Production and characterization of crude proteases from Halobacillus salinus strain DZ28 newly isolated from salt lake sediments in Algeria and their use as detergent bioadditives



Halobacillus salinus, Protease, Process optimization, Laundry detergent, Wash performance


New Algerian Gram-positive, rod-shaped, endospore-forming, salt-philic bacteria (DZ28 strain) that overproduce extracellular alkaline proteases have been isolated from salt lake deposits in Lake Oubeira, El Taref. Strain DZ28 was assigned as Halobacillus salinus DZ28 on the basis of phenotypic properties and 16S rDNA gene sequencing (ripotyping). The maximum protease activity registered after 36 hours of incubation in optimized medium at 30 ° C was 19,000 U / ml in a shaking bottle culture at 160 rpm. The crude extract protease showed optimal activity at 60 ° C temperature and pH 12. It is actively inhibited by PMSF and DIFP, indicating that it belongs to the serine protease family. Interestingly, the crude extract protease was not only very stable to nonionic surfactants and oxidants, but also showed high stability and compatibility with some commercial detergents. It retaining more than 100% of its initial activity after pre-incubation for 1 h at 40°C with ISIS, followed by Pril (98%), Tide (95%) and Dixan (90%). More curiously, the wash overall performance evaluation discovered that it may dispose of blood-stains remove at 40°C for 1 h with low supplementation (500 U/mL). This is the first report of a protease from Halobacillus salinus and has potential as a promising candidate for future applications as a bioadditive for detergent formulations.


Download data is not yet available.

• Annamalai, N., Rajeswari, M. V., Sahu, S.K., & Balasubramanian, T. (2014). Purification and characterization of solvent stable, alkaline protease from Bacillus firmus CAS 7 by microbial conversion of marine wastes and molecular mechanism underlying solvent stability. Process. Biochem, 49 (6): 1012-1019. doi: 10.1016/j.procbio.2014.03.007

• Banik, R. M., & Prakash, M. (2004). Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res, 159 (2): 135-140.

• Baweja, M., Nain, L., Kawarabayasi, Y., & Shukla, P. (2016). Current Technological Improvements in Enzymes toward Their Biotechnological Applications. Front. Microbiol, 7: 965. doi: 10.3389/fmicb.2016.00965

• Baweja, M., Singh, P. K., Sadaf, A., Tiwari, R., Nain, L., Khare, S. K., & Shukla, P. (2017). Cost effective characterization process and molecular dynamic simulation of detergent compatible alkaline protease from Bacillus pumilus strain MP27. Process. Biochem, 58: 199-203.

• Beg, Q., & Gupta, R. (2003). Purification and characterization of an oxidation stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme. Microb. Technol, 32: 294-304.

• Benkiar, A., Nadia, Z. J., Badis, A., Rebzani, F., Soraya, B. T., Rekik, H., Naili, B., Ferradji, F. Z., Bejar, S., & Jaouadi, B. (2013). Biochemical and molecular characterization of a thermo- and detergent-stable alkaline serine keratinolytic protease from Bacillus circulans strain DZ100 for detergent formulations and feather-biodegradation process. Int. Biodeterior. Biodegrad, 83: 129-138.

• Bouacem, K., Bouanane-Darenfed, A., Laribi-Habchi, H., Elhoul, M. B., Hmida-Sayari, A., Hacene, H., Ollivier, B., Fardeau, M. L., Jaouadi, B., & Bejar, S. (2015). Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis. Int. J. Biol. Macromol, 81: 299-307. doi: 10.1016/j.ijbiomac. 2015.08. 011

• Bradford, M. M. (1976). A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72: 248-254.

• Chew, L. Y., Toh, G.T., & Ismail, A. (2019). Chapter 15 - Application of Proteases for the Production of Bioactive Peptides. In: Enzymes in Food Biotechnology, (Ed.) M. Kuddus, Academic Press, pp. 247-261.

• Dos Santos, Aguilar, J. G., & Sato, H.H. (2018). Microbial proteases: production and application in obtaining protein hydrolysates. Food. Res. Int, 103: 253-262.

• Gong, B. L., Mao, R. Q., Xiao, Y., Jia, M. L., Zhong, X. L., Liu, Y., Xu, P. L., & Li, G. (2017). Improvement of enzyme activity and soluble expression of an alkaline protease isolated from oil-polluted mud flat metagenome by random mutagenesis. Enzyme. Microb. Technol, 106: 97-105. doi: 10.1016/j.enzmictec.2017.06.015

• Gupta, R., Beg, Q. K., & Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol, 59(1): 15-32. doi: 10.1007/s00253-002-0975-y

• Gupta, R., Gupta, K., Saxena, R., & Khan, S. (1999). Bleach-stable alkaline protease from Bacillus sp. Biotechnol. Lett, 21(21): 135-138.

• Gurumallesh, P., Alagu, K., Ramakrishnan, B., & Muthusamy, S. (2019). A systematic reconsideration on proteases. Int. J. Biol. Macromol, 128: 254-267. doi: 10.1016/j.ijbiomac.2019.01.081

• Habicher, T., John, A., Scholl, N., Daub, A., Klein, T., Philip, P., & Buchs, J. (2019). Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane-based fed-batch shake flasks. Biotechnol. Bioeng, 116(6):1326-1340. doi: 10.1002/bit.26948

• 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(13): 3366-3373.

• Hadjidj, R., Badis, A., Mechri, S., Eddouaouda, K., Khelouia, L., Annane, R., El Hattab, M., Jaouadi, B. (2018). Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A. Int. J. Biol. Macromol, 114: 1033-1048. doi: 10.1016/j.ijbiomac.2018.03.167

• Jaouadi, B., Abdelmalek, B., Fodil, D., Ferradji, F. Z., Rekik, H., Zarai, N., & Bejar, S. (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(21): 8361-9. doi: 10.1016/j.biortech.2010.05.066

• Jaouadi, B., Ellouz-Chaabouni, S., Ben Ali, M., Ben Messaoud, E., Naili, B., Dhouib, A., & Bejar, S. (2009). Excellent laundry detergent compatibility and high dehairing ability of the Bacillus pumilus CBS alkaline proteinase (SAPB). Biotechnol. Bioprocess. Eng, 14: 503-512.

• Jaouadi, B., Ellouz-Chaabouni, S., Rhimi, M., & Bejar, S. (2008). Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency. Biochim, 90(9): 1291-305. doi: 10.1016/j.biochi.2008.03.004

• Jaouadi, N. Z., Rekik, H., Badis, A., Trabelsi, S., Belhoul, M., Yahiaoui, A. B., Ben Aicha, H., Toumi, A., Bejar, S., & Jaouadi, B. (2013). Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities. PLoS One, 8(10): e76722. doi: 10.1371/journal.pone.0076722

• Jaouadi, N. Z., Jaouadi, B., Aghajari, N., Bejar, S. (2012). The overexpression of the SAPB of Bacillus pumilus CBS and mutated sapB-L31I/T33S/N99Y alkaline proteases in Bacillus subtilis DB430: new attractive properties for the mutant enzyme. Bioresour. Technol, 105: 142-51. doi: 10.1016/j.biortech.2011.11.115

• Joo, H. S., & Chang, C.S. (2005). Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: optimization and some properties. Process. Biochem, 40(3-4): 1263-1270.

• Kim, O.S., & Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S., Chun, J., (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. International Journal of Systematic and Evolutionary Microbiology, 62: 716–721.

• Lee, S., & Jang, D.J. (2001). Progressive rearrangement of subtilisin Carlsberg into orderly and inflexible conformation with Ca (2+) binding. Biophys. J, 81(5): 2972-2988. doi: 10.1016/S0006-3495(01)75937-1

• Liu, X., Kokare, C. (2017). Microbial enzymes of use in industry. In: Biotechnology of Microbial Enzymes. Elsevier, pp. 267-298.

• Maghsoodi, V., Kazemi, A., Nahid, P., Yaghmaei, S., & Sabzevari, M.A. (2013). Alkaline protease production by immobilized cells using Bacillus licheniformis. Sci. Iran, 20(3): 607-610.

• Mechri, S,, Berrouina, M. B. E., Benmrad, M. O., Jaouadi, N. Z., Rekik, H., Moujehed, E., Chebbi, A., Sayadi, S., Chamkha, M., & Bejar, S. (2017a). Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest. Int J Biol Macromol, 94: 221-232. doi: 10.1016/j.ijbiomac.2016.09.112

• Mechri, S., Kriaa, M., Ben Elhoul Berrouina, M., Omrane Benmrad, M., Zaraî Jaouadi, N., Rekik, H., Bouacem, K., Bouanane-Darenfed, A., Chebbi, A., Sayadi, S., Chamkha, M., Bejar, S., & Jaouadi, B. (2017b). Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R. Int. J. Biol. Macromol, 101: 383-397.

• Mechri, S., Bouacem, K., Jabeur, F., Mohamed, S., Addou, N. A., Dab, A., Bouraoui, A., Bouanane-Darenfed, A., Bejar, S., Hacène, H., Baciou, L., Lederer, F., & Jaouadi, B. (2019a). Purification and biochemical characterization of a novel thermostable and halotolerant subtilisin SAPN, a serine protease from Melghiribacillus thermohalophilus Nari2AT for chitin extraction from crab and shrimp shell by-products. Extremophiles, 23(5): 529-547. doi: 10.1007/s00792-019-01105-8

• Mechri, S., Bouacem, K., Zaraî Jaouadi, N., Rekik, H., Ben Elhoul, M., Omrane Benmrad, M., Hacene, H., Bejar, S., Bouanane-Darenfed, A., & Jaouadi, B. (2019b). Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive. Extremophiles, 23: 687-706.

• Nazari, L., & Mehrabi, M. (2019). Purification and characterization of an extracellular thermotolerant alkaliphilic serine protease secreted from newly isolated Bacillus sp. DEM07 from a hot spring in Dehloran, Iran. Biocatal. Agric. Biotechnol, 18: 101053.

• Omrane Benmrad, M., Mechri, S., Zaraî Jaouadi, N., Ben Elhoul, M., Rekik, H., Sayadi, S, Bejar, S., Kechaou, N., Jaouadi, B. (2019). Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest. BMC Biotechnol, 19(1): 43.

• Radha, S., & Gunasekaran, P. (2008). Sustained expression of keratinase gene under PxylA and PamyL promoters in the recombinant Bacillus megaterium MS941. Bioresour. Technol, 99(13): 5528-5537. doi: 10.1016/j.biortech.2007.10.052

• Rahem, F.Z., Badis, A., Zenati, B., Mechri, S., Hadjidj, R., Rekik, H., Eddouaouda, K., Annane, R., & Jaouadi, B. (2021). Characterization of a novel serine alkaline protease from Bacillus atrophaeus NIJ as a thermophilic hydrocarbonoclastic strain and its application in laundry detergent formulations. Algerian J. Env. Sc. Technology, 7: (1) 1707-1724.

• Raval, V. H., Pillai, S., Rawal, C. M., & Singh, S. P. (2014). Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria. Process. Biochem, 49(6): 955-962.

• Rekik, H., Zaraî Jaouadi, N., Gargouri, F., Bejar, W., Frikha, F., Jmal, N., Bejar, S., & Jaouadi, B. (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. doi: 10.1016/j.ijbiomac.2018.10.139

• Sambrook, J., Fritsch, E., & Maniatis, T. (1989). Molecular cloning: A laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

• Singh, J., Batra, N., Sobti, R. C. (2001). Serine alkaline protease from a newly isolated Bacillus sp. SSR1. Process Biochem, 36(8-9): 781-785.

• Steuer, C., Heinonen, K. H., Kattner, L., & Klein, C. D. (2009). Optimization of assay conditions for dengue virus protease: effect of various polyols and nonionic detergents. J. Biomol. Screen, 14(9): 1102-8. doi: 10.1177/1087057109344115

• Takagi, H., Maeda, T., Ohtsu, I., Tsai, Y. C., & Nakamori, S. (1996). Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue. FEBS letters, 395(2-3): 127-132. doi: 10.1016/0014-5793(96)01014-9

• Venugopal, M., & Saramma, A. V. (2006). Characterization of alkaline protease from Vibrio fluvialis strain VM10 isolated from a mangrove sediment sample and its application as a laundry detergent additive. Process. Biochem, 41(6): 1239-1243.

• Vojcic, L., Pitzler, C., Körfer, G., Jakob, F., Ronny, M., Maurer, K. H., & Schwaneberg, U. (2015). Advances in protease engineering for laundry detergents. New. Biotechnol, 32(6): 629-634. doi: 10.1016/j.nbt.2014.12.010

• Yabuuchi, E. (2001). Current topics on classification and nomenclature of bacteria. 7. Taxonomic outline of Archeae and bacteria in the Second Edition of Bergey's Manual of Systematic Bacteriology. Kansenshogaku Zasshi, 75(8): 653-655. doi: 10.11150/kansenshogakuzasshi1970.75.653

• Zhu, X., & Zhang, H. (2019) .Optimization of [CnPy]Cl (n=2,4,6) ionic liquid aqueous two-phase system extraction of papain using response surface methodology with box-behnken design. Process. Biochem, 77: 113-121.



How to Cite

MEDJEKAL, S., GHADBANE, M., BELHADJ, H., & BENDERRADJI, L. (2023). Production and characterization of crude proteases from Halobacillus salinus strain DZ28 newly isolated from salt lake sediments in Algeria and their use as detergent bioadditives. International Journal of Agriculture, Environment and Food Sciences, 7(1).



Research Article