Author(s):
Narendra Kumar Sura, Vignesh S. Lal, Ahalya Rajeev
Email(s):
narendraks@rvce.edu.in
DOI:
10.52711/0974-360X.2024.00644 
Address:
Narendra Kumar Sura1*, Vignesh S. Lal2, Ahalya Rajeev3
2,3Students, Department of Biotechnology, R.V. College of Engineering, Mysore Road, Bangalore-560 059.
1Professor, the Department of Biotechnology, R.V. College of Engineering, Mysore Road, Bangalore-560 059.
*Corresponding Author
Published In:
Volume - 17,
Issue - 9,
Year - 2024
ABSTRACT:
Alkaline serine protease is a proteolytic enzyme having a wide array of industrial applications. These proteases have a high enzymatic activity at a high pH ranging between 8-12 and temperature 38?, hence finding its importance in industries such as detergent industries where pHs reach as high as 10.7 due to the presence of caustic soda and other alkaline materials. Production of alkaline serine protease from Bacillus subtilis MTCC 8601 on establishment of a shake flask fermentation protocol has been carried out in this study. Qualitative analysis using the Biuret test has been conducted, quantification of protease in the crude enzymatic extract was carried out using Lowry’s method of quantitative analysis which was found to be 0.847 mg/ml. Further, the enzymatic activity of the protease has been determined using the high sensitivity Ninhydrin method of amino acid detection for which the results were 3.51 mg/ml when Bovine serum albumin was used as a substrate and 1.419 mg/ml when Gelatin was used as a substrate, the unit of enzyme is found to be 0.445 µg/ mol min and 0.180 µg/ mol min for each substrate respectively.
Cite this article:
Narendra Kumar Sura, Vignesh S. Lal, Ahalya Rajeev. Production of Alkaline Serine Protease from Bacillus subtilis MTCC 8601. Research Journal of Pharmacy and Technology. 2024; 17(9):4161-8. doi: 10.52711/0974-360X.2024.00644
Cite(Electronic):
Narendra Kumar Sura, Vignesh S. Lal, Ahalya Rajeev. Production of Alkaline Serine Protease from Bacillus subtilis MTCC 8601. Research Journal of Pharmacy and Technology. 2024; 17(9):4161-8. doi: 10.52711/0974-360X.2024.00644 Available on: https://rjptonline.org/AbstractView.aspx?PID=2024-17-9-5
REFERENCES:
1. Z. Khan et al. Protease from Bacillus subtilis ZMS-2: Evaluation of production dynamics through Response Surface Methodology and application in leather tannery. Journal of King Saud University–Science. 102643, Mar. 2023, doi: https://doi.org/10.1016/j.jksus.2023.102643.
3. Sneha S, Mrunal Palsokar, Vemula Sai Jahnavi, A. Sarkar, and B. Rao. Isolation, Characterization and Application of Protease Enzyme from Marine Bacteria. Research Journal of Pharmacy and Technology. 2021: 4236–4240 doi: https://doi.org/10.52711/0974-360x.2021.00735.
4. L. Daoud, H. Hmani, M. Ben Ali, M. Jlidi, and M. Ben Ali. An Original Halo-Alkaline Protease from Bacillus halodurans Strain US193: Biochemical Characterization and Potential Use as Bio-Additive in Detergents. Journal of Polymers and the Environment. 2016; 26(1): 23–32 doi: https://doi.org/10.1007/s10924-016-0916-y.
5. K.V Bhaskara Rao, S Veena, S Pooja, Y Shriya, and R Shivram. Novel Actinomycetales bacterium-PV7 isolated from Kanyakumari Marine Sediments: A Prospective Source for Industrial and Pharmaceutical Enzyme Production. Research Journal of Pharmacy and Technology. 2017; 10(5): 1471–1471. doi: https://doi.org/10.5958/0974-360x.2017.00259.1.
6. C. López-Otín and J. S. Bond. Proteases: Multifunctional Enzymes in Life and Disease. Journal of Biological Chemistry. 2008; 283(45): 30433–30437 doi: https://doi.org/10.1074/jbc.r800035200.
7. S. Singh et al. Transmembrane protease serine 4: An emergent diagnostic biomarker and Therapeutic drug target for cancer. Research Journal of Pharmacy and Technology. 2020; 13(7): 3449, 2020, doi: https://doi.org/10.5958/0974-360x.2020.00612.5.
8. Z. Jafari, Ghasem Najafpour Darzi, and H. Zare. Growth Media Optimization for Production of Alkaline Protease from Industrial Wastewater using Bacillus subtilis PTCC 1254. International Journal of Engineering. Transactions C: Aspects. 2023; 36(3); 513–522 doi: https://doi.org/10.5829/ije.2023.36.03c.11.
9. Green Bio-processes. Springer Nature, 2019. doi: https://doi.org/10.1007/978-981-13-3263-0.
10. M. P. Ramakodi, N. Santhosh, P. T., S. V. Mohan, and S. Basha. Production of protease enzyme from slaughterhouse effluent: An approach to generate value-added products from waste. Bioresource Technology Reports. 2020; 12: 100552 doi: https://doi.org/10.1016/j.biteb.2020.100552.
11. A. Fasim, V. S. More, and S. S. More. Large-scale production of enzymes for biotechnology uses. Current Opinion in Biotechnology. 2021; 69: 68–76 doi: https://doi.org/10.1016/j.copbio.2020.12.002.
12. R. Gurunathan, B. Huang, V. K. Ponnusamy, J.S. Hwang, and H.U. Dahms. Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs. Scientific Reports. 2021; 11(1). doi: https://doi.org/10.1038/s41598-021-90375-4.
13. Palsaniya. Optimization of Alkaline Protease production from bacteria isolated from soil. www.academia.edu, Accessed: Jun. 01, 2024. [Online]. https://www.academia.edu/14693384/Optimization_of_Alkaline_Protease_production_from_bacteria_isolated_from_soil.
14. None Shruthi and N. B. Thippeswamy. Isolation of Potential Extracellular Hydrolytic Enzymes producing Fungi from Western Ghats, Karnataka. Research Journal of Pharmacy and Technology. 2020; 13(5): 2081–2081 doi: https://doi.org/10.5958/0974-360x.2020.00374.1.
15. K. Viswanathan and L. Jeyanthi Rebecca. Screening of Marine Actinomycetes for Fibrinolytic Enzymes. Research Journal of Pharmacy and Technology. 2018; 11(10): 4365–4365 doi: https://doi.org/10.5958/0974-360x.2018.00799.0.
16. H. Mukhtar. Industrial Applications and Production Sources of Serine Alkaline Proteases: A Review. Journal of Bacteriology and Mycology: Open Access. 2016; 3(1) doi: https://doi.org/10.15406/jbmoa.2016.03.00051.
17. C. G. Kumar, M. P. Tiwari, and K. D. Jany. Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties. Process Biochemistry. 1999; 34(5); 441–449 doi: https://doi.org/10.1016/s0032-9592(98)00110-1.
18. Isolation and screening of alkaline protease producing bacteria from fermented foods. www.journalijcar.org. https://www.journalijcar.org/issues/isolation-and-screening-alkaline-protease-producing-bacteria-fermented-foods (accessed Jun. 01, 2024).
19. R. Abdulatif Abdulrazaq, W. Saadi Mahmood, B. Alwan, T. Hadi Saleh, S. T. Hashim, and B. A. Laftaah Al-Rubaii. Biological Study of protease produced by clinical isolates of Staphylococcus aureus. Research Journal of Pharmacy and Technology. 2022:. 5415–5420 doi: https://doi.org/10.52711/0974-360x.2022.00912.
20. A. K. Sharma, V. Sharma, J. Saxena, B. Yadav, A. Alam, and A. Prakash. Isolation and Screening of Extracellular Protease Enzyme from Bacterial and Fungal Isolates of Soil. International Journal of Scientific Research in Environmental Sciences. 2015; 3(9): 334–340 doi: https://doi.org/10.12983/ijsres-2015-p0334-0340.
21. G. Pant et al. Production, optimization and partial purification of protease from Bacillus subtilis. Journal of Taibah University for Science. 2015; 9(1): 50–55 doi: https://doi.org/10.1016/j.jtusci.2014.04.010.
22. Bhunia, B., Basak, B. and Dey, A. A Review on Production of Serine Alkaline Protease by Bacillus spp. Journal of Biochemical Technology. 2012; 3: 448-457. www.scirp.org. https://www.scirp.org/reference/referencespapers?referenceid=1561485 (accessed Jun. 01, 2024).
23. Muthu Padmapriya, B. Christudhas Williams. Purification and characterization of neutral protease enzyme from Bacillus subtilis. Journal of Microbiology and Biotechnology Research. 2012.
24. V. F. Soares, L. R. Castilho, E. P. S. Bon, and D. M. G. Freire. High-Yield Bacillus subtilis Protease Production by Solid-State Fermentation. Applied Biochemistry and Biotechnology. 2005; 121(1–3): 311–320 doi: https://doi.org/10.1385/abab:121:1-3:0311.
25. R. P. D. Bank. RCSB PDB - 1WMD: Crystal Structure of alkaline serine protease KP-43 from Bacillus sp. KSM-KP43 (1.30 angstrom, 100 K). www.rcsb.org. https://www.rcsb.org/structure/1wmd (accessed Jun. 01, 2024).
26. “PROCHECK home page,” www.ebi.ac.uk. https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/
27. H. B. Bhatt and S. P. Singh. Cloning, Expression, and Structural Elucidation of a Biotechnologically Potential Alkaline Serine Protease From a Newly Isolated Haloalkaliphilic Bacillus lehensis JO-26. Frontiers in Microbiology. 2020: 11 doi: https://doi.org/10.3389/fmicb.2020.00941.
28. van et al. Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus. Protein Engineering Design and Selection. 1992; 5(5): 405–411 doi: https://doi.org/10.1093/protein/5.5.405.
29. A. Mahmoud, E. Kotb, A. I. Alqosaibi, A. A. Al-Karmalawy, I. S. Al-Dhuayan, and H. Alabkari. In vitro and in silico characterization of alkaline serine protease from Bacillus subtilis D9 recovered from Saudi Arabia. Heliyon. 2021; 7(10): e08148, Oct. 2021, doi: https://doi.org/10.1016/j.heliyon.2021.e08148.
30. F. Matkawala, S. Nighojkar, A. Kumar, and A. Nighojkar. Microbial alkaline serine proteases: Production, properties and applications. World Journal of Microbiology and Biotechnology. 2021; 37(4). doi: https://doi.org/10.1007/s11274-021-03036-z.
31. A. Ariyaei, A. Farhadi, F. Moradian, and G. Rahimi Mianji. Cloning, expression and characterization of a novel alkaline serine protease gene from native Iranian Bacillus sp.; a producer of protease for use in livestock. Gene. 2019; 693:10–15 doi: https://doi.org/10.1016/j.gene.2019.01.020.
32. S. Hari. Screening of Enzymes from Actinomycetes and Fungi isolated from Plastic Dumped Soil. Research Journal of Pharmacy and Technology. 2019; 12(50: 2261 doi: https://doi.org/10.5958/0974-360x.2019.00376.7.
33. R. Singh Chauhan and R. Mani Mishra. Characterization of Alkaline Protease Producing Bacillus Halodurans RSCVS-PF21 Isolated from Poultry Farm Soil. Biosciences Biotechnology Research Asia. 17(2): 385–392 doi: https://doi.org/10.13005/bbra/2841.
34. A. Razzaq et al. Microbial Proteases Applications. Frontiers in Bioengineering and Biotechnology. 2019; 7(110). doi: https://doi.org/10.3389/fbioe.2019.00110.
35. Global Proteases Market, Growth, Trends, Forecasts. www.mordorintelligence.com. https://www.mordorintelligence.com/industry-reports/proteases-market.
36. L. F. Christensen, B. García-Béjar, C. H. Bang-Berthelsen, and E. B. Hansen. Extracellular microbial proteases with specificity for plant proteins in food fermentation. International Journal of Food Microbiology. 2022; 381: 109889 doi: https://doi.org/10.1016/j.ijfoodmicro.2022.109889.
37. G. Das and M. Prasad. Isolation, purification and mass production of protease enzyme from bacillus subtilis. International Research Journal of Microbiology. 2010 Accessed: Jun. 01, 2024. [Online]. Available: https://www.semanticscholar.org/paper/Isolation%2C-purification-and-mass-production-of-from-Das-Prasad/f2e9eeb9afafe158c32df67f81cd088cef36dc3b
38. S. Singh et al. Comparative evaluation of protease production from okara, broken wheat, chickpea and black gram by the soil isolate of Aspergillus terreus. Research Journal of Pharmacy and Technology. 2021; 14(1): 219–224 doi: https://doi.org/10.5958/0974-360x.2021.00038.x.
39. T. Mothe and V. R. Sultanpuram. Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus. 3 Biotech. 2016; 6(1) doi: https://doi.org/10.1007/s13205-016-0377-y.
40. Nutrient Broth. Available: http://www.liofilchem.net/login/pd/ifu/24103_IFU.pdf
41. C. V. Vidhya. Production and Optimization of Extra-cellular protease from Ganoderma sp. Research Journal of Pharmacy and Technology. 2019: 12(4): 1832 doi: https://doi.org/10.5958/0974-360x.2019.00306.8.
42. Amit Sitoke, Reena Singh Chopra, G Pradip Kumar, and C. Chopra. Identification and Characterization of an Alkalophilic Protease from Bacillus Mycoides strain isolated from Industrial Soil of Phagwara, India. Research Journal of Pharmacy and Technology. 2017; 10(10): 3435–3435 doi: https://doi.org/10.5958/0974-360x.2017.00611.4.
43. Srinubabu Gedela, N. Lokeswari, and K. Jayaraju. Screening of Nutritional Parameters for the Production of Protease fromAspergillus Oryzae. E-journal of Chemistry. 2007; 4(2): 208–215 doi: https://doi.org/10.1155/2007/915432.
44. A. Nowotny. Protein Determination by the Biuret Method. Basic Exercises in Immunochemistry. 1979: 168–169 doi: https://doi.org/10.1007/978-3-642-67356-6_50.
45. J. H. Waterborg and H. R. Matthews. The Lowry Method for Protein Quantitation. Proteins. 1984: 1–4 doi: https://doi.org/10.1385/0-89603-062-8:1.
46. P. A. Kendall. Use of the Ninhydrin Reaction for Quantitative Estimation of Amino Groups in Insoluble Specimens. Nature. 1963; 197(4874); 1305–1306 doi: https://doi.org/10.1038/1971305a0.