Obtaining Efficient Mutant from the Wild Type Bacillus subtilis E6-5 by Physical and Chemical Mutagenesis for High Efficiency Protease Production, Optimizing the Mutant's Culture Medium

Abstract

In this study, to enhance protease production, the wild type of Bacillus subtilis E6-5 was mutagenized by random mutagenesis using ultraviolet radiation and ethidium bromide. After combined treatment, several mutants were obtained. Among these mutants, the mutant strain with the largest proteolytic zone diameter (25 mm) was selected and named Bacillus subtilis ATA38. The enzyme production capacity of the obtained mutant was tested and the mutant strain (404 IU/mL at 24 hours) produced 6.7 times more enzyme than the parental strain (60 IU/mL at 32 hours). The effects of some important parameters in the growth medium on enzyme production were examined. The best carbon, organic nitrogen and metal ion were obtained with wheat starch (525 IU/mL), meat extract (850 IU/mL) and KCl+CaCl2 (548 IU/mL), respectively. pH 6.0, 37°C, 200 rpm, inoculum age 18 hours and inoculation amount 1% were obtained as the best physical factors.To further increase the yield, the best nutritional and physical parameters were combined to create a new modified medium. It was determined that the enzyme yield with mutant strain increased 2.7 times in the modified medium (1096 IU/mL) compared to the control (404 U/mL). The mutant strain (1096 IU/mL) showed an 18.2-fold increase in production compared to the wild type (60 IU/mL) in the modified medium. Protease enzyme obtained from ATA38 mutant strain may have great potential in industry for different purposes.

Keywords

• Protease
• Bacillus
• Random mutagenesis
• Optimization

References

1. [1] Shankar, S., Rao, M., and Seeta, L.R., “Purification and characterization of an alkaline protease by a new strain of Beauveria sp.”, Process Biochemistry, 46(2): 579–585, (2011).
2. [2] Das, G., and Prasad, M.P., “Isolation, purification & mass production of protease enzyme from Bacillus subtilis”, International Research Journal of Microbiology, 1(2): 26–31, (2010).
3. [3] Hammami, A., Hamdi, M., Abdelhedi, O., Jridi, M., Nasri, M., and Bayoudh, A., “Surfactant- and oxidant-stable alkaline proteases from Bacillus invictae: characterization and potential applications in chitin extraction and as a detergent additive”, International Journal of Biological Macromolecules, 96: 272–81, (2017).
4. [4] Gupta, R., Beg, Q.K., Khan, S., and Chauhan, B., “An overview on fermentation, downstream processing and properties of microbial alkaline proteases”, Applied Microbiology and Biotechnology, 60: 381–395, (2002).
5. [5] Pant, G., Prakash, A., Pavani, J.V.P., Bera, S., B., Deviram, G.V.N.S., Kumar, A., Panchpuri, M., and Prasuna, R.G., “Production, optimization and partial purification of protease from Bacillus subtilis”, Journal of Taibah University for Science, 9: 50–55, (2015).
6. [6] Azin, D.A., and Noroozi, R.F., “Effect of chemicals on the improved gluconate productivity by an Aspergillus niger strain”, Applied Biochemistry and Biotechnology, 61: 393–397, (2001).
7. [7] Iftikhar, T., Niaz, M., Abbas, S.Q., Zia, M.A., Ashraf, I., Lee, V., and Haq, I., “Mutation induced enhanced biosynthesis of lipases by Rhizopus oligosporus var. Microsporus”, Pakistan Journal of Botany, 42: 1235–1249, (2010).
8. [8] Zia, M.A, Rahman, K., Sheikh, M.A., and Khan, I.A., “Chemically treated strain improvement of Aspergillus niger for enhanced production of glucose oxidase”, International Journal of Agriculture and Biology, 12: 153–154, (2010).

9. [9] Ribeiro, O., Magalhaes, F., Aguiar, T.Q., Wiebe, M., Penttila, M., and Domingues, L., “Random and direct mutagenesis to enhancemprotein secretion in Ashbya gossypii”, Bioengineered, 4: 322–331, (2013).
10. [10] Basavaraju, S., Kathera, C., and Jasti, P.K., “Induction of Alkaline Protease Production by Bacillus mutants through U.V. irradiation”, International Journal of Pharmaceutical Sciences Review and Research, 26(1): 78–83, (2014).
11. [11] Rani, M.R., Prasad, N.N., and Sambasivarao, K.R.S., “Optimization of cultural conditions for the production of alkaline protease from a mutant Aspergillus Flavus AS2”, Asian Journal of Experimental Biological Sciences, 3: 565–576, (2012).
12. [12] Khedr, M.A., Ewais, E.A., and Khalil, K.M.A., “Overproduction of thermophilic α-amylase productivity and Amy E gene sequence of novel Egyptian strain Bacillus licheniformis MK9 and two induced mutants”, Current Science International, 6(2): 364–376, (2017).
13. [13] Suribabu, K., Govardhan, Y.L., and Hemalatha, K.P.J., “Strain Improvement of Brevibacillus borostelensis R1 for Optimization of α-Amylase Production by Mutagens”, Journal of Microbial and Biochemical Technology, 6(3): 123–12, (2014).
14. [14] Khalil, A.B., Ghandi, H., Anfoka, H., and Bdour, S., “Isolation of plasmids present in thermophilic strains from hot springs in Jordan”, World Journal of Microbiology and Biotechnology, 19: 239–241, (2003).
15. [15] Demirkan, E., Kut, D., Sevgi, T., Dogan, M., and Baygin, E., “Investigation of effects of protease enzyme produced by Bacillus subtilis 168 E6-5 and commercial enzyme on physical properties of woolen fabric”, Journal of the Textile Institute, 111: 26–35, (2020).
16. [16] Zong, H., Zhan, Y., Li, X., Peng, L., Feng, F., and Li, D., “A new mutation breeding method for Streptomyces albulus by anatmospheric and room temperature plasma”, African Journal of Microbiology Research, 6(13): 3154–3158, (2012).
17. [17] Quadar, S.A., Shireen, E., Iqbal, S., and Anwar, A., “Optimization of protease production from newly isolated strain of Bacillus sp. PCSIR EA-3”, Indian Journal of Biotechnology, 8: 286–290, (2009).
18. [18] Keay, L., and Wildi, B.S., “Proteinases of the genus Bacillus. I. Neutral proteinases”, Biotechnology and Bioengineering, 12: 179–212, (1970).
19. [19] Laemmli, U.K., “Cleavage of structural proteins during theassembly of the head of bacteriophage T4”, Nature, 227: 680-685, (1970).
20. [20] Vijayaraghavan, P., Lazarus, S., and Vincent, S.G.P., “De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: biosynthesis and properties”, Saudi Journal of Biological Sciences, 21: 27–34, (2014).
21. [21] Chand, P., Aruna, A., Maqsood, A.M., and Rao, L.V., “Novel mutation method for increased cellulase production”, Journal of Applied Microbiology, 98: 318–323, (2005).
22. [22] Solaiman, E.A.M., Hegazy, W.K., and Maysa, E., “Moharam Induction of overproducing alkaline protease Bacillus mutants through UV irradiation”, The Arab Journal of Biothechnology, 8: 49-60, (2005)
23. [23] Nadeem, M., Qazi, J. I., and Baig, S., “Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing”, Brazilian Archives of Biology and Technology, 53: 1015–1025, (2010).
24. [24] Javed, S., Meraj, M., Bukhari, S. A., Irfan, R., and Mahmood, S., “Hyper-production of Alkaline Protease by Mutagenic Treatment of Bacillus subtilis M-9 using Agroindustrial Wastes in Submerged Fermentation”, Journal of Microbial and Biochemical Technology, 5(3): 74–80, (2013).
25. [25] Wang, X.C., Zhao, H.Y., Liu, G., Cheng, X.J., and Feng, H., “Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4”, Genetics and Molecular Research, 15(2): gmr.15027831.
26. [26] Payal, M., Charu, S., and Pradeep, B., “Strain Improvement of Halotolerant Actinomycete for Protease Production by Sequential Mutagenesis”, International Journal of Chemical Sciences, 15(1): 109, (2017).
27. [27] Shikha, S.A., and Darmwal, N.S., “Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate”, Bioresource Technology, 98: 881–885, (2007).
28. [28] Sajedi, R.H., Naderi-Manesh, H., Khajeh, K., Ahmadvand, R., Ranjbar, B., Asoodeh, A., and Moradian, F., “A Ca-independent amylase that is active and stable at low pH from the Bacillus sp. KR-8104”, Enzyme and Microbial Technology, 36: 666–671, (2005).
29. [29] Couto, S.R., and Sanromán, M.A., “Application of solid-state fermentation to food industry- A review”, Journal of Food Engineering, 76: 291–302, (2006).
30. [30] Souza, P.M.D., “Application of microbial a-amylase in industry-a review”, Brazilian Journal of Microbiology, 41: 850–861, (2010).
31. [31] Raju, E.V.N., and Divakar, G., “Bacillus cereus GD 55 Strain Improvement by Physical and Chemical Mutagenesis for Enhanced Production of Fibrinolytic Protease”, International Journal of Pharmaceutical Sciences and Research, 4: 81–93, (2013).
32. [32] Gulsher, M., Nadeem, M., Syed, Q., Irfan, M., and Baig S., “Protease Production from UV Mutated Bacillus subtilis”, Bangladesh Journal of Scientific and Industrial Research, 47(1): 69–76, (2012).
33. [33] Prakasham, R.S., Rao, C.S., and Sarma, P.N., “Green gram husk - An inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation”, Bioresource Technology, 97: 1449–1454, (2006).
34. [34] Nadeem, M., Iqbal J.Q., Shahjahan B., and Qurat-ul-Ain S., “Effect of medium composition on Commercially Important Alkaline Protease Production by Bacillus licheniformis N-2”, Food Technology and Biotechnology, 46: 385–394, (2008).
35. [35] Mukhtar, H., and Haq, I., “Production of alkaline protease by Bacillus subtilis and its application as a depilating agent in leather processing”, Pakistan Journal of Botany, 40(4): 1673–1679, (2008).
36. [36] Darah, I., Han, L.Z., Nuraqilah, Y., Isnaeni., and Lim Sheh, H., “Bacillus licheniformis BT5.9 isolated from Changar hot spring, Malang, Indonesia, as a potential producer of thermostable α-amylase”, Tropical Life Sciences Research, 24(1): 71–84, (2013).
37. [37] El-Enshasy, H.A., Farid M.A., and El-Sayed E.A., “Influence of inoculums type and cultivation conditions on natamycin production by Streptomyces natalensis”, Journal of Basic Microbiology: An International Journal on Biochemistry, Physiology, Genetics, Morphology, and Ecology of Microorganisms, 40(5-6): 33–342, (2020)
38. [38] Sangeetha, P.T., Ramesh, M.N., and Prapulla S.G., “Production of fructosyl transferase by Aspergillus oryzae CFR202 in solid-stse fermentation using agricultural by-product”, Applied Microbiology and Biotechnology, 65(5): 530–537, (2004).
39. [39] Nadeem, M., Qazi, J.I., Baig, S.J., and Syed, Q.A., “Studies on commercially important alkaline protease from Bacillus licheniformis N-2 isolated from decaying organic soil”, Turkish Journal of Biochemistry, 32: 171–177.
40. [40] Shafique, T., Shafique, J., Zahid, S., Kazi, M., Alnemer, O., and Ahmad, A., “Screening, selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease”, Saudi Journal of Biological Sciences, 28: 1494–1501, (2021).
41. [41] El-Safey, E.M. and Abdul-Raouf, U.M., “Production, purification and characterization of protease enzyme from Bacillus subtilis.” International Conferences for Development and the Environment in the Arab World, Assiut, Egypt, March 23–25, 14, (2004).
42. [42] Vijayaraghavan, P., Vijayan, A., Arun, A., Jenisha, J.K. and Vincent, S.G.P., “Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV”, SpringerPlus, 1: 76, (2012).
43. [43] Kato, T., Yamagata, Y., Arai, T. and Ichishima, E., “Purification of a new extracellular 90 kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action”, Bioscience, Biotechnology, and Biochemistry, 56(7): 1166–1168, (1992).
44. [44] Hussain, F., Kamal, S., Rehman, S., Azeem, M., Bibi, I., Ahmed, T., and Iqbal, H.M.N., “Alkaline protease production using response surface methodology, characterization and industrial exploitation of alkaline protease of Bacillus subtilis”, Catalysis Letters, 147: 1204–1213, (2017).
45. [45] Devanadera, M.K.P., Haw, S.V.O., Arzaga, M.J.J., Buenaflor, L.J., Gagarin, T.J.E., Vargas, A.G., Mercado, S.M., and Santiago, L.A., “Optimization, production, partial purification and characterization of neutral and alkaline proteases produced by Bacillus subtilis”, Journal of Microbiology, Biotechnology and Food Sciences, 6(2): 83–838, (2016).