Characterization of Geobacillus stearothermophilus protease for detergent industry

  • I. Iqbal
  • M.N. Aftab
  • M.S. Afzal
  • A. Kaleem
Keywords: Geobacillus stearothermophilus, protease, thermophile, detergent, purification


Cloning of protease gene from a thermophilic strain of Geobacillus stearothermophilus (B-1172) was carried out in E. coli BL 21, and its expression was studied. The expressed protease was purified followed by its identification. A 16.9 folds purification with 55.68% recovery of the protease was achieved by ammonium sulfate precipitation and gel filtration chromatography. The protease specific activity was 120 U mg−1. The purified enzyme remained stable at 90°C at a pH range 6-9. Its interaction with EDTA, different metal ions, inhibitors, surfactants and detergents was also mapped. Its interaction with EDTA showed no significant effect on the activity of the enzyme confirming its metaloprotease nature. Metal ions i.e. Ca2+, Mg2+, Ni2+, Cd2+, Cu2+, Zn2+ showed no significant effect on the stability of protease. Its compatibility was checked with different commercial detergent (6 mg/mL) such as Surf Excel Arial, Bonus, wheel and Shine. It retained more than 80% proteolytic activity in all detergents after incubation at 50°C for 1 h. Wash performance analysis of the protease of G. stearothermophilus showed good results of de-staining of blood sample at various temperatures. Therefore, recombinant protease could prove as good candidate for commercial use in detergents.


Abidi. F., Liman, F. and Nejib, M. (2008). Production of alkaline proteases by Botrytis cinerea using economic raw materials: assay as biodetergent. Process Biochemistry, 43, 1202-1208.
Asmaa, Ali., Hameed, M. and Nooria, A. (2011). Molecular Cloning and expression of Bacillus stearothermophilus protease gene in Escherichia coli. Journal of Biology and Life Science, 2, 26-31
Bhaskar, N., Sudeepa, E.S., Rashmi, H.N. and Tamil, S.A. (2007). Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities. Bioresource Technology, 98, 2758-2764.
Birnboim, H.C. and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 2, 1513-23.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal of Biochemistry, 72, 248–254.
Cavello, A., Hours, R. and Cavalitto, S. (2012). Bioprocessing of “Hair Waste” by Paecilomyces lilacinus as a Source of a Bleach-Stable, Alkaline, and Thermostable Keratinase with Potential Application as a Laundry Detergent. Additive: Characterization and Wash Performance Analysis. Biotechnology Research International, 2,1-12.
Chatterjee, J., Giri, S., Maity, S., Sinha, A., Ranjan, A., Rajshekhtar, Y. and Gupta, S. (2016). Production and characterzation of theromostable alkaline protease of Bacillus subtillis (ATCC 6633) from optimized solid-state fermentation. Biotechnology and Applied Biochemistry, 62, 709-718.
Chen, X.G., Stabnikova, O., Tay, J.H., Wang, J.Y. and Tay, S.T. (2004). Thermoactive extracellular proteases of Geobacillus caldoproteolyticus, sp. nov., from sewage sludge. Extremophiles, 8, 489-498.
Doddapaneni, K.K., Tatineni, R., Vellanki, R.N., Gandu, B., Panyala, N.R., Chakali, B. and Mangamoori, L.N. (2007). Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus. Microbiology Research, 164, 383-390.
Esakkiraj, P., Balraj, M., Avinash, K.L., Repally, A. and Venkatesan, A. (2016). Cloning, expression, characterization and application of protease produced by Bacillus cereus PMW8. RSC Advances, 6, 38611.
Erban, T., Rybanska, D., Harant, B., Hortova, B. and Hurbert, J. (2016). Feces derived allergens of Tyrophagus putrescentiae reared on dried dog food and evidence of the strong nutritional interaction between the mite and bacillus cereus producing protease Bacillolysins and exochitinases. Frontiers in Physiology, 7, 53.
Gupta, R., Beg, Q.K. and Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59, 15-32.
Güracar, S. (2011). Production, purification and characterization of thermostable protease from kaliphilic and thermophilic Geobacillus sp. Dissertation, School of Engineering and Sciences of İzmir Institute of Technology.
Haki, G. and Rakshit, S. (2003). Developments in industrially important thermostable enzymes: a review. Bioresource Technology, 89, 17-34.
Hang-seung, J. and Jan, W.C. (2012). Purification and characterization of a novel alkaline protease from Bacillus horikoshii. Journal of Microbiology and Biotechnology, 22, 58-68.
Hirata, A., Hori, Y., Koga, Y., Okada, J., Sakudo, A., Ikuta, K., Kanaya, S. and Takano, K. (2013). Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein. BMC Biotechnology, 13, 19.
Hutadilok-Towatana, N., Painupong, A. and Suntinannalert, P. (1999). Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719. Journal of Biosciences and Bioengineering, 87, 581-587.
Iqbal, I., Aftab, M.N., Afzal, M., Rehman, U.A., Aftab, S., Zafar, Z., Din, U.D., Khuharo, A.R., Iqbal, J. and Haq, I.U. (2014). Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus. Journal of Basic Microbiology, 52,1-12.
Jellouli, K., Ghorbel-Bellaaj, O., Ayed, H.B., Manni, L., Agrebi, R. and Nasri, M. (2011). Alkaline-protease from Bacillus licheniformis MP1: purification, characterization and potential application as a detergent additive and for shrimp waste deproteinization. Process Biochemistry, 46, 1248-1256.
Jacobs, M., Elkiasson, M., Uhlén, M. and Flock, J.I. (1985). Cloning, sequencing and expression of Subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acid Research, 13, 8913-8926.
Lakshmi, B.K.M., Ratna, Sri, P.V., Ambika, Devi, K. and Hemalatha, K.P.J. (2014). Media optimization of protease production by Bacillus licheniformis and partial characterization of Alkaline protease. International journal of Current Microbiology and Applied Sciences, 3, 650-659
Lika-Ida, E., Rodringues, da Silver, R., Brito de Oliveira, T., Beltramini Souto, T., Leite, J.A., Rodrigues, A. and Cabral, H. (2016). Biochemical properties and evaluation of washing performance in commercial detergent compatibility of two collagenolytic serine peptidase secreted by Aspergillus fischeri and Penicillium citrinum. Preparative Biochemistry and Biotechnology doi: 10.1080/10826068.2016.1224247
Lomate, P.R. and Bonning, B.C. (2016). Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula. Scientific Reports, 6, 27587. 10.1038/srep27587.
Mahajan, C., Basora, N., Singh, S., Di Falco, M., Tsang, A. and Chadha, B.S. (2016). Malbranchea cinnamomea: A theromophilic fungal source of catalytically efficient lignocellulolytic glycosyl source hydrolases and metal dependent enzymes. Bioresource Technolnology 200: 55-63.
Maharaja, P., Nanthini, E., Swarnalatha, S. and Sekaram, G. (2018). Studies on the production of slat tolerant alkaline protease isolated from Proreus mirabilis and its degradation oh Hyper-saline soak liquor. Enviromental Pollution, 77, 439-457.
McDonald, C.E. and Chen, L.L. (1965). The Lowry modification of the folin reagent for determination of proteinase activity. Anal Biochemistry, 10, 175-177.
Megha, K., Purohit, Satya, P. and Singh, A. (2013). Metagenomic alkaline protease from saline habitat: cloning, over-expression and functional attributes. International Journal of Biological Macromolecules, 53, 138–143.
Mei, C. and Jiang, X. (2005). A novel surfactant- and oxidation-stable alkaline protease from Vibrio metschnikovii DL33–51. Process Biochemistry, 40, 2167-2172.
Mohsen, M.S., Asker, M.G., Mahmoud, K.E.S. and Mohamed, S.A.A. (2013). Purification and characterization of two thermostable protease fractions from Bacillus megaterium. Journal of Genetic Engineering and Biotechnology, 11, 103-109.
Muhammad, A., Bokhari, S.A.I., Vernoux, J.P., Ali, M.I., Faryal, R., Desmasures, N. and Imran, M. (2019). Purification, characterization and thermodynamic assessment of an alkaline protease by Geotrichum candidum of dairy origin. Iranian Journal of Biotechnology 17. DOI: 10.21859/ijb.2042
Ochi, H., Takai, T., Shimura, S., Maruyama.N., Nishioka, I., Kamjio, S., Iida, H., Nakae, S., Ogawa, H., Okimura, K. and Ikeda, S. (2017). Skin Treatment with Detergent Promotes Protease Allergen-Dependent Epicutaneous Sensitization in a Manner Different from Tape Stripping in Mice. J Investigative Dermatology, 137, 1578-1582.
Padmapriya, B., Rajeswari, T., Nandita, R. and Raj, F. (2012). Production and Purification of Alkaline Serine Protease from Marine Bacillus Species and Its Application in Detergent Industry. European Journal of American Studies,4, 21-26.
Peek, R.M., Daniel, C. and Monk, C. (1992). Purification and characterization of thermostable proteinease isolated from Thermos sp. Strain Rt 41A, European Journal of Biochemistry, 207, 1035-44.
Purohit, P., Satya, I. and Singh, P. (2014). Cloning, over expression and functional attributes of serine proteases from Oceanobacillus iheyensis. Process Biochemistry 49: 61–68. DOI: 10.1016/j.procbio.2013.07.009
Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A. and Ashraf, M. (2019). Microbial proteases applications. Frontiers in Bioengineering and Biotechnology, 7.
Salem, S., Shabeb, M.S.A. and Amara, A.A. (2005). Optimization of thermophilic protease production in Bacillus mixed cultures under mesophilic conditions. World Journal of Agriculture Science, 3, 375-383.
Sammond, D.W., Kastelowitz, N., Himmel, M.E., Yin, H., Crowley, M.F. and Bomble, Y.J. (2016). Comparing residue clusters from thermophilic and mesophilic enzymes reveals adaptive mechansims. PLos One, 11, e0145848.
Sanatan, P.T., Lomate, P.R., Giri, A.P. and Hivrale, V.K. (2013). Characterization of a chemostable serine alkaline protease from Periplaneta americana. BMC Biochemistry, 14, 32.
Sarmiento, F., Peralta, R. and Blamey, J.M. (2015). Cold and hot extremozymes: industrial relevance and current trends. Frontiers in Bioengineering and Biotechnology, 3, 148.
Savitha, S., Sadhasivam, S., Swaminathan, K. and Lin, F.H. (2011). Fungal protease: production, purification and compatibility with laundry detergents and their wash performance. Journal of Taiwan Institute of Chemistry Engineers, 42, 298-304.
Sharma, M., Gat, Y., Arya, S., Kumar, V., Panghal, A. and Kumar, A. (2019). A Review on Microbial Alkaline Protease: An Essential Tool for Various Industrial Approaches. Industrial Biotechnology, 15, 69-78.
Sookkheo, B., Sinchaikul, S., Phutrakul, S. and Chen, S.T. (2000). Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33. Protein Expressiona Purification, 20, 142-151.
Srinubabu, G., Lokeswari, L. and Jayaraju, K. (2007) Screening of nutritional parameters for the production of protease from Aspergillus oryzae. Journal of Chemistry, 4, 208-215.
Swati, J. and Satyanarayana, T. (2013). Characteristics and applications of a recombinant alkaline serine protease from a novel bacterium Bacillus lehensis. Bioresource Technology, 131l, 76-85.
Thao, T.N., Thi, Q. and Hoang, T.L. (2013). Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800. Microbiology Cell Fact ,12, 79.
Thebti, W., Riahi, Y., Gharsalli, R. and Belhadj, O. (2016). Screening and characterization of thermoactive enzymes of biotechnological interest produced by thermiphilic Bacillus isolated from hot springs in Tunisia. Acta Biochemica Polonica 10.18388/abp.2016_1271.
Traut, R., Casiano, C., Zecherle, N. and Creighton, T. (1989). Protein Function: A practical Approach. Oxford IRL Press. pp. 101-133.
Valls, C., Pujadas, G., Garcia-Vallve, S. and Mulero, M. (2011). Characterization of the protease activity of detergents: laboratory practicals for studying the protease profile and activity of various commercial detergents. Biochemistry and Molicular Biology Education, 39, 280-90. doi: 10.1002/bmb.20488.
Wells, J.A., Ferrari, E., Henner, D.J., Estell, D.A. and Chen, E.Y. (1983). Cloning, sequencing and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acid Research, 11, 7911-7929.
Wu, S., Liu, G., Xhang, D., Li, C. and Sun, C. (2015). Purification and biochemical characterization of an alkaline protease from marine bacteria Pseudo alteromonas sp. 129-1. Journal of Basic Microbiology, 55, 1427-1434
How to Cite
Iqbal, I., Aftab, M., Afzal, M., & Kaleem, A. (2020). Characterization of Geobacillus stearothermophilus protease for detergent industry. Revista Mexicana De Ingeniería Química, 19(Sup. 1), 267-279.

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