Production of Alkaline Protease Using Industrial Waste Effluent as Low-cost Fermentation Substrate

Document Type : Original Article


1 Department of Chemical and Materials Engineering, Imam Khomeini International University-Buin Zahra Higher Education Center of Engineering and Technology, Buein Zahra, Qazvin, Iran

2 Biotechnology Research Laboratory, Faculty of Chemical Engineering, Babol Noshirvani University of Technology, Babol, Iran


Alkaline proteases are the most important groups of commercial enzymes, which have been broadly used in industrial processes. In this study, Bacillus sp. PTCC 1538 was selected as a biological agent to produce alkaline protease. Enzyme production under submerge fermentation using industrial waste effluent was investigated. Since the costs of the raw material plays an important role in the cost of enzyme production, corn steep liquor (CSL) was selected as a low-cost substrate to reduce the cost of enzyme production. Various carbon sources were used as the auxiliary substrates to enhance enzyme production. Results showed that maximum enzyme activity was obtained when wheat bran was used as an auxiliary substrate. Optimal media composition and growth conditions for alkaline protease production were defined. The optimum conditions were found to be pH 8, incubation temperature of 37 °C, CSL inoculum size of 5 v/v %, yeast extract and wheat bran concentrations of 2 and 6 g/l, respectively. CaCl2 was used as an activator to enhance proteolytic activity of the enzyme. Under optimum condition, enzyme activity of 100.7 U/ml was obtained at CaCl2 concentration of 1.5 g/l.


1.    El-Khonezy, M. I., Elgammal, E. W., Ahmed, E. F. and Abd-Elaziz, A. M., 2021. “Detergent Stable Thiol-Dependant Alkaline Protease Produced from the Endophytic Fungus Aspergillus ochraceus Bt21: Purification and Kinetics”, Biocatalysis and Agricultural Biotechnology,
2.    Srividya, S. and Mala, M., 2011. “Influence of process parameters on the production of detergent compatible alkaline protease by a newly isolated Bacillus sp. Y.”, Turkish Journal of Biology, 35(2), pp: 177-182,
3.    Haddar, A., Fakhfakh-Zouari, N., Hmidet, N., Frikha, F., Nasri, M. and Kamoun, A. S., 2010. “Low-cost fermentation medium for alkaline protease production by  Bacillus mojavensis A21 using hulled grain of wheat and sardinella peptone”, Journal of Bioscience and Bioengineering, 110(3), pp: 288-294,
4.    Cao, S., Song, J., Li, H., Wang, K., Li, Y., Li, Y., Lu, F. and Liu, B., 2020. “Improving characteristics of biochar produced from collagen-containing solid wastes based on protease application in leather production”, Waste Management, 105, pp: 531-539,
5.    Dorra, G., Ines, K., Imen, B. S., Laurent, C., Sana, A., Olfa, T., Pascal, C., Thierry, J. and Ferid, L., 2018. “Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2”, International Journal of Biological Macromolecules, 111, pp: 342-351,
6.    Marathe, S. K., Vashistht, M. A., Prashanth, A., Parveen, N., Chakraborty, S. and Nair, S. S., 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”, Journal of Genetic Engineering and Biotechnology, 16(1), pp: 39-46,
7.    Osmolovskiy, A., Kreier, V., Baranova, N. and Egorov, N., 2017. “Properties of extracellular plasmin-like proteases of Aspergillus ochraceus micromycete”, Applied Biochemistry and Microbiology, 53(4), pp: 429-434,
8.    Rai, S. K. and Mukherjee, A. K., 2010. “Statistical optimization of production, purification and industrial application of a laundry detergent and organic solvent-stable subtilisin-like serine protease (Alzwiprase) from Bacillus subtilis DM-04”, Biochemical Engineering Journal, 48(2), pp: 173-180,
9.    Raj, A., Khess, N., Pujari, N., Bhattacharya, S., Das, A. and Rajan, S. S., 2012. “Enhancement of protease production by  Pseudomonas aeruginosa isolated from dairy effluent sludge and determination of its fibrinolytic potential”, Asian Pacific Journal of Tropical Biomedicine, 2(3), pp: S1845-S1851,
10.    Mehmet, N., Diken, G., Yazici, M., Mazlum, Y., Sayin, S. and Söyler, O., 2021. “The changes in Alkaline, Neutral and Acid Protease Activities of ArtemiaEnriched with Commercial Emulsion and Different Additive Combinations”, Aquatic Sciences and Engineering, 36(3), pp: 152-158,
11.    Mukherjee, A. K., Adhikari, H. and Rai, S. K., 2008. “Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrica grass and potato peel as low-cost medium: Characterization and application of enzyme in detergent formulation”, Biochemical Engineering Journal, 39(2), pp: 353-361,
12.    Rajkumar, R., Jayappriyan, K. R. and Rengasamy, R., 2011. “Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries”, Journal of Basic Microbiology, 51(6), pp: 614-624,
13.    Abidi, F., Limam, F. and Nejib, M. M., 2008. “Production of alkaline proteases by Botrytis cinerea using economic raw materials: assay as biodetergent”, Process Biochemistry, 43(11), pp: 1202-1208,
14.    Liang, T.-W., Hsieh, J.-L. and Wang, S.-L., 2012. “Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation”, Carbohydrate Research, 362, pp: 38-46,
15.    Meena, P., Tripathi, A. D., Srivastava, S. and Jha, A., 2013. “Utilization of agro-industrial waste (wheat bran) for alkaline protease production by Pseudomonas aeruginosa in SSF using Taguchi (DOE) methodology”, Biocatalysis and Agricultural Biotechnology, 2(3), pp: 210-216,
16.    Shankar, S., Rao, M. and Laxman, R. S., 2011. “Purification and characterization of an alkaline protease by a new strain of Beauveria sp.”, Process Biochemistry, 46(2), pp: 579-585,
17.    Patel, R., Dodia, M. and Singh, S. P., 2005. “Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization”, Process Biochemistry, 40(11), pp: 3569-3575,
18.    Ahmed, E., Rateb, M., El-Kassem, L. A. and Hawas, U. W., 2017. “Anti-HCV protease of diketopiperazines produced by the Red Sea sponge-associated fungus Aspergillus versicolor”, Applied Biochemistry and Microbiology, 53(1), pp: 101-106,
19.    Pouryafar, F., Najafpour, G., Noshadi, N. and Jahanshahi, M., 2015. “Thermostable alkaline protease production via solid state fermentation in a tray bioreactor using Bacillus licheniformis ATCC 21424”, International Journal of Environmental Research, 9(4), pp: 1127-1134,
20.    Devanadera, M. K. P., Haw, S. V. O., Arzaga, M. J. J., Buenaflor, L. J., Gagarin, T. J. E., Vargas, A. G., Mercado, S. M. and Santiago, L. A., 2021. “Optimization, production, partial purification and characterization of neutral and alkaline proteases produced by Bacillus subtilis”, Journal of Microbiology, Biotechnology and Food Sciences, 2021, ,pp: 832-838,
21.    Noshadi, N., Mohammadi, M., Najafpour, G. and Pouryafar, F., 2017. “Thermostable alpha-amylase from Lignocellulosic Residues Using Bacillus amyloliquefaciens”, International Journal of Engineering, Transactions B: Applications, 30(8), pp: 1110-1117, 
22.    Mahmoodi, M., Najafpour, G. and Mohammadi, M., 2017. “Production of pectinases for quality apple juice through fermentation of orange pomace”, Journal of Food Science and Technology, 54(12), pp: 4123-4128,
23.    Elumalai, P., Lim, J.-M., Park, Y.-J., Cho, M., Shea, P. J. and Oh, B.-T., 2020. “Agricultural waste materials enhance protease production by Bacillus subtilis B22 in submerged fermentation under blue light-emitting diodes”, Bioprocess and Biosystems Engineering, pp: 1-10,
24.    Oskouie, S. F. G., Tabandeh, F., Yakhchali, B. and Eftekhar, F., 2008. “Response surface optimization of medium composition for alkaline protease production by Bacillus clausii”, Biochemical Engineering Journal, 39(1), pp: 37-42,
25.    Rajan, A. and Nair, A. J., 2011. “A comparative study on alkaline lipase production by a newly isolated Aspergillus fumigatus MTCC 9657 in submerged and solid-state fermentation using economically and industrially feasible substrate”, Turkish Journal of Biology, 35(5), pp: 569-574,
26.    Birhanli, E. and Yeşilada, Ö., 2013. “The utilization of lignocellulosic wastes for laccase production under semisolid-state and submerged fermentation conditions”, Turkish Journal of Biology, 37(4), pp: 450-456,
27.    da Silva, O. S., de Almeida, E. M., de Melo, A. H. F. and Porto, T. S., 2018. “Purification and characterization of a novel extracellular serine-protease with collagenolytic activity from Aspergillus tamarii URM4634”, International Journal of Biological Macromolecules, pp,
28.    Watanabe, M., Techapun, C., Kuntiya, A., Leksawasdi, N., Seesuriyachan, P., Chaiyaso, T., Takenaka, S., Maeda, I., Koyama, M. and Nakamura, K., 2017. “Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function”, Journal of Bioscience and Bioengineering, 123(2), pp: 245-251,
29.    El Enshasy, H., Abuoul-Enein, A., Helmy, S. and El Azaly, Y., 2008. “Optimization of the industrial production of alkaline protease by Bacillus licheniformis in different production scales”, Aust. J. Basic Appl. Sci, 2(3), pp: 583-593.
30.    Joo, H.-S. and Chang, C.-S., 2006. “Production of an oxidant and SDS-stable alkaline protease from an alkaophilic Bacillus clausii I-52 by submerged fermentation: Feasibility as a laundry detergent additive”, Enzyme and Microbial Technology, 38(1), pp: 176-183,
31.    Juhasz, T., Szengyel, Z., Reczey, K., Siika-Aho, M. and Viikari, L., 2005. “Characterization of cellulases and hemicellulases produced by Trichoderma reesei on various carbon sources”, Process Biochemistry, 40(11), pp: 3519-3525,
32.    Mikiashvili, N., Wasser, S. P., Nevo, E. and Elisashvili, V., 2006. “Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity”, World Journal of Microbiology and Biotechnology, 22(9), pp: 999-1002,
33.    Hammami, A., Bayoudh, A., Abdelhedi, O. and Nasri, M., 2018. “Low-cost culture medium for the production of proteases by Bacillus mojavensis SA and their potential use for the preparation of antioxidant protein hydrolysate from meat sausage by-products”, Annals of Microbiology, pp: 1-12,
34.    Qureshi, A. S., Khushk, I., Ali, C. H., Chisti, Y., Ahmad, A. and Majeed, H., 2016. “Coproduction of protease and amylase by thermophilic Bacillus sp. BBXS-2 using open solid-state fermentation of lignocellulosic biomass”, Biocatalysis and Agricultural Biotechnology, 8, pp: 146-151,
35.    Obayori, O. S., Adebusoye, S. A., Ilori, M. O., Oyetibo, G. O., Omotayo, A. E. and Amund, O. O., 2010. “Effects of corn steep liquor on growth rate and pyrene degradation by Pseudomonas strains”, Current Microbiology, 60(6), pp: 407-411,
36.    Shahzad, F., Abdullah, M., Chaudhry, A. S., Hashmi, A. S., Bhatti, J. A., Jabbar, M. A., Ali, H. M., Rehman, T., Ali, F. and Sattar, M. M. K., 2017. “Addition of molasses, corn steep liquor, and rice polish as economical sources to enhance the fungal biomass production of wheat straw by Arachniotus sp”, Turkish Journal of Veterinary and Animal Sciences, 41(3), pp: 332-336,
37.    Ramos, P. R., Kamimura, E. S., Pires, N. A. M., Maldonado, R. R. and de Oliveira, A. L., 2021. “Esterification reaction in SC-CO2 catalyzed by lipase produced with corn steep liquor and Minas Frescal cheese whey”, Bioresource Technology Reports, 14pp: 100670,
38.    Anson, M. L., 1938. “The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin”, Journal of General Physiology, 22(1), pp: 79-89.
39.    Folin, O. and Ciocalteu, V., 1927. “On tyrosine and tryptophane determinations in proteins”, Journal of Biological Chemistry, 73(2), pp: 627-650, 
40.    Shivanand, P. and Jayaraman, G., 2009. “Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast”, Process Biochemistry, 44(10), pp: 1088-1094,
41.    Wang, S.-L. and Yeh, P.-Y., 2006. “Production of a surfactant-and solvent-stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007”, Process Biochemistry, 41(7), pp: 1545-1552,
42.    Chauhan, B. and Gupta, R., 2004. “Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14”, Process Biochemistry, 39(12), pp: 2115-2122,
43.    Gupta, R., Beg, Q., Khan, S. and Chauhan, B., 2002. “An overview on fermentation, downstream processing and properties of microbial alkaline proteases”, Applied Microbiology and Biotechnology, 60(4), pp: 381-395,
44.    Kumar, C. G. and Takagi, H., 1999. “Microbial alkaline proteases: from a bioindustrial viewpoint”, Biotechnology Advances, 17(7), pp: 561-594,
45.    Prakasham, R. S., Rao, C. S. and Sarma, P. N., 2006. “Green gram husk—an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation”, Bioresource Technology, 97(13), pp: 1449-1454,
46.    Beg, Q. K., Sahai, V. and Gupta, R., 2003. “Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor”, Process Biochemistry, 39(2), pp: 203-209,
47.    Hakim, A., Bhuiyan, F. R., Iqbal, A., Emon, T. H., Ahmed, J. and Azad, A. K., 2018. “Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes”, Heliyon, 4(6), pp: e00646,
48.    Hussain, F., Kamal, S., Rehman, S., Azeem, M., Bibi, I., Ahmed, T. and Iqbal, H. M., 2017. “Alkaline protease production using response surface methodology, characterization and industrial exploitation of alkaline protease of Bacillus subtilis sp”, Catalysis Letters, 147(5), pp: 1204-1213,
49.    Ramkumar, A., Sivakumar, N., Gujarathi, A. M. and Victor, R., 2018. “Production of thermotolerant, detergent stable alkaline protease using the gut waste of Sardinella longiceps as a substrate: Optimization and characterization”, Scientific Reports, 8(1), pp: 1-15,