A novel alkali and thermotolerant protease from Aeromonas spp. retrieved from wastewater.
Aeromonas spp.
Alkaline serine protease
SDS resistance.
Thermal stability
Wastewater
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
29 10 2024
29 10 2024
Historique:
received:
25
03
2024
accepted:
09
10
2024
medline:
30
10
2024
pubmed:
30
10
2024
entrez:
30
10
2024
Statut:
epublish
Résumé
Enzymes are integral to numerous industrial processes, with a growing global demand for various enzyme types. Protease enzymes, in particular, have proven to be cost-effective, stable, and compatible alternatives to traditional chemical processes in both industrial and environmental applications. In this study, an alkaline protease-producing strain of Aeromonas spp. was isolated from a wastewater treatment plant in Iran. The protease production was confirmed by culturing the strain on casein agar medium. The bacterium was identified through morphological, biochemical, and 16 S rRNA sequencing analyses. The optimal culture medium for bacterial growth and enzyme production was obtained using peptone, salt, yeast extract, galactose, and CaCl₂ at an initial pH of 8. Maximum protease production was achieved after 20 h of incubation at 40 °C. To partially purify the enzyme, the supernatant of the bacterial culture medium was first centrifuged, and the enzyme was precipitated using ammonium sulfate, followed by dialysis. Zymography revealed the production of one type of protease during bacterial growth. The partially purified protease exhibited optimal activity at pH 8.5 and maximum stability at pH 9. The optimum temperature for maximum enzyme activity was observed at 50 °C, with 100% residual activity retained for 1 h at 0 °C. The effect of metal ions on enzyme activity was assessed, revealing that KCl induced the most significant effects (p < 0.0001) on enzyme activity. Chemical amino acid modifiers and inhibitors, such as EDTA, DEPSI, and IAA, did not exhibit significant inhibition. In contrast, PMSF and HNBB significantly (p < 0.0001) reduced enzyme activity, suggesting that the enzyme could be classified as a serine protease. The protease also demonstrated high stability in the presence of 2% SDS, showing no signs inactivation. The alkaline pH optimum, thermal stability, and resistance to SDS exhibited by the protease produced by the Aeromonas strain are particularly promising characteristics that warrant further investigation. Based on preliminary tests and the enzyme's characteristics, this protease can be recommended for various applications, pending further studies.
Identifiants
pubmed: 39472719
doi: 10.1038/s41598-024-76004-w
pii: 10.1038/s41598-024-76004-w
doi:
Substances chimiques
Wastewater
0
Bacterial Proteins
0
alkaline protease
EC 3.4.99.-
Endopeptidases
EC 3.4.-
Alkalies
0
RNA, Ribosomal, 16S
0
Peptide Hydrolases
EC 3.4.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
26000Informations de copyright
© 2024. The Author(s).
Références
Adrio, J. L. & Demain, A. L. Microbial enzymes: tools for biotechnological processes. Biomolecules. 4, 117–139 (2014).
pubmed: 24970208
pmcid: 4030981
doi: 10.3390/biom4010117
Sathishkumar, R., Ananthan, G. & Arun, J. Production, purification and characterization of alkaline protease by ascidian associated Bacillus subtilis GA CAS8 using agricultural wastes. Biocatal. Agric. Biotechnol. 4, 214–220 (2015).
doi: 10.1016/j.bcab.2014.12.003
Raveendran, S. et al. Applications of microbial enzymes in food industry. Food Technol. Biotechnol. 56, 16–30 (2018).
pubmed: 29795993
pmcid: 5956270
doi: 10.17113/ftb.56.01.18.5491
Rao, M. B., Tanksale, A. M., Ghatge, M. S. & Deshpande, V. V. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597–635 (1998).
pubmed: 9729602
pmcid: 98927
doi: 10.1128/MMBR.62.3.597-635.1998
Raval, V. H., Pillai, S., Rawal, C. M. & Singh, S. P. Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria. Process. Biochem. 49, 955–962 (2014).
doi: 10.1016/j.procbio.2014.03.014
Gemechu, G., Masi, C., Tafesse, M. & Kebede, G. A review on the bacterial alkaline proteases. J. Xidian Univ. 4, 632–634 (2020).
Gupta, R., Beg, Q. & Lorenz, P. Bacterial alkaline proteases: Molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59, 15–32 (2002).
pubmed: 12073127
doi: 10.1007/s00253-002-0975-y
Kembhavi, A. A., Kulkarni, A. & Pant, A. Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM 64. Appl. Biochem. Biotechnol. 38, 83–92 (1993).
pubmed: 8346907
doi: 10.1007/BF02916414
Shikha, Sharan, A. & Darmwal, N. S. Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate. Bioresour Technol. 98, 881–885 (2007).
doi: 10.1016/j.biortech.2006.03.023
Nilegaonkar, S. S., Zambare, V. P., Kanekar, P. P., Dhakephalkar, P. K. & Sarnaik, S. S. Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour Technol. 98, 1238–1245 (2007).
pubmed: 16782331
doi: 10.1016/j.biortech.2006.05.003
Liu, Z. & Smith, S. R. Enzyme recovery from biological wastewater treatment. Waste Biomass Valorization. 12, 4185–4211 (2021).
doi: 10.1007/s12649-020-01251-7
Lagarde, D. et al. High-throughput screening of thermostable esterases for industrial bioconversions. Org. Process. Res. Dev. 6, 441–445 (2002).
doi: 10.1021/op020019h
Abusham, R. A., Rahman, R. N. Z. R. A., Salleh, A. & Basri, M. 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, (2009).
Zeldes, B. M. et al. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals. Front. Microbiol. 6, (2015).
Jisha, V. N. et al. Versatility of microbial proteases. Adv. Enzyme Res. 1, 39–51 (2013).
doi: 10.4236/aer.2013.13005
Hashmi, S., Iqbal, S., Ahmed, I. & Janjua, H. A. Production, optimization, and partial purification of alkali-thermotolerant proteases from Newly Isolated Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12. Processes. 10, (2022).
Aleem, B. et al. Random mutagenesis of super Koji (Aspergillus oryzae): Improvement in production and thermal stability of α-amylases for maltose syrup production. BMC Microbiol. 18, 1–3 (2018).
doi: 10.1186/s12866-018-1345-y
Mall, K. B., Emtiazi, G. & Nahvi, I. Production of alkaline protease by Bacillus cereus and Bacillus polymixa in new industrial culture mediums and its immobilization. Afr. J. Microbiol. Res. 3, 491–497 (2009).
Kunitz, M. Crystalline soybean trypsin inhibitor II. J. Gen. Physiol. 30, 291–310 (1947).
pubmed: 19873496
pmcid: 2142836
doi: 10.1085/jgp.30.4.291
Bradford, M. M. 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 (1976).
pubmed: 942051
doi: 10.1016/0003-2697(76)90527-3
Bergey, D. H. Bergey’s Manual of Determinative Bacteriology: A Key for the Identification of Organisms of the Class Schizomycetes 1860–1937 (The Williams & Wilkins Company, 1923).
Schaeffer, A. B. & Fulton, M. D. A simplified method of staining endospores. Science. 77, 94–194 (1933).
doi: 10.1126/science.77.1990.194
Kotb, E. et al. Isolation, screening, and identification of alkaline protease-producing bacteria and application of the most potent enzyme from Bacillus sp. Mar64. Fermentation 9, (2023).
Englard, S. & Seifter, S. [22] Precipitation techniques. Methods Enzymol. 182, 285–300 (1990).
pubmed: 2314242
doi: 10.1016/0076-6879(90)82024-V
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1974).
doi: 10.1038/227680a0
Walker, J. M. The Protein Protocols Handbook (Springer Science & Business Media, 1996).
Drancourt, M. et al. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J. Clin. Microbiol. 38, 3623–3630 (2000).
pubmed: 11015374
pmcid: 87447
doi: 10.1128/JCM.38.10.3623-3630.2000
Mignard, S. & Flandrois, J. P. 16S rRNA sequencing in routine bacterial identification: A 30-month experiment. J. Microbiol. Methods 67, 574–581 (2006).
pubmed: 16859787
doi: 10.1016/j.mimet.2006.05.009
Woo, P. C. Y. et al. Actinomyces hongkongensis sp. nov. a novel Actinomyces species isolated from a patient with pelvic actinomycosis. Syst. Appl. Microbiol. 26, 518–522 (2003).
pubmed: 14666979
doi: 10.1078/072320203770865819
Martin-Carnahan, A., Joseph, S. W. & Order, X. I. I. Aeromonadales ord. Nov. In Bergey’s Manual of Systematic Bacteriology (eds Brenner, D. J., Krieg, N. R., Staley, J. T. & Garrity, G. M.) 556–578 (Williams & Wilkins: Philadelphia, PA, USA, (2005).
doi: 10.1007/0-387-28022-7_12
Huys, G. & The family Aeromonadaceae. The Prokaryotes: Gammaproteobacteria (eds. Rosenberg, E., DeLong, E.F., Lory, S., Stackebrandt, E., Thompson, F.) 27–57 (Springer-Verlag Berlin, Heidelberg, (2014).
Fernández-Bravo, A. & Figueras, M. J. An update on the genus Aeromonas: Taxonomy, epidemiology, and pathogenicity. Microorganisms 8, 129 (2020).
pubmed: 31963469
pmcid: 7022790
doi: 10.3390/microorganisms8010129
Liu, P. V. & Hsieh, H. C. Inhibition of protease production of various bacteria by ammonium salts: Its effect on toxin production and virulence. J. Bacteriol. 99, 406–413 (1969).
pubmed: 4309097
pmcid: 250031
doi: 10.1128/jb.99.2.406-413.1969
Singh, L., Ram, M. S., Agarwal, M. K. & Alam, S. I. Characterization of Aeromonas Hydrophila strains and their evaluation for biodegradation of night soil. World J. Microbiol. Biotechnol. 16, 625–630 (2000).
doi: 10.1023/A:1008968712682
O’Reilly, T. & Day, D. F. Effects of cultural conditions on protease production by Aeromonas hydrophila. Appl. Environ. Microbiol. 45, 1132–1135 (1983).
pubmed: 6342534
pmcid: 242419
doi: 10.1128/aem.45.3.1132-1135.1983
Horz, H. P., Barbrook, A., Field, C. B. & Bohannan, B. J. M. Ammonia-oxidizing bacteria respond to multifactorial global change. Proc. Natl. Acad. Sci. U S A. 101, 15136–15141 (2004).
pubmed: 15469911
pmcid: 524064
doi: 10.1073/pnas.0406616101
Sun, B. et al. The fermentation optimization for alkaline protease production by Bacillus subtilis BS-QR-052. Front. Microbiol. 14, 1301065 (2023).
pubmed: 38169798
pmcid: 10758460
doi: 10.3389/fmicb.2023.1301065
Kuypers, M. M. M., Marchant, H. K. & Kartal, B. The microbial nitrogen-cycling network. Nat. Rev. Microbiol. 16, 263–276 (2018).
pubmed: 29398704
doi: 10.1038/nrmicro.2018.9
Suleiman, A. D., Rahman, N. A., Yusof, H. M., Shariff, F. M. & Yasid, N. A. Effect of cultural conditions on protease production by a thermophilic Geobacillus thermoglucosidasius SKF4 isolated from Sungai Klah hot spring park, Malaysia. Molecules. 25, 2609 (2020).
pubmed: 32512695
pmcid: 7321352
doi: 10.3390/molecules25112609
Mokashe, N., Chaudhari, A. & Patil, U. Optimal production and characterization of alkaline protease from newly isolated halotolerant Jeotgalicoccus Sp. Biocatal. Agric. Biotechnol. 4, 235–243 (2015).
doi: 10.1016/j.bcab.2015.01.003
Adinarayana, K., Ellaiah, P. & Prasad, D. S. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus Subtilis PE-11. AAPS PharmSciTech. 4, 440–448 (2003).
pmcid: 2750649
doi: 10.1208/pt040456
Akkale, C. & Production Optimization and partial characterization of alkaline protease from Bacillus subtilis spp. subtilis NRRL B-3384 and B-3387. Hittite J. Sci. Eng. 10, 135–144 (2023).
doi: 10.17350/HJSE19030000300
Sharma, K. M., Kumar, R., Panwar, S. & Kumar, A. Microbial alkaline proteases: optimization of production parameters and their properties. J. Genet. Eng. Biotechnol. 5, 115–126 (2017).
doi: 10.1016/j.jgeb.2017.02.001
Bhattacherjee, R. et al. Structural-genetic insight and optimization of protease production from a novel strain of Aeromonas veronii CMF, a gut isolate of Chrysomya megacephala. Arch. Microbiol. 203, 2961–2977 (2021).
pubmed: 33772325
doi: 10.1007/s00203-021-02282-x
Ratzke, C. & Gore, J. Modifying and reacting to the environmental pH can drive bacterial interactions. PLoS Biol. 16, (2018).
Popoff, M. & Genus, I. I. I. Aeromonas. In Bergey’s Manual of Systematic Bacteriology (eds. Krieg, N.R. and Holt, J.G). 545–548 (Williams & wilkins. Baltimore 1984).
Palumbo, S. A., Morgan, R. L. & Buchanan, R. L. Influence of temperature, NaCI, and PH on the growth of Aeromonas Hydrophila. J. Food Sci. 50, 1417–1421 (1985).
doi: 10.1111/j.1365-2621.1985.tb10490.x
Nadeem, M., Qazi, J. I., Baig, S. & Syed, Q-U-A. Effect of medium ccomposition on commercially important alkaline protease production by Bacillus licheniformis N-2. Food Technol. Biotechnol. 46, 388–394 (2008).
Ward, O. & Moo-young, M. Thermostable enzymes. Biotech 6, 39–69 (1988).
Deng, A., Wu, J., Zhang, Y., Zhang, G. & Wen, T. Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001. Bioresour Technol. 101, 7100–7106 (2010).
doi: 10.1016/j.biortech.2010.03.130
Patel, R., Dodia, M. & Singh, S. P. Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization. Process. Biochem. 40, 3569–3575 (2005).
doi: 10.1016/j.procbio.2005.03.049
Patel, R. K., Dodia, M. S., Joshi, R. H. & Singh, S. P. Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. Process. Biochem. 41, 2002–2009 (2006).
doi: 10.1016/j.procbio.2006.04.016
Ibrahim, A. S. S., Al-Salamah, A. A., Elbadawi, Y. B., El-Tayeb, M. A. & Shebl Ibrahim, S. S. Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes. Electron. J. Biotechnol. 18, 236–243 (2015).
doi: 10.1016/j.ejbt.2015.04.001
Kumar, C. G. & Takagi, H. Microbial alkaline proteases: From a bioindustrial viewpoint. Biotechnol. Adv. 17, 561–594 (1999).
pubmed: 14538129
doi: 10.1016/S0734-9750(99)00027-0
Reddy, L. V. A., Wee, Y. J. & Ryu, H. W. Purification and characterization of an organic solvent and detergent-tolerant novel protease produced by Bacillus sp. RKY3. J. Chem. Technol. Biotechnol. 83, 1526–1533 (2008).
doi: 10.1002/jctb.1946
Laishram, S. & Pennathur, G. Purification and characterization of a membrane-unbound highly thermostable metalloprotease from Aeromonas Caviae. Arab. J. Sci. Eng. 41, 2107–2116 (2016).
doi: 10.1007/s13369-015-1849-9
Singh, L. Simultaneous production of amylase and protease by Aeromonas hydrophila PC5, a psychrotrophic bacterium. J. Gen. Appl. Microbiol. 40, 339–348 (1994).
doi: 10.2323/jgam.40.339
Ramkumar, A., Sivakumar, N., Gujarathi, A. M. & Victor, R. Production of thermotolerant, detergent stable alkaline protease using the gut waste of Sardinella longiceps as a substrate: Optimization and characterization. Sci. Rep. 8, 12442 (2018).
pubmed: 30127443
pmcid: 6102305
doi: 10.1038/s41598-018-30155-9
Cho, S. J. et al. Purification and characterization of extracellular temperature-stable serine protease from Aeromonas hydrophila. J. Microbiol. 41, 207–211 (2003).
Toma, C., Ichinose, Y. & Iwanaga, M. Purification and characterization of an Aeromonas caviae metalloprotease that is related to the Vibrio cholerae hemagglutinin/protease. FEMS Microbiol. Lett. 170, 237–242 (1999).
pubmed: 9919673
doi: 10.1111/j.1574-6968.1999.tb13379.x
Datta, S., Menon, G., Varughese, B. & Datta Amity, S. Production, characterization and immobilization of partially purified surfactant-detergent and alkali-thermo stable protease from newly isolated Aeromonas Caviae. Prep. Biochem. Biotechnol. 47, 49–56 (2017).
doi: 10.1080/10826068.2016.1244688
Karunakaran, T. & Devi, B. G. Proteolytic activity of Aeromonas Caviae. J. Basic. Microbiol. 35, 241–247 (1995).
pubmed: 7473065
doi: 10.1002/jobm.3620350409
Goda, D. A., Bassiouny, A. R., Monem, A., Soliman, N. M., Abdel-Fattah, Y. R. & N. A. & Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization. Sci. Rep. 11, 14543 (2021).
pubmed: 34267231
doi: 10.1038/s41598-021-93279-5
Cui, H., Wang, L. & Yu, Y. Production and characterization of alkaline protease from a high yielding and moderately halophilic strain of SD11 marine bacteria. J. Chem. 3, 1–8 (2015).
doi: 10.1155/2015/798304
Kalwasińska, A., Jankiewicz, U., Felföldi, T., Burkowska-But, A. & Brzezinska, M. S. Alkaline and halophilic protease production by Bacillus luteus H11 and its potential industrial applications. Food Technol. Biotechnol. 56, 553–561 (2018).
pubmed: 30923452
pmcid: 6399708
doi: 10.17113/ftb.56.04.18.5553
Strohalm, M., Kodíček, M. & Pechar, M. Tryptophan modification by 2-hydroxy-5-nitrobenzyl bromide studied by MALDI-TOF mass spectrometry. Biochem. Biophys. Res. Commun. 312, 811–816 (2003).
pubmed: 14680838
doi: 10.1016/j.bbrc.2003.11.004
Divakar, K., Priya, J. D. A. & Gautam, P. Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01. J. Mol. Catal. B Enzym. 66, 311–318 (2010).
doi: 10.1016/j.molcatb.2010.06.008
Borhani, M. S., Etemadifar, Z., Emtiazi, G. & Jorjani, E. A statistical approach for production improvement of a neutral protease from a newly isolated strain of Aeromonas Hydrophila. Iran. J. Sci. Technol. Trans. Sci. 42, 1771–1778 (2018).
doi: 10.1007/s40995-017-0444-1