Bacillus subtilis has been a reliable platform for the expression of extracellular proteases for several decades. Although a majority of Bacillus subtilis subspecies express proteases, the amount of secreted enzyme varies depending on the strain and environmental conditions used. Here, two Bacillus subtilis spp. subtilis strains, NRRL B-3384 and NRRL B-3387, from the ARS Culture collection (NRRL), were compared for secreted protease activity. The highest activity was found in strain NRRL B-3384, and proteolysis occurred at temperatures as high as 80°C and across a broad range of pH, with maximum activity at pH 9.0 and 60°C indicating the presence of a thermostable alkaline protease. To our knowledge, this is the first study to evaluate protease production in Bacillus subtilis spp. subtilis strains NRRL B-3384 and B3387 and suggests that NRRL B-3384 may have utility in the production of enzymes for industrial use.
Author would like to thank Dr. Hasan Buğra ÇOBAN from Dokuz Eylül University, for his helpful discussions for improving this article and Dr. İrem DENİZ CAN from Manisa Celal Bayar University for her support in laboratory facilities and equipment. Author would like to thank Prof. Dr. Theodore G. CLARK from Cornell University for his valuable comments and editing the final version of the manuscript.
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fermentation. Preparative Biochemistry and Biotechnology.
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1. Jegannathan KR, Nielsen PH. Environmental assessment of enzyme
use in industrial production–a literature review. Journal of cleaner
production. 2013;42:228-40.
2. Singh S, Bajaj BK. Bioprocess optimization for production of
thermoalkali-stable protease from Bacillus subtilis K-1 under solidstate
fermentation. Preparative Biochemistry and Biotechnology.
2016;46(7):717-24.
3. Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, et al.
Microbial proteases applications. Frontiers in bioengineering and
biotechnology. 2019;7:110.
4. Singhal P, Nigam V, Vidyarthi A. Studies on production,
characterization and applications of microbial alkaline proteases.
International Journal of Advanced Biotechnology and Research.
2012;3(3):653-69.
5. Sharma KM, Kumar R, Panwar S, Kumar A. Microbial alkaline
proteases: Optimization of production parameters and their
properties. Journal of Genetic Engineering and Biotechnology.
2017;15(1):115-26.
6. Pihlanto A. Antioxidative peptides derived from milk proteins.
International dairy journal. 2006;16(11):1306-14.
7. Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. Molecular and
biotechnological aspects of microbial proteases. Microbiology and
molecular biology reviews. 1998;62(3):597-635.
8. Gupta R, Beg Q, Lorenz P. Bacterial alkaline proteases: molecular
approaches and industrial applications. Applied microbiology and
biotechnology. 2002;59(1):15-32.
9. Contesini FJ, Melo RRd, Sato HH. An overview of Bacillus
proteases: from production to application. Critical reviews in
biotechnology. 2018;38(3):321-34.
10. Helal M, Amer H, Abdelwahed N, Ghobashy M. Physiological and
microbiological studies on production of alkaline protease from
locally isolated Bacillus subtilis. Aust J Basic Appl Sci. 2012;6:193-
203.
11. Kalwasińska A, Jankiewicz U, Felföldi T, Burkowska-But A,
Swiontek Brzezinska M. Alkaline and halophilic protease
production by Bacillus luteus H11 and its potential industrial
applications. Food technology and biotechnology. 2018;56(4):553-
61.
12. Joo HS, Chang CS. Oxidant and SDS‐stable alkaline protease from a
halo‐tolerant Bacillus clausii I‐52: enhanced production and simple
purification. Journal of applied microbiology. 2005;98(2):491-7.
13. Ibrahim KS, Muniyandi J, Pandian SK. Purification and
characterization of manganese-dependent alkaline serine protease
from Bacillus pumilus TMS55. Journal of microbiology and
biotechnology. 2011;21(1):20-7.
14. Baweja M, Tiwari R, Singh PK, Nain L, Shukla P. An alkaline
protease from Bacillus pumilus MP 27: functional analysis of
its binding model toward its applications as detergent additive.
Frontiers in microbiology. 2016;7:1195.
15. Haddar A, Bougatef A, Agrebi R, Sellami-Kamoun A, Nasri M.
A novel surfactant-stable alkaline serine-protease from a newly
isolated Bacillus mojavensis A21. Purification and characterization.
Process Biochemistry. 2009;44(1):29-35.
16. Saggu SK, Mishra PC. Characterization of thermostable alkaline
proteases from Bacillus infantis SKS1 isolated from garden soil.
PloS one. 2017;12(11):e0188724.
17. Anbu P. Characterization of solvent stable extracellular protease
from Bacillus koreensis (BK-P21A). International Journal of
Biological Macromolecules. 2013;56:162-8.
18. Singh J, Vohra R, Sahoo DK. Enhanced production of alkaline
proteases by Bacillus sphaericus using fed-batch culture. Process
Biochemistry. 2004;39(9):1093-101.
19. Moon SH, Parulekar SJ. A parametric study ot protease production
in batch and fed‐batch cultures of Bacillus firmus. Biotechnology
and bioengineering. 1991;37(5):467-83.
20. Ferreira L, Ramos M, Dordick J, Gil M. Influence of different silica
derivatives in the immobilization and stabilization of a Bacillus
licheniformis protease (Subtilisin Carlsberg). Journal of Molecular
Catalysis B: Enzymatic. 2003;21(4-6):189-99.
21. Yilmaz B, Baltaci MO, Sisecioglu M, Adiguzel A. Thermotolerant
alkaline protease enzyme from Bacillus licheniformis A10:
purification, characterization, effects of surfactants and organic
solvents. Journal of enzyme inhibition and medicinal chemistry.
2016;31(6):1241-7.
22. Pohl S, Harwood CR. Heterologous protein secretion by Bacillus
species: from the cradle to the grave. Advances in applied
microbiology. 2010;73:1-25.
23. Cui W, Han L, Suo F, Liu Z, Zhou L, Zhou Z. Exploitation
of Bacillus subtilis as a robust workhorse for production of
heterologous proteins and beyond. World Journal of Microbiology
and Biotechnology. 2018;34(10):1-19.
24. Abusham RA, Rahman RNZR, Salleh AB, Basri M. Optimization of
physical factors affecting the production of thermo-stable organic
solvent-tolerant protease from a newly isolated halo tolerant
Bacillus subtilis strain Rand. Microbial Cell Factories. 2009;8(1):1-9.
25. ÇOBAN HB. Axinella damicornis süngerinden izole edilen sucul
bakterilerin proteaz üretkenliklerinin araştırılması ve üretilen
proteaz enziminin kısmi karakterizasyonu. Mediterranean
Agricultural Sciences.33(2):223-9.
26. Vaithanomsat P, Malapant T, Apiwattanapiwat W. Silk degumming
solution as substrate for microbial protease production. Agriculture
and Natural Resources. 2008;42(3):543-51.
27. Prasad R, Abraham TK, Nair AJ. Scale up of production in a
bioreactor of a halotolerant protease from moderately halophilic
Bacillus sp. isolated from soil. Brazilian Archives of Biology and
Technology. 2014;57(3):448-55.
28. VijayAnand S, Hemapriya J, Selvin J, Kiran S. Production and
optimization of haloalkaliphilic protease by an extremophile-
Halobacterium sp. Js1, isolated from thalassohaline environment.
Global J Biotechnol Biochem. 2010;5(1):44-9.
29. Ali N, Ullah N, Qasim M, Rahman H, Khan SN, Sadiq A, et al.Molecular characterization and growth optimization of halotolerant protease producing Bacillus subtilis Strain BLK-1.5 isolated
from salt mines of Karak, Pakistan. Extremophiles. 2016;20(4):395-
402.
30. Adinarayana K, Ellaiah P, Prasad DS. Purification and partial
characterization of thermostable serine alkaline protease from
a newly isolated Bacillus subtilis PE-11. Aaps Pharmscitech.
2003;4(4):440-8.
31. Nihalani D, Satyanarayana T. Isolation and characterization of
extracellular alkaline enzyme producing bacteria. Indian journal
of microbiology New Delhi. 1992;32(4):443-9.
32. Gessesse A, Gashe BA. Production of alkaline protease by
an alkaliphilic bacteria isolated from an alkaline soda lake.
Biotechnology Letters. 1997;19(5):479-81.
33. Pant G, Prakash A, Pavani J, Bera S, Deviram G, Kumar A, et al.
Production, optimization and partial purification of protease
from Bacillus subtilis. Journal of Taibah University for Science.
2015;9(1):50-5.
34. Khan I, Gupta P, Vakhlu J. Thermo-alkaliphilic halotolerant
detergent compatible protease (s) of Bacillus tequilensis MTCC
9585. African Journal of Microbiology Research. 2011;5(23):3968-
75.
35. Abd Rahman RNZ, Geok LP, Basri M, Salleh AB. Physical factors
affecting the production of organic solvent-tolerant protease
by Pseudomonas aeruginosa strain K. Bioresource technology.
2005;96(4):429-36.
36. Gouda MK. Optimization and purification of alkaline proteases
produced by marine Bacillus sp. MIG newly isolated from
Eastern Harbour of Alexandria. Polish Journal of microbiology.
2006;55(2):119.
37. Asha B, Palaniswamy M. Optimization of alkaline protease
production by Bacillus cereus FT 1 isolated from soil. J App Pharm
Sci. 2018;8(02):119-27.
38. Ahmed M, Rehman R, Siddique A, Hasan F, Ali N, Hameed A.
Production, purification and characterization of detergent-stable,
halotolerant alkaline protease for eco-friendly application in
detergents’ industry. Int J Biosci. 2016;8(2),:47-65.
39. Sharmin S, Hossain MT, Anwar M. Isolation and characterization
of a protease producing bacteria Bacillus amovivorus and
optimization of some factors of culture conditions for protease
production. J Biol Sci. 2005;5(3):358-62.
40. Shumi W, Hossain MT, Anwar M. Proteolytic activity of a
bacterial isolate Bacillus fastidiosus den Dooren de Jong. J biol Sci.
2004;4(3):370-4.
41. Chantawannakul P, Oncharoen A, Klanbut K, Chukeatirote E,
Lumyong S. Characterization of proteases of Bacillus subtilis strain
38 isolated from traditionally fermented soybean in Northern
Thailand. Science Asia. 2002;28(4):241-5.
42. Rehman R, Ahmed M, Siddique A, Hasan F, Hameed A, Jamal
A. Catalytic role of thermostable metalloproteases from Bacillus
subtilis KT004404 as dehairing and destaining agent. Applied
biochemistry and biotechnology. 2017;181(1):434-50.
43. Frankena J, Koningstein GM, van Verseveld HW, Stouthamer AH.
Effect of different limitations in chemostat cultures on growth
and production of exocellular protease by Bacillus licheniformis.
Applied microbiology and biotechnology. 1986;24(2):106-12.
44. Nascimento WCAd, Martins MLL. Production and properties of
an extracellular protease from thermophilic Bacillus sp. Brazilian
journal of microbiology. 2004;35:91-6.
45. Abdulrahman A, Yasser M. Production and some properties of
protease produced by Bacillus licheniformis isolated from Tihamet
Aseer, Saudi Arabia. Pakistan Journal of Biological Sciences
(Pakistan). 2004.
46. Colantuono A, D'Incecco P, Fortina MG, Rosi V, Ricci G, Pellegrino
L. Milk substrates influence proteolytic activity of Pseudomonas
fluorescens strains. Food Control. 2020;111:107063.
47. Sharma AK, Sharma V, Saxena J, Yadav B, Alam A, Prakash A.
Optimization of protease production from bacteria isolated from
soil. Appl Res J. 2015;1(7):388-94.
48. Uyar F, Porsuk I, Kizil G, Yilmaz EI. Optimal conditions for
production of extracellular protease from newly isolated Bacillus
cereus strain CA15. EurAsian Journal of BioSciences. 2011;5.
49. Akhavan Sepahy A, Jabalameli L. Effect of culture conditions on the
production of an extracellular protease by Bacillus sp. isolated from
soil sample of Lavizan Jungle Park. Enzyme research. 2011;2011.
50. Shafee N, Aris SN, Rahman R, Basri M, Salleh AB. Optimization
of environmental and nutritional conditions for the production
of alkaline protease by a newly isolated bacterium Bacillus cereus
strain 146. J Appl Sci Res. 2005;1(1):1-8.
51. Mehta V, Thumar J, Singh S. Production of alkaline protease
from an alkaliphilic actinomycete. Bioresource technology.
2006;97(14):1650-4.
52. Kebabci Ö, Cihangir N. Full Length Research Paper Isolation of
protease producing novel Bacillus cereus and detection of optimal
conditions. African Journal of Biotechnology. 2011;10(7):1160-4.
53. Yang J-K, Shih L, Tzeng Y-M, Wang S-L. Production and purification
of protease from a Bacillus subtilis that can deproteinize crustacean
wastes☆. Enzyme and microbial technology. 2000;26(5-6):406-13.
54. Priest FG. Extracellular enzyme synthesis in the genus Bacillus.
Bacteriological reviews. 1977;41(3):711-53.
55. Prestidge L, Gage V, Spizizen J. Protease activities during the
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