Purification of alkaline serine protease from local Bacillus subtilis M33 by two steps: a novel organic solvent and detergent tolerant enzyme

Year 2019, Volume: 32 Issue: 1, 116 - 129, 01.03.2019

Munteha Nur Sonuc Karaboga , Elif Logoglu

Abstract

Alkaline proteases are important from an industrial perspective due to
their wide scale applications and obtained from different sources. In this
study, an alkaline protease from a newly isolated Bacillus subtilis M33
was purified by ammonium sulfate precipitation and DEAE cellulose anion
exchange chromatography with %38.66 yield and 15.50 fold. The molecular mass of
purified enzyme was determined approximately 39 kDa by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS PAGE) and gel filtration
chromatography. The enzyme exhibited pH and temperature optima of 10.0 and 55°C
respectively, and was stable between a wide pH range of 8.0 and 11.0 for 7
days. Some spesific protease inhibitors such as phenylmethyl sulfonyl fluoride
(PMSF) completely inhibited the enzyme activity. However, the protease activity
was increased in the presence of 2-mercaptoethanol and dithiothreitol,
suggesting it to be a thiol-dependent serine protease.  The enzyme was also stable towards laboratory
bleaches (H²O²), surfactants (Tween 80, Triton X-100,
SDS) and organic solvents such as benzene, toluene, acetone. Different
commercially avaliable detergents were used to study the compatibility of the
purified alkaline protease. The kinetic parameters Km and Vmax of the protease
were determined by measuring the protease activity casein as a substrate 0.706
mg/ml, 3000 μM.min^-1 respectively.

Keywords

Bacillus subtilis, Alkaline protease, Bacillus subtilis, Enzyme purification, DEAE

References

• [1] B. Turk, Targeting Proteases: Successes, Failures and Future Prospects, Nat Rev Drug Discov. 5 (2006) 785-99.
• [2] M.B. Rao, A.M. Tanksale, M.S. Ghatge, Molecular and Biotechnological Aspects of Microbial Protease, Microbiol Mol Biol Rev. 62(1998) 597–635.
• [3] C.L Otin, J.S. Bond, Proteases: Multifunctional Enzymes in Life and Disease, J Biol Chem. 283(2008) 30433-30437.
• [4] D.P.N Ramakrishna, R.N. Gopi, S.V. Rajagopal, Purification and Properties of an Extra Cellular Alkaline Protease Produced By Bacillus subtilis (MTTC N0-10110). Int J Biochem Biotechnol. 6(2010), 493-504.
• [5] A. Gessesse, R. Hatti-Kaul, B.A. Gashe, B. Mattiasson, Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather, Enzyme Microb Technol. 32(2003) 519–524.
• [6] C. Subba Rao, T. Sathish, P. Ravichandra, R.S. Prakasham, Characterization of thermo- and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications, Process Biochem. 44 (2009) 262–268.
• [7] S.K Rai, A.K. Mukherjee, Ecological significance and some biotechnological application of an organic solvent stable alkaline serine protease from Bacillus subtilis strain DM-04, Bioresour Technol. 100(2009)2642–2645.
• [8] J. Singh, R.M. Vohra, D.K. Sahoo, Alkaline protease from a new obligate alkalophilic isolate of Bacillus sphaericus, Biotechnol Lett. 21(1999)921– 924.
• [9] R. Gupta, Q.K. Beg, P. Lorenz, Bacterial alkaline proteases: molecular approaches and industrial applications, Appl Microbiol Biotechnol. 59 (2002) 15 – 32.
• [10] K. Shah, K. Mody, J. Keshri, B. Jha, Purification and characterization of a solvent, detergent and oxidizing agent tolerant protease from Bacillus cereus isolated from the Gulf of Khambhat, J Mol Catal B Enzym. 67 (2010)85–91.
• [11] H.S Joo, C.G Kumar, G.C Park, S.R Paik, C.S. Chang, Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties, J Appl Microbiol. 95(2003)267–272.
• [12] L. Manni, K. Jellouli, O. Ghorbel-Bellaaj, R. Agrebi, A. Haddar Sellami- Kamoun, A. Nasri, An Oxidant- and Solvent-Stable Protease Produced by Bacillus cereus SV1: Application in the Deproteinization of Shrimp Wastes and as a Laundry Detergent Additive, Appl Biochem Biotechnol. 160(2010)2308–2321.
• [13] K. Adinarayana, P. Ellaiah, D.S. Prasad, Purification and Partial Characterization of Thermostable Serine Alkaline Protease from a Newly Isolated Bacillus subtilis PE-11, Pharm Sci Tech. 4(2003)1-9.
• [14] Q.K Beg, R. Gupta, Purification and characterization of an oxidation stable, thiol-dependent serine alkaline protease from Bacillus mojavensis, Enzyme Microb Technol. 32(2003)294-304.
• [15] D. Kumar, V. Kumar, A. K. Verma, P. Singh, A. Dubey, Characterization and immobilization of partially purified alkaline protease extracted from rhizospheric soil bacterium, Bacillus megaterium strain EN-2 and Bacillus subtilis strain EN-3, African Journal of Microbiology Research 8(2014) 33-39.
• [16] R.N.Z.R Rahman, L.P. Geok, M. Basri, A.B. Salleh, An organic solvent stable alkaline protease from Pseudomonas aeruginosa strain K: Enzyme purification and characterization, Enzyme Microb Technol. 39 (2006)1484–1491.
• [17] N. Tekin, A. Coler, C. Basar, B. Karaca, C. Cokmus, A Comparative Study: Taxonomic Grouping of Alkaline Protease Producing Bacilli, Pol J Microbial.66(2017)39-56.
• [18] L.V.A. Reddy, Y.J Wee, H.W. Ryu, Purification and characterization of an organic solvent and detergent-tolerant novel protease produced by Bacillus sp. RKY3, J Chem Technol Biotechnol. 83(2008)1526–1533.
• [19] R. Sareen, P. Mishra, Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37, Appl Microbiol Biotechnol. 79(2008)399–405.
• [20] H. Takami, T. Akiba, K. Horikoshi, Production of extremely thermostable alkaline protease from Bacillus sp. No. AH-101, Appl Microbiol Biotechnol. 30(1989)120-124.
• [21] M. Yilmaz, H. Soran, and Y. Beyatli, Antimicrobial activities of some Bacillus spp. strains isolated from the soil, Microbiological Research. 161 (2006)127-131.
• [22] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem. 72(1976) 248-254.
• [23] U.K. Laemmli, Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 227(1970) 680 - 685.
• [24] C.G Kumar, H. Takagi, Microbial alkaline proteases: From a bioindustrial viewpoint. Biotechnol Adv. 17(1999)561-594.
• [25] M. Arulmani, K. Aparanjini, K. Vasanthi, P. Arumugam, M. Arivuchelvi, T. Kalaichelvan, Purification and partial characterization of serine protease from thermostable alkalophilic Bacillus laterosporus-AK1, World J Microbiol Biotechnol. 23(2007)475–481.
• [26] T. Maruthiah, P. Esakkiraj, Purification and characterization of moderately halophilic alkaline serine protease from marine Bacillus subtilis AP-MSU, Biocatalysis and Agricultural Biotechnology. 2(2013)116-119.
• [27] I. Abdelnasser, A. Salamah, Detergent-, solvent- and salt-compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization, Extremophiles. 5(2015)961-971.
• [28] S. Tanskul, K. Hiraga, K. Takada, S. Rungratchote, P. Suntinanalert, K. Oda, An alkaline serine-proteinase from a bacterium isolated from Bat Feces: purification and characterization, Biosci. Biotechnol. Biochem. 73(2009)2393-2398.
• [29] Y. Toyokawa, H. Takahara, A. Reungsang, M. Fukuta, Y. Hachimine, S. Tachibana, M. Yasuda, Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce, Appl Microbiol Biotechnol. 86(2010)1867–1875.
• [30] S.K. Rai, A.K. Mukherjee, 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(2010)173–180.
• [31] M.S. Dodia, C.M. Rawal, H.G. Bhimani, Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp, AH-6. J Ind Microbiol Biotechnol. 35(2008)121– 131.
• [32] V. Zambare Purification and characterization of neutral serine protease from Bacillus sp, Asiatic J. Biotechnol. Resour. 3(2010)183–192.
• [33] H.Uchida, D. Kondo, S. Yamashita, T. Tanaka, L.H. Tran, H. Nagano, T. Uwajima, Purification and properties of a protease produced by Bacillus subtilis CN2 isolated from a Vietnamese fish sauce. World Journal of Microbiology & Biotechnology, 20(2004)579-582.
• [34] D. Kazan, A. A. Denizci, M. N. Kerimak Oner, A. Erarslan, Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42., J Ind Microbiol Biotechnol 32(2005)335 344.
• [35] A. Gupta, I. Roy, S.P. Singh, S.K. Khare, M.N Gupta, One-step purification and characterization of an alkaline protease from haloalkaliphilic Bacillus sp. Journal of Chromatography A, 1075(2005) 103–108.
• [36] Ghafoor, A.; Hasnain, S. Purification and characterization of an extracellular protease from Bacillus subtilis EAG-2 strain isolated from ornamental plant nursery, Polish Journal of Microbiology. 59(2010)107-112.