DOI: https://doi.org/10.14710/jksa.27.8.395-402

Characterization of a Halostable Metalloprotease from the Halophilic Bacterium Bacillus clausii J1G-0%B

Chemistry Department, Faculty of Sciences and Mathematics, Diponegoro University, Jl. Prof. Soedarto, SH., Tembalang, Semarang, Indonesia

Received: 18 Jun 2024; Revised: 25 Aug 2024; Accepted: 26 Aug 2024; Published: 31 Aug 2024.
Open Access Copyright 2024 Jurnal Kimia Sains dan Aplikasi under http://creativecommons.org/licenses/by-sa/4.0.

Citation Format:
Cover Image
Abstract

Protein plays a crucial role as a biocatalyst in various industries, particularly in breaking down proteins into amino acids. The demand for proteases capable of functioning under extreme conditions, such as high salinity, temperature, and pH, is increasing. To address this, the exploration of bacteria that produce stable proteases in such environments is essential. Bacillus clausii J1G-0%B, a halophilic bacterium isolated from Madura salt ponds, thrives in salinity levels of 0-20% NaCl. This study aims to obtain and characterize the protease produced by Bacillus clausii J1G-0%B, focusing on its activity and stability under extreme conditions. The research involved screening, production, and purification of the protease using ammonium sulfate fractionation and dialysis. Protease activity was measured using the Kunitz method, and protein content was determined using the Lowry method. Characterization included optimizing enzymatic conditions (pH, temperature, NaCl concentration), identifying metalloprotease types, and analyzing enzyme kinetics and thermodynamics. The study successfully produced protease using a halophilic medium with casein and 5% NaCl. After 96 hours of incubation, the protease exhibited a specific activity of 654.737 U/mg. Optimal activity was observed at pH 7, 50°C, and 10% NaCl, with stability between 2.5% and 15% NaCl concentration. Enzyme kinetics revealed a high affinity for casein, with a KM value of 0.164 mg/mL and Vmax of 13.182 µmol/mL·min. Thermodynamic analysis indicated high stability, as shown by a positive ΔGi value (+105.84 kJ/mol), a low inactivation constant (ki = 0.0031 min-1), and a long half-life (t½ = 223.548 minutes). EDTA chelation tests confirmed that the protease is a metalloprotease. The halostable protease from Bacillus clausii J1G-0%B shows significant potential for industrial applications and bioremediation in high-salinity environments.

Fulltext View|Download
Keywords: Halophilic bacteria; Bacillus clausii; halostable protease; high salinity; Madura salt ponds

Article Metrics:

Article Info
Section: Research Articles
Language : EN
Recent articles
Synthesis of NiFe₂O₄ Magnetic Using Artocarpus altilis Leave Extract for Photocatalytic Degradation of Methylene Blue Dye and Antibacterial Applications The Effect of Mole Ratio Natural-CaCO₃/KF in the Synthesis of KCaF₃ Perovskite Base Catalyst Green Synthesis of Gold Nanoparticles Using Basella alba Leaf Extract and Their Antioxidant Activity More recent articles
Most cited articles
Isolasi Bakteri Termofilik Sumber Air Panas Gedongsongo dengan Media Pengaya Minimal YT (Yeast Tripton) serta Identifikasi Genotipik dan Fenotipik Penggunaan Zeolit Alam Terdealuminasi Sebagai Adsorben Senyawa Aromatik Uji Aktivitas Antioksidan Senyawa Flavonoid dari Ekstrak Etanol Daun Johar (Senna siamea Lamk) Utilization of Rice Husk Cellulose as a Magnetic Nanoparticle Biocomposite Fiber Source for the Absorption of Manganese (Mn2+) Ions in Peat Water Pengaruh Konsentrasi Substrat Maltosa terhadap Potensial Listrik Baterai Lactobacillus bulgaricus (MFC) More cited articles
  1. Hongli Yao, Shuangping Liu, Tiantian Liu, Dongliang Ren, Zhilei Zhou, Qilin Yang, Jian Mao, Microbial-derived salt-tolerant proteases and their applications in high-salt traditional soybean fermented foods: a review, Bioresources and Bioprocessing, 10, (2023), 82 https://doi.org/10.1186/s40643-023-00704-w
  2. Janos K. Lanyi, Joann Stevenson, Studies of the Electron Transport Chain of Extremely Halophilic Bacteria: IV. Role of Hydrophobic Forces in The Structure of Menadione Reductase, Journal of Biological Chemistry, 245, 16, (1970), 4074-4080 https://doi.org/10.1016/S0021-9258(18)62887-X
  3. Aharon Oren, Halophilic Microorganisms and their Environments, Springer Science & Business Media, 2006,
  4. Shiladitya DasSarma, Priya DasSarma, Halophiles, in: eLS, 2012, https://doi.org/10.1002/9780470015902.a0000394.pub3
  5. Rahmad Budiharjo, Purbowatiningrum Ria Sarjono, Mukhammad Asy’ari, Pengaruh Konsentrasi NaCl Terhadap Aktivitas Spesifik Protease Ekstraseluler dan Pertumbuhan Bakteri Halofilik Isolat Bittern Tambak Garam Madura, Jurnal Kimia Sains dan Aplikasi, 20, 3, (2017), 142-145 https://doi.org/10.14710/jksa.20.3.142-145
  6. Galuh Dwi Arum, Mukhammad Asy'ari, Nies Suci Mulyani, Effect of Storage of Yellow Pigment from Halophilic Bacillus clausii J1G-0%B on Antioxidant Activity, Jurnal Kimia Sains dan Aplikasi, 25, 11, (2022), 399-404 https://doi.org/10.14710/jksa.25.11.399-404
  7. Tsutomu Yamaguchi, Yukiko Yamashita, Imao Takeda, Hisashi Kiso, Proteolytic Enzymes in Green Asparagus, Kiwi Fruit and Miut: Occurrence and Partial Characterization, Agricultural and Biological Chemistry, 46, 8, (1982), 1983-1986 https://doi.org/10.1080/00021369.1982.10865376
  8. Roberto Umaña, Reevaluation of the method of Kunitz for the assay of proteolytic activities in liver and brain homogenates, Analytical Biochemistry, 26, 3, (1968), 430-438 https://doi.org/10.1016/0003-2697(68)90204-2
  9. Oliver H. Lowry, Nira J. Rosebrough, A. Lewis Farr, Rose J. Randall, Protein measurement with the Folin phenol reagent, Journal of Biological Chemistry, 193, 1, (1951), 265-275
  10. Nanik Suwarso, Yandri A. S., Sutopo Hadi, Peningkatan Kestabilan Enzim Protease dari Bacillus subtilis ITBCCB148 dengan Modifikasi Kimia Menggunakan Sitrakonat Anhidrida, Jurnal Analis Kesehatan, 5, 1, (2016), 475-482
  11. Sonika Gupta, Parul Sharma, Kamal Dev, Anuradha Sourirajan, Halophilic Bacteria of Lunsu Produce an Array of Industrially Important Enzymes with Salt Tolerant Activity, Biochemistry Research International, 2016, 1, (2016), 9237418 https://doi.org/10.1155/2016/9237418
  12. Chris E. Petersen, Investigations in the Biology 1151 Laboratory, Stipes Pub., 2005,
  13. Ashutosh Kumar Hemker, Loc Thai Nguyen, Deepti Salvi, Chapter 3 - Effect of high-pressure technologies on enzyme activity and stability, in: B.R.d.C. Leite Júnior, A.A.L. Tribst (Eds.) Effect of High-Pressure Technologies on Enzymes, Academic Press, 2023, https://doi.org/10.1016/B978-0-323-98386-0.00008-7
  14. B. A. Kornbrust, T. Forman, I. Matveeva, 19 - Applications of enzymes in breadmaking, in: S.P. Cauvain (Ed.) Breadmaking (Second Edition), Woodhead Publishing, 2012, https://doi.org/10.1533/9780857095695.2.470
  15. David L. Nelson, Albert L. Lehninger, Michael M. Cox, Lehninger Principles of Biochemistry, Macmillan, 2008,
  16. Dian Herasari, Arifa Rahmatika Salsabilla, Indah Parwatih, Aspita Laila, Mulyono Mulyono, Suharso Suharso, Karakterisasi Enzim Protease dari Bakteri Klebsiella sp. Indigen Tanah di Bandar Lampung, Analit: Analytical Environmental Chemistry, 7, 01, (2022), 35-53
  17. Natividad Ortega, Silvia De Diego, José M. Rodríguez-Nogales, Manuel Perez-Mateos, María D. Busto, Kinetic behaviour and thermal inactivation of pectinlyase used in food processing, International Journal of Food Science & Technology, 39, 6, (2004), 631-639 https://doi.org/10.1111/j.1365-2621.2004.00822.x
  18. K. Miyanaga, H. Unno, 2.05 - Reaction Kinetics and Stoichiometry, in: M. Moo-Young (Ed.) Comprehensive Biotechnology (Second Edition), Academic Press, Burlington, 2011, https://doi.org/10.1016/B978-0-08-088504-9.00085-4
  19. Zhen Yang, Michael Domach, Robert Auger, Fang Xiao Yang, Alan J. Russell, Polyethylene glycol-induced stabilization of subtilisin, Enzyme and Microbial Technology, 18, 2, (1996), 82-89 https://doi.org/10.1016/0141-0229(95)00073-9
  20. Zübeyde Baysal, Yasemin Bulut, Murat Yavuz, Çetin Aytekin, Immobilization of α-amylase via adsorption onto bentonite/chitosan composite: Determination of equilibrium, kinetics, and thermodynamic parameters, Starch - Stärke, 66, 5-6, (2014), 484-490 https://doi.org/10.1002/star.201300133
  21. Neeta Kulkarni, Abhay Shendye, Mala Rao, Molecular and biotechnological aspects of xylanases, FEMS Microbiology Reviews, 23, 4, (1999), 411-456 https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
  22. W. A. Hidayani, S. Setiasih, S. Hudiyono, Determination of the effect of EDTA and PCMB on purified bromelain activity from pineapple core and in vitro antiplatelet activity, IOP Conference Series: Materials Science and Engineering, 763, (2020), 012054 https://doi.org/10.1088/1757-899X/763/1/012054
  23. Ji-Wei Wu, Xiu-Lan Chen, Extracellular metalloproteases from bacteria, Applied Microbiology and Biotechnology, 92, 2, (2011), 253-262 https://doi.org/10.1007/s00253-011-3532-8
  24. Jen-Kuo Yang, Ing-Lung Shih, Yew-Min Tzeng, San-Lang Wang, Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes☆, Enzyme and Microbial Technology, 26, 5, (2000), 406-413 https://doi.org/10.1016/S0141-0229(99)00164-7
  25. Anna Lopata, Balázs Jójárt, Éva V. Surányi, Enikő Takács, László Bezúr, Ibolya Leveles, Ábris Á. Bendes, Béla Viskolcz, Beáta G. Vértessy, Judit Tóth, Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity, Biomolecules, 9, 10, (2019), 621 https://doi.org/10.3390/biom9100621

Last update:

No citation recorded.

Last update: 2024-11-04 11:17:24

No citation recorded.