.org/10.1016/j.ijbiomac.2016.06.025
  45. Correa APF, Daroit, DJ, Coelho J, Meira SM, Lopes FC, Segalin J, et al. 2011. Antioxidant, antihypertensive and antimicrobial properties of ovine milk caseinate hydrolyzed with a microbial protease. J. Sci. Food Agric. 91: 2247-2254.
  46. Abidi F, Limam F, Nejib MM. 2008. Production of alkaline proteases by Botrytis cinerea using economic raw materials: assay as biodetergent. Proc. Biochem. 43: 1202-1208. https://doi.org/10.1016/j.procbio.2008.06.018
  47. Patil U, Mokashe N, Chaudhari A. 2016. Detergent-compatible, organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: purification and characterization. Prep. Biochem. Biotechnol. 46: 56-64. https://doi.org/10.1080/10826068.2014.979205
  48. Ekchaweng K, Khunjan U, Churngchow N. 2017. Molecular cloning and characterization of three novel subtilisin-like serine protease genes from Hevea brasiliensis. Physiol. Mol. Plant Pathol. 97: 79-95. https://doi.org/10.1016/j.pmpp.2016.12.007
  49. Li G, Xu L, Zhang H, Liu J, Yan J, Yan Y. 2020. De novo designed esterase with p-nitrophenyl acetate hydrolysis activity. Molecules 25: 4658
  50. Godde C, Sahm K, Brouns SJ, Kluskens LD, van der Oost J, de Vos WM, et al. 2005. Cloning and expression of islandisin, a newthermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli. Appl. Environ. Microbiol. 71: 3951-3958. https://doi.org/10.1128/AEM.71.7.3951-3958.2005
  51. Terada I, Kwon ST, Miyata Y, Matsuzawa H, Ohta T. 1990. Unique precursor structure of an extracellular protease, aqualysin I, with NH2-and COOH-terminal pro-sequences and its processing in Escherichia coli. J. Biol. Chem. 265: 6576-6581. https://doi.org/10.1016/S0021-9258(19)39186-0
  52. Siezen RJ, Kuipers OP, de Vos WM. 1996. Comparison of lantibiotic gene clusters and encoded proteins. Antonie Van Leeuwenhoek 69: 171-184. https://doi.org/10.1007/BF00399422
  53. Barbieri G, Albertini AM, Ferrari E, Sonenshein AL, Belitsky BR. 2016. Interplay of CodY and ScoC in the regulation of major extracellular protease genes of Bacillus subtilis. J. Bacteriol. 198: 907-920. https://doi.org/10.1128/JB.00894-15
  54. Jacobs M, Eliasson M, Uhlen M, Flock JI.1985. Cloning, sequencing and expression of subtilisin carlsberg from Bacillus licheniformis. Nucleic Acids Res. 13: 8913-8926 https://doi.org/10.1093/nar/13.24.8913
  55. Kaur I, Kocher GS, Gupta VK. 2012. Molecular cloning and nucleotide sequence of the gene for an alkaline protease from Bacillus circulans MTCC 7906. Indian J. Microbiol. 52: 630-637. https://doi.org/10.1007/s12088-012-0297-4
  56. Iqbal I, Aftab MN, Afzal M, Ur-Rehman A, Aftab S, Zafar A, et al. 2015. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus. J. Basic Microbiol. 55: 160-171. https://doi.org/10.1002/jobm.201400190
  57. Forgarty WM, Griffin PJ, Joyce AM. 1974. Enzymes of Bacillus species. L Proc. Biochem. 9: 11-18.
  58. Hao JH, Sun M. 2015. Purification and characterization of a cold alkaline protease from a psychrophilic Pseudomonas aeruginosa HY1215. Appl. Biochem. Biotechnol. 175: 715-722. https://doi.org/10.1007/s12010-014-1315-2
  59. Basavaraju S, Kathera C, Jasti PK. 2017. Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant. J. Microbiol. Biotechnol. Res. 7: 1-12. https://doi.org/10.24896/jmbr.2017741
  60. Manavalan T, Manavalan A, Ramachandran S, Heese K. 2020. Identification of a novel thermostable alkaline protease from Bacillus megaterium-TK1 for the detergent and leather industry. Biology 9: 472.
  61. Steele DB, Fiske MJ, Steele BP, Kelley VC. 1992. Production of a low molecular weight, alkaline active, thermostable protease by a novel spiral-shaped bacterium,Kurthia spiroforme sp. nov. Enzyme Microb. Technol. 14: 358-360. https://doi.org/10.1016/0141-0229(92)90003-7
  62. Kwon YT, Kim JO, Moon SY, Lee HH, Rho HM. 1994. Extracellular alkaline protease from alkalophilic Vibrio metschnikovii strain RH530. Biotechnol. Lett. 16: 413-418. https://doi.org/10.1007/BF00245062
  63. Rahman RNZ, Razak CNA, Ampon A, Bashir M, Yunus WMZ, Saleh AB.1994. Purification and characterization of a heat stable alkaline protease from Bacillus sterothermophillus F1. Appl. Microbiol. Biotechnol. 40: 822-827. https://doi.org/10.1007/BF00173982
  64. Zhu W, Cha D, Cheng G, Peng Q, Shen P. 2007. Purification and characterization of a thermostable protease from a newly isolated Geobacillus sp. YMTC 1049. Enzyme Microb. Technol. 40: 1592-1597. https://doi.org/10.1016/j.enzmictec.2006.11.007
  65. Peek K, Daniel RM, Monk C, Parker L, Coolbear T. 1992. Purification and characterisation of a thermostable proteinase isolated from Thermus sp. strain Rt41A. Eur. J. Biochem. 207: 1035-1044. https://doi.org/10.1111/j.1432-1033.1992.tb17140.x
  66. Lakshmi BKM, Kumar DM, Hemalatha KPJ. 2018. Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8. J. Genet. Eng. Biotechnol. 16: 295-304. https://doi.org/10.1016/j.jgeb.2018.05.009
  67. Chang C, Gong S, Liu Z, Yan Q, Jiang Z. 2021. High level expression and biochemical characterization of an alkaline serine protease from Geobacillus stearothermophilus to prepare antihypertensive whey protein hydrolysate. BMC Biotechnol. 21: 1-13. https://doi.org/10.1186/s12896-020-00660-9
  68. Barzkar N, Homaei A, Hemmati R, Patel S. 2018. Thermostable marine microbial proteases for industrial applications: scopes and risks. Extremophiles 22: 335-346. https://doi.org/10.1007/s00792-018-1009-8
  69. Saiki T, Kimura R, Arima K. 1972. Isolation and characterization of extremely thermophilic bacteria from hot springs. Agric. Biol.Chem. 36: 2357-2366. https://doi.org/10.1080/00021369.1972.10860589
  70. Ahmad A, Mishra R. 2022. Structural and functional adaptation in extremophilic microbial α-amylases. Biophys. Rev. 14: 499-515. https://doi.org/10.1007/s12551-022-00931-z
  71. 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. Inter. J. Biol. Macromol. 153: 680-696. https://doi.org/10.1016/j.ijbiomac.2020.03.006
  72. Kumar CG, Takagi H. 1999. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol. Adv. 17: 561-594. https://doi.org/10.1016/S0734-9750(99)00027-0
  73. Veloorvalappil NJ, Robinson BS, Selvanesan P, Sasidharan S, Kizhakkepawothail NU, Sreedharan S, et al. 2013. Versatility of microbial proteases. Adv. Enzy. Res. 1: 39-51. https://doi.org/10.4236/aer.2013.13005
  74. Kumar A, Sharma A, Kaur G, Makkar P, Kaur J. 2017. Functional characterization of hypothetical proteins of Mycobacterium tuberculosis with possible esterase/lipase signature: a cumulative in silico and in vitro approach. J. Biomol. Struct. Dyn. 35: 1226-1243. https://doi.org/10.1080/07391102.2016.1174738
  75. Takami H, Akiba T, Horikoshi K. 1990. Characterisation of an alkaline protease from Bacillus sp. no. AH-101. Appl. Microbiol. Biotechnol. 33: 519-23. https://doi.org/10.1007/BF00172544
  76. Kaur I, Sharma AD, Joshi N, Kocher GS. 2020. Alkaline proteases: a review on production optimization parameters and their physicochemical properties. Res. Rev. Biotechnol. Biosc. 7: 33-39.
  77. Horikoshi K. 1971. Production of alkaline enzymes by alkalophilic microorganisms: part i. alkaline protease produced by Bacillus no. 221. Agric. Biol. Chem. 35: 1407-1414. https://doi.org/10.1080/00021369.1971.10860094
  78. Rama Devi P, Babu C, Vasudhevan I, Felcial S, Lakshmanan G. 2018. Purification and characterization of protease enzyme from seaweed, Gracilaria fergusonii. Int. J. Curr. Res. Life Sc. 7: 2801-2804.
  79. Fujiwara N, Masui A, Imanaka T. 1993. Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp. J. Biotechnol. 30: 245-56. https://doi.org/10.1016/0168-1656(93)90117-6
  80. Kocher GS. 2018.Biochemical characterization of alkaline protease from Bacillus circulans Mtcc 7906. Res. Rev. Biotechnol. Biosci. 5: 35-45.
  81. Mohanty B. 2020. Characterization of digestive acidic and alkaline proteolytic enzyme (proteases) from the visceral wastes of Chinese major carp, Cyprinus carpio (Linnaeus, 1758). J. Entomol. Zool. Stud. 8: 1862-1869.
  82. Kudryashova EV, Mozhaev VV, Balny C. 1998. Catalytic activity of thermolysin under extremes of pressure and temperature: modulation by metal ions. Biochem. Biopys. Acta 1386: 199-210. https://doi.org/10.1016/S0167-4838(98)00055-7
  83. Jellouli K, Ghorbel-Bellaaj O, Ayed HB, Manni L, Agrebi R, Nasri M. 2011.Alkaline-protease from Bacillus licheniformis MP1: purification, characterization and potential application as a detergent additive and for shrimp waste deproteinization. Proc. Biochem. 46: 1248-1256. https://doi.org/10.1016/j.procbio.2011.02.012
  84. Kannan Y, Koga Y, Inoue Y, Haruki M, Takagi M, Imanaka T. et al. 2001. Activesubtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence. Appl. Environ. Microbiol. 67: 2445-2452. https://doi.org/10.1128/AEM.67.6.2445-2452.2001
  85. Rao CS, Sathish T, Ravichandra P, Prakasham RS. 2009. Characterization of thermo-and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Proc. Biochem. 44: 262-268. https://doi.org/10.1016/j.procbio.2008.10.022
  86. Kumar CG, 2002. Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus. Lett. Appl. Microbiol. 34: 13-17. https://doi.org/10.1046/j.1472-765x.2002.01044.x
  87. Lee JK, Kim YO, Kim HK, Park SY, Oh TK. 1996. Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. Biosci. Biotechnol. Biochem. 60: 840-846. https://doi.org/10.1271/bbb.60.840
  88. Gohel SD, Singh SP. 2015. Thermodynamics of a Ca2+-dependent highly thermostable alkaline protease from a haloalkliphilic actinomycete. Int. J. Biol. Macromol. 72: 421-429. https://doi.org/10.1016/j.ijbiomac.2014.08.008
  89. Karray A, Alonazi M, Horchani H, Ben Bacha A. 2021. A novel thermostable and alkaline protease produced from Bacillus stearothermophilus isolated from olive oil mill sols suitable to industrial biotechnology. Molecules 26: 1139.
  90. Pan T, Lin S. 1991. Fermentative production of alkaline protease as detergent additive. J. Chin. Biochem. Soc. 20: 49-60. https://doi.org/10.1108/eb005874
  91. Zhu B, Chen M, Yin H, Du Y, Ning L. 2016. Enzymatic hydrolysis of alginate to produce oligosaccharides by a new purified endo-type alginate lyase. Mar. Drugs 14: 108
  92. Mienda BS, Yahya A, Galadima IA, Shamsir MS. 2014. An overview of microbial proteases for industrial applications. Res. J. Pharm. Biol. Chem. Sci. 5: 388-396.
  93. Jellouli K, Bayoudh A, Manni L, Agrebi R, Nasri M.2008.Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 on shrimp wastes. Appl. Microbiol. Biotechnol. 79: 989-999. https://doi.org/10.1007/s00253-008-1517-z
  94. Rahman RNZA Geok LP, Basri M, Salleh AB. 2006. An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: enzyme purification and characterization. Enzy. Microb. Technol. l39: 1484-1491. https://doi.org/10.1016/j.enzmictec.2006.03.038
  95. Gupta A, Roy I, Khare SK, Gupta MN.2005. Purification and characterization of a solvent-stable protease from Pseudomonas aeruginosa PseA. J. Chromatogr. 1069: 155-161 https://doi.org/10.1016/j.chroma.2005.01.080
  96. Abusham RA, Rahman RNZR, Salleh AB, Basri M. 2009. Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand. Microb. Cell Fact. 8: 1-9. https://doi.org/10.1186/1475-2859-8-1
  97. Yilmaz B, Baltaci MO, Sisecioglu M, Adiguzel A. 2016. Thermotolerant alkaline protease enzyme from Bacillus licheniformis A10: purification, characterization,effects of surfactants and organic solvents. J. Enzyme Inhib. Med. Chem. 31: 1241-1247 https://doi.org/10.3109/14756366.2015.1118687
  98. Yang S, Zhai L, Huang L, Meng D, Li J, Hao, Z.et al. 2020. Mining of alkaline proteases from Bacillus altitudinis W3 for desensitization of milk proteins: their heterologous expression, purification, and characterization. Int. J. Biol. Macromol. 153: 1220-1230 https://doi.org/10.1016/j.ijbiomac.2019.10.252
  99. Doukyu N, Ogino H. 2010.Organic solvent tolerant enzymes. Biochem. Eng. J. 48: 270-282. https://doi.org/10.1016/j.bej.2009.09.009
  100. Sittipol D, Saelao P, Lohnoo T, Lerksuthirat T, Kumsang Y, Yingyong W, et al. 2019. Cloning, expression, purification and characterization of a thermo- and surfactant-stable proteasemfrom Thermomonospora curvata. Biocatal. Agric. Biotechnol. 19: 101111.
  101. Vieille C, Zeikus GJ. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65: 1-43. https://doi.org/10.1128/MMBR.65.1.1-43.2001
  102. Gey MH, Unger KK. 1995. Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms. J. Chromatogr. B. 166: 188-193. https://doi.org/10.1016/0378-4347(94)00562-J
  103. Chen J, Wesley ES. 2004.Replacement of staphylococcal nuclease hydrophobic core residues with those from thermophilic homologues indicates packing is improved in some thermostable proteins. J. Mol. Biol. 344: 271-280. https://doi.org/10.1016/j.jmb.2004.09.008
  104. Anbu P, Hur BK, Lee CG. 2013. Isolation and characterization of a novel oxidant- and surfactant-stable extracellular alkaline protease from Exiguobacterium profundum BK-P23. Biotechnol. Appl. Biochem. 60: 155-161. https://doi.org/10.1002/bab.1059
  105. Bose A, Chawdhary V, Keharia H, Subramanian RB. 2014. Production and characterization of a solvent-tolerant protease from a novel marine isolate Bacillus tequilensis P15. Annal. Microbiol. 64: 343-354. https://doi.org/10.1007/s13213-013-0669-y
  106. Annamalai N, Rajeswari MV, Balasubramanian T. 2013. Extraction, purification and application of thermostable and halostable alkaline protease from Bacillus alveayuensis CAS 5 using marine wastes. Food Bioprod. Process 92: 335-342.
  107. Niyonzima FN, More S. 2014. Purification and properties of detergent-compatible extracellular alkaline protease from Scopulariopsis spp. Pre. Biochem. Biotechnol. 44: 738-759. https://doi.org/10.1080/10826068.2013.854254
  108. Marathe SK, Vashistht MA, Prashanth A, Parveen N, Chakraborty S. Nair SS. 2018. Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis, Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples. J. Gen. Eng. Biotechnol. 16: 39-46. https://doi.org/10.1016/j.jgeb.2017.10.001
  109. Mala M, Srividya S. 2010. Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated Bacillus species Y. Ind. J. Microbiol. 50: 309-317. https://doi.org/10.1007/s12088-010-0024-y
  110. Mechri S, Berrouina MBE, Benmrad MO, Jaouadi NZ, Rekik H, Moujehed E, et al. 2017. Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest. Int. J. Biol. Macromol. 94: 221-232. https://doi.org/10.1016/j.ijbiomac.2016.09.112
  111. Xu Y, Xu F, Ding X, Qian K, Li L. 2019. Cloning, secretory expression, partial characterization, and structural modeling of an alkaline protease from Bacillus subtilis D-2. BioRes. 14: 5301-5315. https://doi.org/10.15376/biores.14.3.5301-5315
  112. Kim Y, Bae JH, Oh BK, Lee WH, Choi JW 2002. Enhancement of proteolytic enzyme activity excreted from Bacillus stearothermophilus for a thermophilic aerobic digestion process. Bioresour. Technol. 82: 157-164. https://doi.org/10.1016/S0960-8524(01)00177-8
  113. Nam GW, Lee DW, Lee HS, Lee NJ, Kim,BC, Choe, EA, et al. 2002. Native feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase- producing thermophilic anaerobe. Arch. Microbiol. 178: 538-547. https://doi.org/10.1007/s00203-002-0489-0
  114. Vemula M, Balakrishnan K, Banerjee S, Guruprasad L. 2018. Mycobacterium tuberculosis PE1 and PE2 proteins carrying conserved α/β-serine hydrolase domain are esterases. Progress Biophys. Mol. Biol. 140: 90-102. https://doi.org/10.1016/j.pbiomolbio.2018.04.012
  115. Patel S. 2017. A critical review on serine protease: key immune manipulator and pathology mediator. Allergol. Immunopathol. 45: 579-591. https://doi.org/10.1016/j.aller.2016.10.011
  116. Salihi A, Asoodeh A, Aliabadian, M. 2017. Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93. Int. J. Biol. Macromol. 94: 827-835. https://doi.org/10.1016/j.ijbiomac.2016.06.023
  117. Mothe T, Sultanpuram VR. 2016. Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus. 3 Biotech 6: 53.
  118. Adinarayana K, Ellaiah P, Prasad DS. 2003. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS PharmSciTech 4: 56-60. https://doi.org/10.1208/pt040456
  119. Jayakumar R, Jayashree S, Annapurna B, Seshadri S. 2012. Characterization of thermostable serine alkaline protease from an alkaliphilic strain Bacillus pumilus MCAS8 and its applications. Appl. Biochem. Biotechnol. 168: 1849-1866 https://doi.org/10.1007/s12010-012-9902-6
  120. Sari E, Logoglub E, Oktemer A. 2015. Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34. Biomed. Chromatogr. 29: 1356-1363. https://doi.org/10.1002/bmc.3431
  121. Farhadian S, Asoodeh A, Lagzian M. 2015. Purification, biochemical characterization and structural modeling of a potential htrA-like serine protease from Bacillus subtilis DR8806. J. Mol. Catal. B: Enzym. 115: 51-58. https://doi.org/10.1016/j.molcatb.2015.02.001
  122. Iqbal M, Asgher M, Bashir F. 2018. Purification and kinetic characterization of alkaline protease for UV-90 Mutant of Bacillus Subtilis. J. Biochem. Anal. Stud. 3: 2576- 5833. https://doi.org/10.16966/2576-5833.112
  123. Ito S, Kobayashi T, Ara K, Ozaki K, Kawai S. 1998. Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics and structure. Extremophiles 21: 185-190.
  124. Kobayashi T, Hakamada Y, Adachi S, Hitomi J, Yoshimatsu T, Koike K. et al. 1995. Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl. Microbiol. Biotechnol. 43: 473-481. https://doi.org/10.1007/BF00218452
  125. Kalisz HM. 1988. Microbial proteinases. Adv. Biochem. Eng. Biotechnol. 36: 1-65. https://doi.org/10.1007/BFb0047944
  126. Solanki P, Putatunda C, Kumar A, Bhatia R, Walia A. 2021. Microbial proteases: ubiquitous enzymes with innumerable uses. 3 Biotech. 11: 1-25. https://doi.org/10.1007/s13205-021-02928-z
  127. Masui A, Yasuda M, Fujiwara N, Ishikawa H. 2004. Enzymatic hydrolysis of gelatin layer on used film using thermostable alkaline protease for the recovery of silver and PET Film. Biotechnol. Progress 20: 1267-1269. https://doi.org/10.1021/bp030058s
  128. Cavello IA, Crespo JM, Garcia SS, Zapiola JM, Luna MF, Cavalitto SF. 2015. Plant growth promotion activity of keratinolytic fungi growing on a recalcitrant waste known as "Hair Waste". Biotechnol. Res. Int. 2015: 952921.