The worldwide demand for industrially essential enzymes is growing due to their eco-friendly and sustainable applications in broad industrial sectors. The main objective of this investigation is intended to isolate potent alkalophilic native bacteria from local habitats for the cost-effective production of an enzyme alkaline protease. For screening of alkalophilic bacteria, bacterial samples were taken from various natural alkaline habitats, and bacterial cultures were screened on agar plate having skimmed milk as protein substrate by using the protein hydrolysis method. The bacteria showing maximum proteolytic potential were identified by microscopic, biochemical, and 16S rDNA analysis. Additionally, the culture components and other medium parameters have been optimized for higher enzyme production. High yield alkaline protease was obtained with conditions of 1% inoculum, pH 9.0, and at 37°C temperature, with 1% sugarcane molasses as the best suitable carbon substrate. Partial pur¬ification of the enzyme was performed and characterized for their optimum activity on various parameters (temperatures and pH range). The approx. molecular weight of the enzyme was estimated to be ~35 kDa. This study demonstrates the production of an industrially important enzyme (alkaline protease) from a newly isolated native Bacillus spp. Economical production of this enzyme can be commercially applied in vast industrial sectors like agriculture, textile, food, detergent, and leather industries.
| Published in | International Journal of Microbiology and Biotechnology (Volume 6, Issue 4) |
| DOI | 10.11648/j.ijmb.20210604.15 |
| Page(s) | 137-146 |
| Creative Commons |
This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited. |
| Copyright |
Copyright © The Author(s), 2024. Published by Science Publishing Group |
Agro-industrial Residues, Alkaline Protease, Bacillus Spp, Cost-effective, Sugarcane Molasses
| [1] | C. Zhou, H. Qin, X. Chen, Y. Zhang, Y. Xue, and Y. Ma, “A novel alkaline protease from alkaliphilic Idiomarina sp. C9-1 with potential application for eco-friendly enzymatic dehairing in the leather industry,” Sci. Rep., no. May, pp. 1–18, 2018. |
| [2] | M. D. Pastor, G. S. Lorda, A. Balatti, S. Rosa, and L. Pampa, “PROTEASE OBTENTION USING BACILLUS SUBTILIS 3411 AND AMARANTH SEED MEAL MEDIUM AT DIFFERENT AERATION RATES,” pp. 6–9, 2001. |
| [3] | A. Burhan, U. Nisa, C. Gökhan, C. Ömer, A. Ashabil, and G. Osman, “Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6,” Process Biochem., 2003. |
| [4] | R. Gupta, Q. Beg, and P. Lorenz, “Bacterial alkaline proteases: Molecular approaches and industrial applications,” Appl. Microbiol. Biotechnol., vol. 59, no. 1, pp. 15–32, 2002. |
| [5] | N. Annamalai, M. V. Rajeswari, and T. Balasubramanian, “Extraction, purification and application of thermostable andhalostable alkaline protease from Bacillus alveayuensis CAS 5using marine wastes,” Food Bioprod. Process., 2014. |
| [6] | V. Jisha et al., “Versatility of microbial proteases,” Adv. Enzym. Res., vol. 01, no. 03, pp. 39–51, 2013, doi: 10.4236/aer.2013.13005. |
| [7] | S. Lin, M. Zhang, J. Liu, and G. S. Jones, “Construction and application of recombinant strain for the production of an alkaline protease from Bacillus licheniformis,” J. Biosci. Bioeng., vol. 119, no. 3, pp. 284–288, 2015. |
| [8] | K. Adinarayana, P. Ellaiah, and D. S. Prasad, “Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11.,” AAPS PharmSciTech, vol. 4, no. 4, p. E56, 2003. |
| [9] | L. A. I. De Azeredo, L. R. Castilho, S. G. F. Leite, R. R. R. Coelho, and D. M. G. Freire, “Protease production by Streptomyces sp. isolated from Brazilian Cerrado soil: Optimization of culture medium employing statistical experimental design,” 2003. |
| [10] | K. Kathiresan and S. Manivannan, “Production of alkaline protease by Streptomyces sp., isolated from coastal mangrove sediment,” Res. J. Environ. Sci., 2007. |
| [11] | S. Ç. Aksoy, A. Uzel, and E. E. Hameş Kocabaş, “Extracellular serine proteases produced by Thermoactinomyces strains from hot springs and soils of West Anatolia,” Ann. Microbiol., 2012. |
| [12] | D. H. Petrova, S. A. Shishkov, and S. S. Vlahov, “Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: Purification and some properties,” J. Basic Microbiol., 2006. |
| [13] | S. Bakhtiar, R. J. Estiveira, and R. Hatti-Kaul, “Substrate specificity of alkaline protease from alkaliphilic feather-degrading Nesterenkonia sp. AL20,” Enzyme Microb. Technol., 2005. |
| [14] | S. D. Gohel and S. P. Singh, “Characteristics and thermodynamics of a thermostable protease from a salt-tolerant alkaliphilic actinomycete,” Int. J. Biol. Macromol., 2013. |
| [15] | G. Raut, S. Vetal, R. Biao, X. Y. Liu, L. Zhang, and C. Kokare, “Purification and characterization of organic solvent and detergent stable protease isolated from marine Saccharopolyspora sp. A9: Application of protease for wound healing,” Biochem. Eng. J., 2012. |
| [16] | M. Schallmey, A. Singh, and O. P. Ward, “Developments in the use of Bacillus species for industrial production,” Can. J. Microbiol., vol. 50, no. 1, pp. 1–17, 2004. |
| [17] | K. Jellouli et al., “Molecular and biochemical characterization of an extracellular serine-protease from Vibrio metschnikovii J1,” J. Ind. Microbiol. Biotechnol., 2009. |
| [18] | J. S. Mukherjee et al., “Programmes and principles in treatment of multidrug-resistant tuberculosis,” Lancet, vol. 363. pp. 474–481, 2004. |
| [19] | B. K. Lakshmi, P. Ratna sri, K. Ambika Devi, and K. Hemalatha, “Screening, optimization of production and partial characterization of alkaline protease from haloalkaliphilic Bacillus sp,” Int. J. Res. Eng. Technol., vol. 3, no. 2, pp. 435–443, 2014. |
| [20] | R. N. Z. R. A. Rahman, M. Basri, and A. B. Salleh, “Thermostable alkaline protease from Bacillus stearothermophilus F1; nutritional factors affecting protease production,” Ann. Microbiol., 2003. |
| [21] | N. A. Razak, M. Y. A. Samad, M. Basri, W. M. Z. W. Yunus, K. Ampon, and A. B. Salleh, “Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production,” World J. Microbiol. Biotechnol., 1994. |
| [22] | B. Bhunia, B. Basak, and A. Dey, “A review on production of serine alkaline protease by Bacillus spp.,” J. Biochem. Tech., vol. 3, no. 4, pp. 448–457, 2012. |
| [23] | K. Saeki et al., “A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease,” Extremophiles, vol. 6, no. 1, pp. 65–72, 2002. |
| [24] | N. Saitou and M. Nei, “The neighbor-joining method: a new method for reconstructing phylogenetic trees.,” Mol. Biol. Evol., vol. 4, no. 4, pp. 406–425, 2017. |
| [25] | K. Krishnaveni, D. J. Mukesh Kumar, M. D. Balakumaran, S. Ramesh, and P. T. Kalaichelvan, “Production and optimization of extracellular Alkaline Protease from Bacillus subtilis isolated from dairy effluent,” Der Pharm. Lett., vol. 4, no. 1, pp. 98–109, 2012. |
| [26] | M. M. Bradford, “Bradford assay for protein quantification,” Anal. Biochem., 1976. |
| [27] | C. E. McDonald and L. L. Chen, “The Lowry modification of the Folin reagent for determination of proteinase activity,” Anal. Biochem., 1965. |
| [28] | S. D. Tambekar and D. H. Tambekar, “Optimization of the production and partial charaterization of an extracellular alkaline protease from thermo-halo-alkalophilic Lonar lake bacteria,” Biosci. Discov., vol. 4, no. 1, pp. 30–38, 2013. |
| [29] | V. Bhaskar et al., “Optimization of production of subtilisin in solid substrate fermentation using response surface methodology,” J. Biotechnol., vol. 7, no. 13, pp. 2286–2291, 2008. |
| [30] | C. N. Aguilar, G. Gutiérrez-sánchez, A. Plilia, R. Rodríguez-herrera, J. L. Martínez-hernandez, and J. C. Contreras-esquivel, “No Title,” vol. 4, no. 4, pp. 354–366, 2008. |
| [31] | S. A. Jani, Y. M. Parekh, T. N. Parmar, and T. J. Dalwadi, ‘Screening and Characterization of Alkaline Protease Producing Bacillus Strain B-4 Bacillus flexus and Study of its Potential for Alkaline Protease Production’, vol. 5, no. 5, pp. 767–787, 2016. |
| [32] | K. Aruna, J. Shah, and R. Birmole, ‘Production and Partial Characterization of Alkaline Protease from Bacillus Tequilensis Strains Csgab0139 Isolated from Spoilt Cottage Cheese.’, Int. J. Appl. Biol. an Pharm. Technol., vol. 5, no. 3, pp. 201–221, 2014. |
| [33] | F. M. Olajuyigbe, ‘Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04 grown on groundnut (Arachis hypogaea) meal’, Adv. Enzym. Res., vol. 01, no. 04, pp. 112–120, 2013. |
| [34] | S. A. Ul Qadar, E. Shireen, S. Iqbal, and A. Anwar, ‘Optimization of protease production from newly isolated strain of Bacillus sp. PCSIR EA-3’, Indian J. Biotechnol., vol. 8, no. 3, pp. 286–290, 2009. |
| [35] | A. Sumantha, C. Larroche, and A. Pandey, ‘Microbiology and industrial biotechnology of food-grade proteases: A perspective’, Food Technol. Biotechnol., vol. 44, no. 2, pp. 211–220, 2006. |
| [36] | A. S. Qureshi, M. A. Bhutto, I. Khushk, and M. U. Dahot, ‘Optimization of cultural conditions for protease production by Bacillus subtilis EFRL 01’, African J. Biotechnol., vol. 10, no. 26, pp. 5173–5181, 2011. |
| [37] | R. S. Prakasham, C. S. Rao, and P. N. Sarma, ‘Green gram husk-an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation’, Bioresour. Technol., vol. 97, no. 13, pp. 1449–1454, 2006. |
| [38] | K. Ash, A. Milton Lall, K. Rao, and P. W. Ramteke, ‘Production and Optimization of an Alkaline Protease from Acinetobacter variabilis Isolated from Soil Samples’, Int. J. Agric. Environ. Biotechnol. Cit. IJAEB, vol. 11, no. 2, pp. 379–386, 2018. |
| [39] | R. N. Z. R. A. Rahman, M. Basri, and A. B. Salleh, ‘Thermostable alkaline protease from Bacillus stearothermophilus F1 ; nutritional factors affecting protease production’, Ann. Microbiol., 2003. |
| [40] | P. K. Sarker, S. A. Talukdar, P. Deb, S. M. A. Sayem, and K. Mohsina, ‘Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003’, Springerplus, 2013. |
| [41] | S. Kaur, R. M. Vohra, M. Kapoor, Q. K. Beg, and G. S. Hoondal, ‘Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2’, World J. Microbiol. Biotechnol., vol. 17, no. 2, pp. 125–129, 2001. |
| [42] | S. Puri, Q. K. Beg, and R. Gupta, ‘Optimization of alkaline protease production from Bacillus sp. by Response surface methodology’, Curr. Microbiol., vol. 44, no. 4, pp. 286–290, 2002. |
| [43] | C. G. G. Kumar, ‘Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus’, Lett. Appl. Microbiol., vol. 34, no. 1, pp. 13–17, 2002. |
| [44] | I. Saadoun, R. Rawashdeh, T. Dayeh, Q. Ababneh, and A. Mahasneh, ‘Isolation, characterization and screening for fiber hydrolytic enzymes-producing streptomycetes of Jordanian forest soils’, Biotechnology, vol. 6, no. 1. pp. 120–128, 2007. |
| [45] | G. Lakshmi and N. N. Prasad, ‘Purification and Characterization of Alkaline Protease from a Mutant Bacillus licheniformis Bl8’, vol. 9, no. 1, pp. 15–23, 2015. |
| [46] | J. Singh, N. Batra, and R. Sobti, ‘Serine alkaline protease from a newly isolated Bacillus sp. SSR1’, Process Biochem., vol. 36, no. 8, pp. 781–785, 2001. |
| [47] | D. young Yum, H. C. Chun, D. H. Bai, and D. H. Oh, ‘Purification and Characterization of Alkaline Serine Protease from an Alkalophilic Streptomyces sp’, Biosci. Biotechnol. Biochem., vol. 58, no. 3, pp. 470–474, 1994. |
| [48] | P. Sarker, S. Talukdar, P. Deb, S. Sayem, and K. Mohsina, ‘Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003’, Springerplus, vol. 2, no. 1, p. 506, 2013. |
| [49] | Yu, Jeong-Hyeon & Jin, Hyun-Seok & Choi, Woo-Young & Yoon, Min-Ho, ‘Production and Characterization of an Alkaline Protease from Bacillus licheniformis MH31’, J. Appl Biol Chem., vol. 49, no. 4. pp. 135-139, 2006. |
| [50] | P. Sathyavrathan and S. Krithika, ‘Production and Optimization of Protease from Bacillus licheniformis NRRL-NRS-1264 using cheap source substrates by submerged (SmF) and solid-state fermentation (SSF)’, Int. J. ChemTech Res., vol. 6, no. 1, pp. 286–292, 2014. |
| [51] | W. C. A. Do Nascimento and M. L. Leal Martins, ‘Production and properties of an extracellular protease from thermophilic Bacillus sp’, Brazilian J. Microbiol., vol. 35, no. 1–2, pp. 91–96, 2004. |
| [52] | F. Pouryafar, G. D. Najafpour, N. Noshadi, and M. Jahanshahi, ‘Thermostable alkaline protease production via solid state fermentation in a tray bioreactor using Bacillus licheniformis ATCC 21424’, Int. J. Environ. Res., vol. 9, no. 4, pp. 1127–1134, 2015. |
APA Style
Ranjeet Singh, Ravindra Meena, Harsh Kumar, Banwari Lal. (2021). Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated Bacillus Bacteria. International Journal of Microbiology and Biotechnology, 6(4), 137-146. https://doi.org/10.11648/j.ijmb.20210604.15
ACS Style
Ranjeet Singh; Ravindra Meena; Harsh Kumar; Banwari Lal. Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated Bacillus Bacteria. Int. J. Microbiol. Biotechnol. 2021, 6(4), 137-146. doi: 10.11648/j.ijmb.20210604.15
AMA Style
Ranjeet Singh, Ravindra Meena, Harsh Kumar, Banwari Lal. Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated Bacillus Bacteria. Int J Microbiol Biotechnol. 2021;6(4):137-146. doi: 10.11648/j.ijmb.20210604.15
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Download@article{10.11648/j.ijmb.20210604.15,
author = {Ranjeet Singh and Ravindra Meena and Harsh Kumar and Banwari Lal},
title = {Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated Bacillus Bacteria},
journal = {International Journal of Microbiology and Biotechnology},
volume = {6},
number = {4},
pages = {137-146},
doi = {10.11648/j.ijmb.20210604.15},
url = {https://doi.org/10.11648/j.ijmb.20210604.15},
eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijmb.20210604.15},
abstract = {The worldwide demand for industrially essential enzymes is growing due to their eco-friendly and sustainable applications in broad industrial sectors. The main objective of this investigation is intended to isolate potent alkalophilic native bacteria from local habitats for the cost-effective production of an enzyme alkaline protease. For screening of alkalophilic bacteria, bacterial samples were taken from various natural alkaline habitats, and bacterial cultures were screened on agar plate having skimmed milk as protein substrate by using the protein hydrolysis method. The bacteria showing maximum proteolytic potential were identified by microscopic, biochemical, and 16S rDNA analysis. Additionally, the culture components and other medium parameters have been optimized for higher enzyme production. High yield alkaline protease was obtained with conditions of 1% inoculum, pH 9.0, and at 37°C temperature, with 1% sugarcane molasses as the best suitable carbon substrate. Partial pur¬ification of the enzyme was performed and characterized for their optimum activity on various parameters (temperatures and pH range). The approx. molecular weight of the enzyme was estimated to be ~35 kDa. This study demonstrates the production of an industrially important enzyme (alkaline protease) from a newly isolated native Bacillus spp. Economical production of this enzyme can be commercially applied in vast industrial sectors like agriculture, textile, food, detergent, and leather industries.},
year = {2021}
}
TY - JOUR T1 - Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated Bacillus Bacteria AU - Ranjeet Singh AU - Ravindra Meena AU - Harsh Kumar AU - Banwari Lal Y1 - 2021/12/24 PY - 2021 N1 - https://doi.org/10.11648/j.ijmb.20210604.15 DO - 10.11648/j.ijmb.20210604.15 T2 - International Journal of Microbiology and Biotechnology JF - International Journal of Microbiology and Biotechnology JO - International Journal of Microbiology and Biotechnology SP - 137 EP - 146 PB - Science Publishing Group SN - 2578-9686 UR - https://doi.org/10.11648/j.ijmb.20210604.15 AB - The worldwide demand for industrially essential enzymes is growing due to their eco-friendly and sustainable applications in broad industrial sectors. The main objective of this investigation is intended to isolate potent alkalophilic native bacteria from local habitats for the cost-effective production of an enzyme alkaline protease. For screening of alkalophilic bacteria, bacterial samples were taken from various natural alkaline habitats, and bacterial cultures were screened on agar plate having skimmed milk as protein substrate by using the protein hydrolysis method. The bacteria showing maximum proteolytic potential were identified by microscopic, biochemical, and 16S rDNA analysis. Additionally, the culture components and other medium parameters have been optimized for higher enzyme production. High yield alkaline protease was obtained with conditions of 1% inoculum, pH 9.0, and at 37°C temperature, with 1% sugarcane molasses as the best suitable carbon substrate. Partial pur¬ification of the enzyme was performed and characterized for their optimum activity on various parameters (temperatures and pH range). The approx. molecular weight of the enzyme was estimated to be ~35 kDa. This study demonstrates the production of an industrially important enzyme (alkaline protease) from a newly isolated native Bacillus spp. Economical production of this enzyme can be commercially applied in vast industrial sectors like agriculture, textile, food, detergent, and leather industries. VL - 6 IS - 4 ER -
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