Isolation, Purification and Characterization of new cold active subtilisin-like protease from Bacillus sp. strain EL-GU1

Yıl 2019, , 2057 - 2073, 31.07.2019

Elif Guduk , Gulhan Yasar Unzile Guven Gulhan Fatih Aktaş

https://doi.org/10.29130/dubited.537340

Cited By: 1

Öz

Proteases are hydrolytic enzymes that slice peptide bonds between amino
acid residues and these enzymes have various industrial applications including
detergent, food, pharmaceutical, leather and diagnostic reagent industries.
Among them, alkaline proteases, the most commercialized enzymes in the
industry, are of particular interest due to their potential applications in the
detergent industry as cleaning additives. In this study, a novel alkaline
protease from Bacillus sp. strain EL-GU1 was
reported showing highest activity at pH 6 and 25°C. The novel protease was
purified by using ammonium sulfate precipitation and identified by 16S rDNA
sequencing. Highest activity was observed as 3300 µmol/min^-1mg^-1 when casein used as a
substrate. Kinetic parameters of the enzyme were determined; K_M, V_max,
k_cat and catalytic efficiency values were calculated as 1.4 mM, 1
mM/s, 2.10^-7 s^-1, 0.14 10^-7 s^-1M^-1,
respectively. These results indicated that the novel cold active protease from Bacillus sp. strain EL- GU1 can be a good
candidate for the detergent industry

Anahtar Kelimeler

Bacillus sp., protease, enzyme characterization, cold active and alkali protease

Soğuk Aktif ve Alkali Subtilisin Benzeri Proteaz Enziminin Bacillus sp. strain EL-GU1’ den İzolasyonu, Saflaştırılması ve Karekterizasyonu

Öz

One
of the important hydrolytic enzymes are proteases that slice peptide bonds
between amino acid residues. Proteases have various industrial applications
including detergent, food, pharmaceutical, leather and diagnostic reagent
industries. Among them, the most commercialized enzymes are alkaline proteases
in the industry. Due to their potential applications in the detergent industry
as cleaning additives, they are of particular interest. In this study, a novel
protease from Bacillus sp. strain EL-GU1 was
reported showing highest activity at pH 6 and 20°C. The novel protease was
purified by using ammonium sulfate precipitation and identified by 16S rDNA
sequencing. Highest activity was observed as 3300 µmol/min^-1mg^-1 when casein used as a
substrate. Kinetic parameters of the enzyme were determined; K_M, V_max,
k_cat and catalytic efficiency values were calculated as 1.4 mM, 1
mM/s, 2.10^-7 s^-1, 0.14 10^-7 s^-1M^-1,
respectively. These results indicated that the novel cold active protease from Bacillus sp. strain EL- GU1 can be a
good candidate for the detergent industry.

 

Anahtar Kelimeler

Bacillus sp., proteaz, enzim karakterizasyonu, soğuk aktif ve alkali proteaz

Kaynakça

• [1] K. Jellouli, O. Ghorbel Bellaaj, H. Ayed, L. Manni, R. Agrebi, M. Nasri. “Alkaline-protease from Bacillus licheniformis MP1: Purification, characterization and potential application as a detergent additive and for shrimp waste deproteinization”, Process Biochemistry, vol. 46, pp. 1248–1256, 2011.
• [2] M. B. Rao, A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande, “Molecular and biotechnological aspects of microbial proteases,” Molecular and Biotechnological Aspects of Microbial Proteases, vol. 62, no. 3, pp. 597-635, 1998.
• [3] R. Gupta, Q. K. Beg, P. Lorenz, “Bacterial alkaline proteases: molecular approaches and industrial applications,” Applied Microbiology and Biotechnology, vol. 59, pp. 1-15, 2002b.
• [4] S. Ito, T. Kobayashi, K. Ara, K. Ozaki, S. Kawai, Y. Hatada, “Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures,” Extremophiles, vol. 2, pp. 185, 1998.
• [5] K. Horikoshi, “Alkaliphiles – from an industrial point of view,” FEMS Microbiology Reviews vol. 18, pp. 259-270, 1996.
• [6] F. B. Rebah, and N. Miled, “Fish Processing Wastes for Microbial Enzyme Production: A Review,” 3 Biotech, vol. 3, no. 4, pp. 255–265, 2013.
• [7] M. H. Kalisz, “Microbial proteinases,” in Enzyme Studies, Advances in Biochemical Engineering/Biotechnology, vol. 36, Berlin, Heidelberg: Springer, 1988, pp. 17–55.
• [8] B. Poldermans, “Proteolytic enzymes” in W. Gerhartz(ed.), Proteolytic Enzymes in Industry: Production and Applications, Weinheim, Germany, VCH Publishers, 1990, pp. 108–123.
• [9] F.W. Studier, “Protein production by auto-induction in high-density shaking cultures,” Protein Expression and Purification, vol. 41, pp. 207–234, 2005.
• [10] M. Kunitz, “Isolation of a crystalline protein compound of trypsin and of soybean trypsin inhibitor,” The Journal of General Physiology, vol. 30, no. 4, pp. 311–320, 1947.
• [11] P. Wingfield, “Protein precipitation using ammonium sulfate,” Current protocols in protein science, Appendix 3: Appendix 3F, 200 1.
• [12] M. M. S. Asker, M. G. Mahmoud, K. El Shebwy, M. S. Abd el Aziz, “Purification and characterization of two thermostable protease fractions from Bacillus megaterium,” Journal of Genetic Engineering and Biotechnology, vol. 11, pp. 103–109, 2013.
• [13] B. Liu, N. Zhang, C. Zhao, B. Lin, L. Xie, Y. Huang, “Characterization of a recombinant hermostable xylanase from hot spring thermophilic Geobacillus sp. TC-W7,” J. Microbiol. Biotechnol. vol. 22, pp. 1388–1394, 2012.
• [14] N. Muhammad, J. Qazi, Q . Syed, and M. Gulsher, “Purification and Characterization of an Alkaline Protease from Bacillus Licheniformis UV-9 for Detergent Formulations,” Songklanakarin Journal of Science and Technology, vol. 35, no. 2, pp. 187-195, 2013.
• [15] A. Anwar, S. A. Ul Qader, A. Raiz, S. Iqbal and A. Azhar “Calcium Alginate: A Support Material for Immobilization of Proteases from Newly Isolated Strain of Bacillus Subtilis KIBGE-HAS.” World Applied Sciences Journal, vol. 7, no. 10, pp. 1281–1286, 2009.
• [16] A. Haddar, A. Sellami-Kamoun, N. Fakhfakh-Zouari, N. Hmidet, M. Nasri, “Characterization of Detergent Stable and Feather Degrading Serine Proteases from Bacillus Mojavensis A21,” Biochemical Engineering Journal, vol. 51, pp. 53–63, 2010.
• [17] J. Swati, and T. Satyanarayana. “Biotechnology of Cold-Active Proteases,” Biology vol. 2, pp. 755–783, 2013.
• [18] B. Johnvesly, and G. R. Naik. “Studies on Production of Thermostable Alkaline Protease from Thermophilic and Alkaliphilic Bacillus Sp. JB-99 in a Chemically Defined Medium,” Process Biochemistry, vol. 37, pp. 139-144, 2001.
• [19] Abdelnasser S.S. Ibrahim, Ali, A. Al-Salamah, Yahya, B. Elbadawi, Mohammed, A. El-Tayeb, and Shebl S. S. Ibrahim, “Production of Extracellular Alkaline Protease by New Halotolerant Alkaliphilic Bacillus Sp. NPST-AK15 Isolated from Hyper Saline Soda Lakes,” Electronic Journal of Biotechnology, vol. 18, pp. 236-243, 2015.
• [20] A. Gerze, D. Omay, and Y. Guvenilir, “Partial Purification and Characterization of Protease Enzyme from Bacillus subtilis megatherium,” Applied Biochemistry and Biotechnology, vol. 121, pp. 335-345, 2005.
• [21] M. Thirumalai et al. “Purification and Characterization of Moderately Halophilic Alkaline Serine Protease from Marine Bacillus subtilis AP-MSU 6.” Biocatalysis and Agricultural Biotechnology, vol. 2, pp. 116-119, 2013.
• [22] V. Ponnuswamy, S. Lazarus, and S. G. P. Vincent. “De-Hairing Protease Production by an Isolated Bacillus cereus strain AT under Solid-State Fermentation Using Cow Dung: Biosynthesis and Properties,” Saudi Journal of Biological Sciences, vol. 21, pp. 27-34, 2014.
• [23] R. Dalal. “Screening and Isolation of Protease Producing Bacteria from Soil Collected from Different Areas of Burhanpur Region (MP) India,” International Journal of Current Microbiology and Applied Sciences, vol. 4, pp. 597-606, 2015.
• [24] S. Aftab, S. Ahmed, S. Saeed, and Sheikh Ajaz Rasool. “Screening, Isolation and Characterization of Alkaline Protease Producing Bacteria from Soil,” Pakistan Journal of Biological Sciences, vol. 9, pp. 2122-2126, 2016.
• [25] Asha A. Kembhavi, A. Kulkarni, and A. Pant, “Salt-Tolerant and Thermostable Alkaline Protease from Bacillus subtilis NCIM No. 64,” Applied Biochemistry and Biotechnology, vol. 38, 1993.
• [26] L. Milad, and A. Asoodeh. “An Extremely Thermotolerant, Alkaliphilic Subtilisin-like Protease from Hyperthermophilic Bacillus sp. MLA64,” International Journal of Biological Macromolecules, vol. 51, pp. 960-967, 2012.
• [27] C. G. Kumar, “Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Bacillus pumilus,” Letters in Applied Microbiology, vol.34, pp. 13-17, 2002.
• [28] S. Kaur, R. M. Vohra, M. Kapoor, Q. K. Beg, G. S. Hoondal, “Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2.” World Journal of Microbiology and Biotechnology, vol. 17, pp. 125-129, 2001.
• [29] W. J. Kim, and S. M. Kim. “Purification and Characterization of Bacillus subtilis JM-3 Protease from Anchovy Sauce,” Journal of Food Biochemistry, vol. 29, pp. 591-610, 2005.
• [30] F. X. Ting, S. G. You, and S. M. Kim. “Characterization of a Salt-Tolerant Acid Protease Produced by Bacillus megaterium KLP-98 and Its Potential as a Fermentation Starter for the Manufacture of Fish Sauce.” Journal of Food Biochemistry, vol. 32, pp. 279-298, 2008.
• [31] J. K. Gopal, S. Kumar, and S. Vinar, “Production of Moderately Halotolerant, SDS Stable Alkaline Protease from Bacillus cereus MTCC 6840 Isolated from Lake Nainital, Uttaranchal State, India,” Brazilian Journal of Microbiology, vol. 38, pp. 773-779, 2007.
• [32] B. Selmen, O. Hamdi, and A. Landoulsi. “Enzymatic Dehairing of Goat Skins Using Alkaline Protease from Bacillus sp. SB12,” Protein Expression and Purification, vol. 121, pp. 9-16, 2016.
• [33] F. Shaghayegh, A. Asoodeh, and M. Lagzian. “Purification, Biochemical Characterization and Structural Modeling of a Potential HtrA-like Serine Protease from Bacillus subtilis DR8806,” Journal of Molecular Catalysis B: Enzymatic, vol. 115, pp. 51-58, 2015.
• [34] B. Khushboo, V. Chaturvedi, and R. Bhatt. “Simultaneous Production of Detergent Stable Keratinolytic Protease, Amylase and Biosurfactant by Bacillus subtilis PF1 Using Agro Industrial Waste,” Biotechnology Reports, vol. 10, pp. 94-104, 2016.
• [35] F. M. Olajuyigbe, and A. M. Falade, “Purification and Partial Characterization of Serine Alkaline Metalloprotease from Bacillus brevis MWB-01,” Bioresources and Bioprocessing, vol. 1, no. 1, pp. 8, 2014.
• [36] P. H. A. Sneath and R. R. Sokal, Numerical Taxonomy, Freeman, San Francisco, 1973.
• [37] K. Tamura, M. Nei, and S. Kumar, “Prospects for inferring very large phylogenies by using the neighbor-joining method,” Proceedings of the National Academy of Sciences (USA) vol. 101, pp. 11030-11035, 2004.
• [38] S. Kumar, G. Stecher, and K. Tamura, “MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets,” Molecular Biology and Evolution, vol. 33, pp. 1870-1874, 2016.
• [39] G. Yasar, U. G. Guven, E. Guduk & F. Aktas, “Partial purification and characterization of the novel halotolerant and alkalophilic laccase produced by a new isolate of Bacillus subtilis LP2,” Biocatalysis and Biotransformation, vol. 37:4, pp. 268-277, 2019.
• [40] M. Okuda, N. Sumitomo, Y. Takimura, A. Ogawa, K. Saeki, S. Kawai, T. Kobayashi, S. Ito, “A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular- mass alkaline proteases from Bacillus spp.” Extremophiles, vol. 4, 229-235, 2008.
• [41] E. S. Son, J. I. Kim, “Multicatalytic alkaline serine protease from the psychrotrophic Bacillus amyloliquefaciens S94.” J. Microbiol., vol. 41, 58-62, 2003.
• [42] 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.” Applied Microbiol. Biotechnol., vol. 86, 1867-1875, 2010.
• [43] Briki, S., Hamdi, O., & Landoulsi, A. “Enzymatic dehairing of goat skins using alkaline protease from Bacillus sp. SB12.” Protein Expression and Purification, vol. 121, pp. 9-16, 2016.
• [44] The Republic of Turkey Mineral Research and Exploration Institute, Report on Geological Survey of Trabzon Area, Northeastern Turkey, Phase 1, M.T.A. vol. 125, Ankara, 1974.