Optimization and Partial Characterization of <i>Bacillus</i> Protease Isolated from Soil and Agro-industrial Wastes

Daniel Yimer Legesse

doi:doi:10.11648/j.ijnfs.20170601.16

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Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes

Daniel Yimer Legesse

Published in International Journal of Nutrition and Food Sciences (Volume 6, Issue 1)

Received: 3 November 2016 Accepted: 2 December 2016 Published: 20 January 2017

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Abstract

Proteases from microbial sources possess almost all the characteristics desired for their biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from soils and agro-industrial wastes, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Thus, the optimum protease production time for these 3 isolates, was found to be 36 h isolated from industrial waste, manure, and soil, respectively. The optimum temperature of protease production for both PS-3 and PI-3 was 40°C. Whereas 37°C was the optimum for PM-1. In all cases, pH 7 was the optimum for production of protease. Furthermore, 0.6 M NaCl concentration was found to give better protease activity than the media containing no NaCl in all 3 isolates,. Among the metallic ions, media containing Mn²⁺ performed better than Cu²⁺, Mg²⁺, Fe²⁺, and Zn²⁺ for PS-3 and PM-1, whereas Mg²⁺ was the best for PI-3. Studies on the effect of pH on the stability of protease enzymes revealed that the crude enzyme had a maximum stability at pH 9.0 for isolates PS-3 (36.5 and 26.5 U/ml) and PI-3 (29.7 and 22.7 U/ml), while for isolate PM-1 maximum stability was achieved at pH 8 with values corresponding to 18.2 and 15.5 U/ml, respectively. These proteases are also stable at 75°C for PS-3 (42.9 and 33.4 U/ml) and PI-3 (46.8 and 42.1 U/ml), while they showed maximum activity and stability at 50°C for PM-1 (22.5 and 23.1 U/ml, respectively). Pre-incubation at temperatures above 70°C for PS-3 and PI-3 and 50°C for PM-1 resulted in reduction of enzyme activity, indicating that the proteases are thermally unstable. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease were better in 1 mM than in 5 mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for PS-3 (4.3 U/ml) and PM-1 (3.0 U/ml), whereas human hair was better for PI-3 (3.9 U/ml). Since protease was produced from readily available complex substrates and agro-industrial wastes, the 3 Bacillus species appear to have substantial potential for application in various proteolytic processes. Thus, identification of the 3 Bacillus isolates at a molecular level and purification as well as detailed characterization of the types of the proteases are recommended for effective utilization in different area of applications.

Published in	International Journal of Nutrition and Food Sciences (Volume 6, Issue 1)
DOI	10.11648/j.ijnfs.20170601.16
Page(s)	31-38
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), 2017. Published by Science Publishing Group

Keywords

Bacillus sp., Enzyme Assay, Gelatin Hydrolysis, Protease, Agro-industrial Wastes

References

[1]	Walsh, G. Proteins Biochemistry and Biotechnology. John Wiley & Sons, Ltd., England. 2002; 547p.
[2]	Whitford, D. Protein Structure and Function. John Wiley & Sons, Ltd., England. 2005; 528p.
[3]	Nadeem, M. Biotechnological production of alkaline protease for industrial use. PhD Thesis. University of Punjab, Lahore, Pakistan. 2009; 208p.
[4]	Sumantha, A., C. Larroche and A. Pandey. Microbiology and Industrial Biotechnology of Food-Grade Proteases: A Perspective. Food Technol. Biotechnol. 2006; 44 (2): 211–220.
[5]	Nirmal, N. P., Shankar S. and Laxman R. S. Fungal Proteases: An Overview. J. Biotech and Biosci, 2011; 1 (1): 1-40.
[6]	Sevinc, N. and E. Demirkan. Production of Protease by Bacillus sp. N-40 Isolated from Soil and Its Enzymatic Properties. J. Biol. Environ. Sci., 2011; 5 (14), 95-103
[7]	Ikram. N. Enhanced production of thermostable bacterial proteases and their applications. PhD. Thesis. University of Punjab. Pakistan. 2008; 158p
[8]	Gupta, R., Q. K. Beg, S. Khan and B. Chauhan. An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol. 2002b; 60 (4): 381-95.
[9]	Rao, M. B, Aparna T. M, Mohini G. S., Vasanti D. V. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev 1998; 62: 597-635.
[10]	Genckal, H. and Tari, C. Alkaline protease production from alkalophilic Bacillus sp. isolated from natural habitats. Enzyme Microb. Technol., 2006; 39: 703-710.
[11]	Alnahdi, H. S. Isolation and screening of extracellular proteases produced by new Isolated Bacillus sp. Journal of Applied Pharmaceutical Science 2012; 2 (9): 071-074.
[12]	Vishwanatha, T, Spoorthi N. J., Reena, V., Divyashree, B. C., Siddalingeshwara, K. G., Karthic, J and Sudipta K. M. Screening of Substrates for Protease Production from Bacillus licheniformisInternat. Journal of Engineering Science and Technology: 2010; 2(11): 6550-6554.
[13]	Ghaemi. F. S, Tabandeh. F, Yakhchali. B and Eftekhar. F. Enhancement of alkaline protease production by Bacillus clausii using Taguchi experimental design. African Journal of Biotechnology. 2007; 6 (22): 2559-2564.
[14]	Holt, J. G., N. R. Krieg, P. H. A. Sneath and J. T. Staley. Bergey’s Manual of Determinative Bacteriology. Nineteenth edition, Williams and wilkins company, Baltimore, MD, USA, 1994; pp: 255-273.
[15]	Hema, T. A and Shiny, M. Production of Protease Enzyme from Bacillus Clausii Sm3. IOSR Journal of Pharmacy and Biological Sciences. 2012; 1: 37-40
[16]	Folin, O., and Ciocalteu, V. Enzymatic Assay of Protease Casein as a Substrate J. Biol. Chem. 1929; 73: 627
[17]	Muthu, P. and Christudhas W. Purification and characterization of neutral protease enzyme from Bacillus Subtilis. J. Microbiol. Biotech. Res. 2012; 2 (4): 612-618
[18]	Agrawal, R., Singh R., Verma A., Panwar P. and Verma A. K. Partial Purification and Characterization of Alkaline Protease from Bacillus sp. Isolated from Soil. World Journal of Agricultural Sciences. 2012; 8 (1): 129-133.
[19]	Oliveira, A. N., Oliveira, L. A. and Andrade S. Production and Some Properties of Crude Alkaline Proteases of Indigenous Central Amazonian Rhizobia Strains. Brazilian Archives of Biology and Technology. 2010; 53: 1185-1195.
[20]	Udandi B., Rajendran R., Palanivel K. Vinayaga S. and Manoharan M. Optimization of Protease Enzyme Production Using Bacillus Sp. isolated from different Wastes. Botany Research International 2009; 2 (2): 83-87.
[21]	Al-Shehri, A.; Mostafa, M. and Yasser, S. Production and some properties of protease produced by Bacillus licheniformis isolated from Tihamet Asser, Saudi Arabia. Pak. J. Biol. Sci., 2004; 7, 1631-1635.
[22]	Qadar, S. A. U., E. Shireen, S. Iqbal and A. Anwar. Optimization of Protease production from newly isolated strains of Bacillus sp. PCSIR EA-3. Indian Journal of Biotechnology 2009; 8: 286-290.
[23]	Josephine S., Ramya V., Devi N., Ganapa B., Siddalingeshwara K. G., Venugopal. N and Vishwanatha T. Isolation, production and characterization of protease from Bacillus Sp isolated from soil sample. J. Microbiol. Biotech. Res., 2012; 2 (1): 163-168
[24]	Rai SK, Roy JK, and Mukherjee AK. Characterization of a detergent-stable alkaline rotease from a novel thermophilic strain Paenibacillus tezpurensis sp. Nov. AS-S24-II. Appl. Micrbiol. Biotechnol. 2010; 85: 1437-1450.
[25]	Huang, Q., Y. Peng, X. Li, H. Wang, and Y. Zhang. Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr. Microbiol. 2003; 46: 169-173.
[26]	Deng, A., J. Wua, Y. Zhang, G. Zhang, T. Wena. Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001 Bioresource Technology 2010; 101: 7100–7106
[27]	Kumara, M. S. Kashyap, N. S., Vijay. R., Rahul. T, Anuradha. M. Production and optimization of extra cellular Protease from bacillus sp. Isolated from soil. International Journal of Advanced Biotechnology and Research. 2012; 3: 564-569.
[28]	Akel H, Al-Quadan F, Yousef TK. Characterization of a purified thermosatble protease from hyperthermophilic Bacillus strain HUTBS71. Eur. J. Sci. Res. 2009; 31: 280-288.
[29]	Kumar, D. J. M. P. Venkatachalam, N. Govindarajan, M. D. Balakumaran and P. T. Kalaichelvan. Production and Purification of Alkaline Protease from Bacillus sp. MPTK 712 Isolated from Dairy Sludge. Global Veterinaria 2012; 8 (5): 433-439.
[30]	Gessesse, A. The use of nug meal as a low-cost substrate for the production of alkaline protease by the alkaliphilic Bacillus sp. AR-009 and some properties of the enzyme” Bioresource Technology, 1997; 62: 59-61.
[31]	Enshasy, E. H., Abuol-Enein, A., Helmy, S. and Azaly, E. Optimization of The industrial production of alkaline protease by Bacillus licheniformis in different production scales. Australian Journal of Basic and Applied Sciences. 2008; 2: 583-593.

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Daniel Yimer Legesse. (2017). Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes. International Journal of Nutrition and Food Sciences, 6(1), 31-38. https://doi.org/10.11648/j.ijnfs.20170601.16

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Daniel Yimer Legesse. Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes. Int. J. Nutr. Food Sci. 2017, 6(1), 31-38. doi: 10.11648/j.ijnfs.20170601.16

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Daniel Yimer Legesse. Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes. Int J Nutr Food Sci. 2017;6(1):31-38. doi: 10.11648/j.ijnfs.20170601.16

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@article{10.11648/j.ijnfs.20170601.16,
  author = {Daniel Yimer Legesse},
  title = {Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes},
  journal = {International Journal of Nutrition and Food Sciences},
  volume = {6},
  number = {1},
  pages = {31-38},
  doi = {10.11648/j.ijnfs.20170601.16},
  url = {https://doi.org/10.11648/j.ijnfs.20170601.16},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijnfs.20170601.16},
  abstract = {Proteases from microbial sources possess almost all the characteristics desired for their biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from soils and agro-industrial wastes, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Thus, the optimum protease production time for these 3 isolates, was found to be 36 h isolated from industrial waste, manure, and soil, respectively. The optimum temperature of protease production for both PS-3 and PI-3 was 40°C. Whereas 37°C was the optimum for PM-1. In all cases, pH 7 was the optimum for production of protease. Furthermore, 0.6 M NaCl concentration was found to give better protease activity than the media containing no NaCl in all 3 isolates,. Among the metallic ions, media containing Mn2+ performed better than Cu2+, Mg2+, Fe2+, and Zn2+ for PS-3 and PM-1, whereas Mg2+ was the best for PI-3. Studies on the effect of pH on the stability of protease enzymes revealed that the crude enzyme had a maximum stability at pH 9.0 for isolates PS-3 (36.5 and 26.5 U/ml) and PI-3 (29.7 and 22.7 U/ml), while for isolate PM-1 maximum stability was achieved at pH 8 with values corresponding to 18.2 and 15.5 U/ml, respectively. These proteases are also stable at 75°C for PS-3 (42.9 and 33.4 U/ml) and PI-3 (46.8 and 42.1 U/ml), while they showed maximum activity and stability at 50°C for PM-1 (22.5 and 23.1 U/ml, respectively). Pre-incubation at temperatures above 70°C for PS-3 and PI-3 and 50°C for PM-1 resulted in reduction of enzyme activity, indicating that the proteases are thermally unstable. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease were better in 1 mM than in 5 mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for PS-3 (4.3 U/ml) and PM-1 (3.0 U/ml), whereas human hair was better for PI-3 (3.9 U/ml). Since protease was produced from readily available complex substrates and agro-industrial wastes, the 3 Bacillus species appear to have substantial potential for application in various proteolytic processes. Thus, identification of the 3 Bacillus isolates at a molecular level and purification as well as detailed characterization of the types of the proteases are recommended for effective utilization in different area of applications.},
 year = {2017}
}

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TY  - JOUR
T1  - Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes
AU  - Daniel Yimer Legesse
Y1  - 2017/01/20
PY  - 2017
N1  - https://doi.org/10.11648/j.ijnfs.20170601.16
DO  - 10.11648/j.ijnfs.20170601.16
T2  - International Journal of Nutrition and Food Sciences
JF  - International Journal of Nutrition and Food Sciences
JO  - International Journal of Nutrition and Food Sciences
SP  - 31
EP  - 38
PB  - Science Publishing Group
SN  - 2327-2716
UR  - https://doi.org/10.11648/j.ijnfs.20170601.16
AB  - Proteases from microbial sources possess almost all the characteristics desired for their biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from soils and agro-industrial wastes, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Thus, the optimum protease production time for these 3 isolates, was found to be 36 h isolated from industrial waste, manure, and soil, respectively. The optimum temperature of protease production for both PS-3 and PI-3 was 40°C. Whereas 37°C was the optimum for PM-1. In all cases, pH 7 was the optimum for production of protease. Furthermore, 0.6 M NaCl concentration was found to give better protease activity than the media containing no NaCl in all 3 isolates,. Among the metallic ions, media containing Mn2+ performed better than Cu2+, Mg2+, Fe2+, and Zn2+ for PS-3 and PM-1, whereas Mg2+ was the best for PI-3. Studies on the effect of pH on the stability of protease enzymes revealed that the crude enzyme had a maximum stability at pH 9.0 for isolates PS-3 (36.5 and 26.5 U/ml) and PI-3 (29.7 and 22.7 U/ml), while for isolate PM-1 maximum stability was achieved at pH 8 with values corresponding to 18.2 and 15.5 U/ml, respectively. These proteases are also stable at 75°C for PS-3 (42.9 and 33.4 U/ml) and PI-3 (46.8 and 42.1 U/ml), while they showed maximum activity and stability at 50°C for PM-1 (22.5 and 23.1 U/ml, respectively). Pre-incubation at temperatures above 70°C for PS-3 and PI-3 and 50°C for PM-1 resulted in reduction of enzyme activity, indicating that the proteases are thermally unstable. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease were better in 1 mM than in 5 mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for PS-3 (4.3 U/ml) and PM-1 (3.0 U/ml), whereas human hair was better for PI-3 (3.9 U/ml). Since protease was produced from readily available complex substrates and agro-industrial wastes, the 3 Bacillus species appear to have substantial potential for application in various proteolytic processes. Thus, identification of the 3 Bacillus isolates at a molecular level and purification as well as detailed characterization of the types of the proteases are recommended for effective utilization in different area of applications.
VL  - 6
IS  - 1
ER  -

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Daniel Yimer Legesse

Ethiopian Institute of Agricultural Research (EIAR), Holeta Agricultural Research Center (HARC), National Agricultural Biotechnology Laboratory, Microbial Biotechnology Research, Addis Ababa, Ethiopia

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Daniel Yimer Legesse. (2017). Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes. International Journal of Nutrition and Food Sciences, 6(1), 31-38. https://doi.org/10.11648/j.ijnfs.20170601.16

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ACS Style

Daniel Yimer Legesse. Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes. Int. J. Nutr. Food Sci. 2017, 6(1), 31-38. doi: 10.11648/j.ijnfs.20170601.16

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AMA Style

Daniel Yimer Legesse. Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes. Int J Nutr Food Sci. 2017;6(1):31-38. doi: 10.11648/j.ijnfs.20170601.16

Copy | Download

@article{10.11648/j.ijnfs.20170601.16,
  author = {Daniel Yimer Legesse},
  title = {Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes},
  journal = {International Journal of Nutrition and Food Sciences},
  volume = {6},
  number = {1},
  pages = {31-38},
  doi = {10.11648/j.ijnfs.20170601.16},
  url = {https://doi.org/10.11648/j.ijnfs.20170601.16},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijnfs.20170601.16},
  abstract = {Proteases from microbial sources possess almost all the characteristics desired for their biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from soils and agro-industrial wastes, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Thus, the optimum protease production time for these 3 isolates, was found to be 36 h isolated from industrial waste, manure, and soil, respectively. The optimum temperature of protease production for both PS-3 and PI-3 was 40°C. Whereas 37°C was the optimum for PM-1. In all cases, pH 7 was the optimum for production of protease. Furthermore, 0.6 M NaCl concentration was found to give better protease activity than the media containing no NaCl in all 3 isolates,. Among the metallic ions, media containing Mn2+ performed better than Cu2+, Mg2+, Fe2+, and Zn2+ for PS-3 and PM-1, whereas Mg2+ was the best for PI-3. Studies on the effect of pH on the stability of protease enzymes revealed that the crude enzyme had a maximum stability at pH 9.0 for isolates PS-3 (36.5 and 26.5 U/ml) and PI-3 (29.7 and 22.7 U/ml), while for isolate PM-1 maximum stability was achieved at pH 8 with values corresponding to 18.2 and 15.5 U/ml, respectively. These proteases are also stable at 75°C for PS-3 (42.9 and 33.4 U/ml) and PI-3 (46.8 and 42.1 U/ml), while they showed maximum activity and stability at 50°C for PM-1 (22.5 and 23.1 U/ml, respectively). Pre-incubation at temperatures above 70°C for PS-3 and PI-3 and 50°C for PM-1 resulted in reduction of enzyme activity, indicating that the proteases are thermally unstable. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease were better in 1 mM than in 5 mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for PS-3 (4.3 U/ml) and PM-1 (3.0 U/ml), whereas human hair was better for PI-3 (3.9 U/ml). Since protease was produced from readily available complex substrates and agro-industrial wastes, the 3 Bacillus species appear to have substantial potential for application in various proteolytic processes. Thus, identification of the 3 Bacillus isolates at a molecular level and purification as well as detailed characterization of the types of the proteases are recommended for effective utilization in different area of applications.},
 year = {2017}
}

Copy | Download

TY  - JOUR
T1  - Optimization and Partial Characterization of Bacillus Protease Isolated from Soil and Agro-industrial Wastes
AU  - Daniel Yimer Legesse
Y1  - 2017/01/20
PY  - 2017
N1  - https://doi.org/10.11648/j.ijnfs.20170601.16
DO  - 10.11648/j.ijnfs.20170601.16
T2  - International Journal of Nutrition and Food Sciences
JF  - International Journal of Nutrition and Food Sciences
JO  - International Journal of Nutrition and Food Sciences
SP  - 31
EP  - 38
PB  - Science Publishing Group
SN  - 2327-2716
UR  - https://doi.org/10.11648/j.ijnfs.20170601.16
AB  - Proteases from microbial sources possess almost all the characteristics desired for their biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from soils and agro-industrial wastes, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Thus, the optimum protease production time for these 3 isolates, was found to be 36 h isolated from industrial waste, manure, and soil, respectively. The optimum temperature of protease production for both PS-3 and PI-3 was 40°C. Whereas 37°C was the optimum for PM-1. In all cases, pH 7 was the optimum for production of protease. Furthermore, 0.6 M NaCl concentration was found to give better protease activity than the media containing no NaCl in all 3 isolates,. Among the metallic ions, media containing Mn2+ performed better than Cu2+, Mg2+, Fe2+, and Zn2+ for PS-3 and PM-1, whereas Mg2+ was the best for PI-3. Studies on the effect of pH on the stability of protease enzymes revealed that the crude enzyme had a maximum stability at pH 9.0 for isolates PS-3 (36.5 and 26.5 U/ml) and PI-3 (29.7 and 22.7 U/ml), while for isolate PM-1 maximum stability was achieved at pH 8 with values corresponding to 18.2 and 15.5 U/ml, respectively. These proteases are also stable at 75°C for PS-3 (42.9 and 33.4 U/ml) and PI-3 (46.8 and 42.1 U/ml), while they showed maximum activity and stability at 50°C for PM-1 (22.5 and 23.1 U/ml, respectively). Pre-incubation at temperatures above 70°C for PS-3 and PI-3 and 50°C for PM-1 resulted in reduction of enzyme activity, indicating that the proteases are thermally unstable. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease were better in 1 mM than in 5 mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for PS-3 (4.3 U/ml) and PM-1 (3.0 U/ml), whereas human hair was better for PI-3 (3.9 U/ml). Since protease was produced from readily available complex substrates and agro-industrial wastes, the 3 Bacillus species appear to have substantial potential for application in various proteolytic processes. Thus, identification of the 3 Bacillus isolates at a molecular level and purification as well as detailed characterization of the types of the proteases are recommended for effective utilization in different area of applications.
VL  - 6
IS  - 1
ER  -

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