Year 2020, Volume 48 , Issue 4, Pages 355 - 365 2020-07-06

Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions

Elif DEMİRKAN [1] , Kübra ÖZDEMİR [2]


In this study is aimed the improvement of Bacillus subtilis E6-5 for high protease production by EMS treatment as chemical mutagen. The 82 mutants were screened on skim milk agar plates and, the mutant with maximum protease activity was named KE20. The mutant was quantitatively tested, and showed 9.2 times more protease activity than the parental strain. The effects of nutritional and physical factors on the protease production of mutant KE20 were investigated. The best carbon and nitrogen sources were corn starch and skim milk. No effect was observed when metal ions were used alone. In the physical parameters, the best results were obtained at 37°C, pH 7.0, 150 rpm, 3% as inoculum size and 18 has inoculum age. A new medium was made by optimizing the incubation conditions and enzyme yield enhanced 79% compared to basal medium. The mutant KE20 strain may have a prominent potential for protease production on an industrial scale.
Bacillus, Protease, EMS Mutagenesis, Optimization
  • Reference1 D.S. Ningthoujam, P. Kshetri, P. Sanasam, S. Nimaichand, Screening identification of best producers and optimization of extracellular proteases from moderately halophilic alkali thermotolerant indigenous actinomycetes, World Appl. Sci. J., 7 (2009) 907–916.
  • Reference2 K.M. Sharma, R. Kumar, S. Panwar, A. Kumar, Microbial alkaline proteases: optimization of production parameters and their properties, J. Genet. Eng. Biotechnol., 15 (2017) 115–126.
  • Reference3 A. Belmessikh, H. Boukhalfa, A. Mechakra-Maza, Statistical optimization of culture medium for neutral protease production by Aspergillus oryzae comparative study between solid and submerged fermentations on tomato pomace, J. Taiwan Inst. Chem. Eng., 44 (2013) 377-385.
  • Reference4 Z. Chi, C. Ma, P. Wang, Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans, Bioresour. Tech., 98 (2007) 534-538.
  • Reference5 E. Bach, V. Sant’Anna, D.J. Daroit, Production one-step purification and characterization of a keratinolytic protease from Serratia marcescens P3, Process Biochem., 47 (2012) 2455–2462.
  • Reference6 F.J. Contesini, R.R. Melo, H.H. Sato, An overview of Bacillus proteases from production to application, Crit. Rev. Biotechnol., 38 (208) 321-334.
  • Reference7 R. Gupta, Q.K. Beg, P. Lorenz, Bacterial alkaline proteases molecular approaches and industrial applications, App. Microbiol. Biotech., 59 (2002) 15-32.
  • Reference8 S. Singh, V. Sharma, M.L. Soni, S. Das, Biotechnological applications of industrially important amylase. Int. J. Pharma. Bio. Sci., 2 (2011) 487-496.
  • Reference9 D.A. Azin, R.F. Noroozi, Effect of chemicals on the improved gluconate productivity by an Aspergillus niger strain, Appl. Biochem. Biotech. Appl. Biochem., 61 (2001) 393-397.
  • Reference10 M. Munazzah, A.J. Khalil-ur-Rahman, A. Muhammad, R. Ibraheem, J. Sadia, J. Nazish, Bacillus subtilis improvement through UV and chemical mutagenesis for indigenously hyperproduced urate oxidase, Pak. J. Life. Soc. Sci., 10 (2012) 123-129.
  • Reference11 O. Ribeiro, F. Magalhaes, T.Q. Aguiar, M. Wiebe, M. Penttila, L. Domingues, Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii, Bioengineered, 4 (2013) 322–331.
  • Reference12 E.A.M. Soliman, W.K. Hegazy, E.M. Maysa, Induction of overproducing alkaline protease Bacillus mutants through UV irradiation. Arab. J. Biotech., 8 (2004) 49-60.
  • Reference13 M. Nadeem, J.I. Qazi, Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing, Braz. Arch. Biol. Technol., 53 (2010) 1015-1025.
  • Reference14 N. Thakur, T.C. Bhalla, D. Kumar, Systemic mutagenesis of Bacillus sp. APR-4 for enhanced production of thermostable and alkaline protease, Biol. Forum., 9 (2017) 54-60.
  • Reference15 E. Demirkan, D. Kut, T. Sevgi, M. Dogan, E. Baygin, Investigation of effects of protease enzyme produced by Bacillus subtilis 168 E6-5 and commercial enzyme on physical properties of woolen fabric, J. Text. I., 111 (2020) 26-35.
  • Reference16 H. Zong, Y. Zhan, X. Li, L. Peng, F. Feng, D. Li, A new mutation breeding method for Streptomyces albulus by an atmospheric and room temperature plasma, Afr. J. Microbiol. Res., 6 (2012) 3154-3158.
  • Reference17 S.A. Quadar, E. Shireen, S. Iqbal, A. Anwar, Optimization of protease production from newly isolated strain of Bacillus sp. PCSIR EA-3. Indian J. Biotechnol., 8 (2009) 286-290.
  • Reference18 L. Keay, B.S. Wildi, Proteinases of the genus Bacillus. I. Neutral proteases, Biotechnol. Bioeng., 12 (1970) 179-212.
  • Reference19 J.R. Dutta, R. Banerjee, Isolation and characterization of a newly isolated Pseudomonas mutant for protease production, Braz. Arch. Biol. Technol., 49 (2006) 37-47.
  • Reference20 B.M. Rao, M.A. Tanksale, S.M. Ghathe, V.V Deshpande, Molecular and biotechnological aspects of microbial proteases, Microbiol. Mol. Biol. Rev., 62 (1998) 597-635.
  • Reference21 I. Haq, S. Ali, M.M. Javed, U. Hameed, A. Saleem, F. Adnan, M.A. Qadeer, Production of alpha amylase from a randomly induced mutant strain of Bacillus amyloliquefaciens and its application as a desizer in textile industry, Pak. J. Bot., 42 (2010) 473-484.
  • Reference22 N. Rakariyatham, B. Butr-Indr, H. Niamsup, L. Shank, Improvement of myrosinase activity of Aspergillus sp.NR4617 by chemical mutagenesis, Electron. J. Biotechn., 9 (2006) 379- 385.
  • Reference23 I.M. Meraz, T. Choudhary, M.M. Hoq, Optimization of mutation conditions of Bacillus sp. to increase the yield of alkaline protease, FEMS Microbial. Lett., 66 (2005) 239- 244.
  • Reference24 Shikha, A. Sharan, N.S. Darmwal, Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate, Bioresour. Technol., 98 (2007) 881-885.
  • Reference25 M. Nadeem, Q. Syed, A. Liaquat, S. Baig, A. Kashmiri, Study on biosynthesis of alkaline protease by mutagenized culture of Bacillus pumilus, Pak. J. Food Sci., 20 (2011) 24-30.
  • Reference26 E.V.N. Raju, and G. Divakar, Bacillus cereus GD 55 Strain Improvement by Physical and Chemical Mutagenesis for Enhanced Production of Fibrinolytic Protease, Int. J. Pharm. Sci. Res., 4 (2013) 81-93.
  • Reference27 M. Hussain, A.K. Chaudhary, H. Bashir, Enhance production of alkaline protease by chemical mutagenesis of Bacillus species, Biocell, 43 (2019) 312-322.
  • Reference28 S. Mrudula, B.A. Apsana, K. Ashwitha, P.K. Pindi, Enhanced Production of Alkaline Protease By Bacillus subtilis in Submerged Fermentation, Int. J. Pharm. Bio. Sci., 3 (2012) 619-631.
  • Reference29 S. Mehrotra, P.K. Gaur, N.S. Darmwai, The production of alkaline protease by a Bacillus species isolate, Bioresour. Technol., 67 (1999) 201- 203
  • .Reference30 S.S. Mabrouk, A.M. Hashem, N.M.A EI-Shhayeb, A.S. Ismail, A.F. Abdel-Fattah, Optimization of alkaline protease productivity by Bacillus licheniformis ATCC21415, Bioresour. Technol., 69 (1999) 155-159.
  • Reference31 H. Mukhtar, I. Haq, Optimization of volume of fermentation medium for the production of alkaline protease by an ems mutant strain of Bacillus subtilis, Pak. J. Bot., 39 (2007) 2705-2715.
  • Reference32 X.W. Leng, Y. Xu, Improvement of acid protease production by a mixed culture of Aspergillus nigerand, Aspergillus oryzaeusing solid state fermentation technique, Afr. J. Biotechnol., 10 (2011) 6824- 6829.
  • Reference33 E. Fawzi, Comparative study of two purified inulinases from Thermophilethielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 grown on Cichorium intybus, Braz. J. Microbiol., 42 (2011) 633-649.
  • Reference34 Y. Muhammad, M. Irfan, Z.U. Khokhar, Q.A. Syed, S. Baig, A. Iqbal, Enhanced production of protease by mutagenized strain of Aspergillus oryzae in solid substrate fermentation of rice bran, Sci. Int. 22 (2010) 119-123.
  • Reference35 T.V. Sivakumar, T. Ramasubramanian, P. Shankar, Screening of keratinolytic bacteria Bacillus cereus from the feather dumping soil of sivakasi, Insight Bacteriol., 1 (2012) 1-6.
  • Reference36 M. Kuddus, Enzymes in Food Biotechnology, production, applications, and future prospects, 1st Edition, Academic Press, San Diego-California, U.S.A., (2018).
  • Reference37 H. Mukhtar, I. Haq, Production of alkaline protease by Bacillus subtilis and its application as a depilating agent in leather processing, Pak. J. Bot., 40 (2008) 1673-1679.
  • Reference38 A. Latif, M. Iqbal, M. Asgher, Ethyl Methane Sulfonate Chemical Mutagenesis of Bacillus subtilis for Enhanced Production of Protease, Org. Med. Chem. Int. J., 5 (2018) 555664.
  • Reference39 J.R. Dutta, P.K. Dutta, R. Banerjee, Optimization of culture parameters for extracellular protease production from a newly isolated Pseudomonas sp. using response surface and artificial neural network models, Process Biochem, 39 (2004) 2193-2198.
Primary Language en
Subjects Engineering
Journal Section Articles
Authors

Orcid: 0000-0002-5292-9482
Author: Elif DEMİRKAN (Primary Author)
Institution: BURSA ULUDAĞ ÜNİVERSİTESİ
Country: Turkey


Orcid: 0000-0002-3750-8292
Author: Kübra ÖZDEMİR
Institution: BURSA ULUDAĞ ÜNİVERSİTESİ
Country: Turkey


Dates

Publication Date : July 6, 2020

Bibtex @research article { hjbc697070, journal = {Hacettepe Journal of Biology and Chemistry}, issn = {2687-475X}, eissn = {2687-475X}, address = {Hacettepe Üniversitesi Fen Fakültesi, 06532, Beytepe/ ANKARA/ TÜRKİYE}, publisher = {Hacettepe University}, year = {2020}, volume = {48}, pages = {355 - 365}, doi = {10.15671/hjbc.697070}, title = {Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions}, key = {cite}, author = {Demirkan, Elif and Özdemir, Kübra} }
APA Demirkan, E , Özdemir, K . (2020). Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions . Hacettepe Journal of Biology and Chemistry , 48 (4) , 355-365 . DOI: 10.15671/hjbc.697070
MLA Demirkan, E , Özdemir, K . "Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions" . Hacettepe Journal of Biology and Chemistry 48 (2020 ): 355-365 <https://dergipark.org.tr/en/pub/hjbc/issue/55838/697070>
Chicago Demirkan, E , Özdemir, K . "Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions". Hacettepe Journal of Biology and Chemistry 48 (2020 ): 355-365
RIS TY - JOUR T1 - Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions AU - Elif Demirkan , Kübra Özdemir Y1 - 2020 PY - 2020 N1 - doi: 10.15671/hjbc.697070 DO - 10.15671/hjbc.697070 T2 - Hacettepe Journal of Biology and Chemistry JF - Journal JO - JOR SP - 355 EP - 365 VL - 48 IS - 4 SN - 2687-475X-2687-475X M3 - doi: 10.15671/hjbc.697070 UR - https://doi.org/10.15671/hjbc.697070 Y2 - 2020 ER -
EndNote %0 Hacettepe Journal of Biology and Chemistry Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions %A Elif Demirkan , Kübra Özdemir %T Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions %D 2020 %J Hacettepe Journal of Biology and Chemistry %P 2687-475X-2687-475X %V 48 %N 4 %R doi: 10.15671/hjbc.697070 %U 10.15671/hjbc.697070
ISNAD Demirkan, Elif , Özdemir, Kübra . "Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions". Hacettepe Journal of Biology and Chemistry 48 / 4 (July 2020): 355-365 . https://doi.org/10.15671/hjbc.697070
AMA Demirkan E , Özdemir K . Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions. HJBC. 2020; 48(4): 355-365.
Vancouver Demirkan E , Özdemir K . Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions. Hacettepe Journal of Biology and Chemistry. 2020; 48(4): 355-365.
IEEE E. Demirkan and K. Özdemir , "Ethyl Methanesulfonate (EMS) Mutation for Hyper Protease Production by Bacillus subtilis E6-5 and Optimization of Culture Conditions", Hacettepe Journal of Biology and Chemistry, vol. 48, no. 4, pp. 355-365, Jul. 2020, doi:10.15671/hjbc.697070