Screening Streptomycin Resistant Mutations from Gamma Ray Irradiated Bacillus subtilis B5 for Selection of Potential Mutants with high Production of Protease
Main Article Content
Abstract
Abstract: The suspensions of Bacillus subtilis B5, a rather high protease production strain, in logarithmic growth phase were irradiated under gamma Cobalt-60 source at Hanoi Irradiation Center. After treatment, the irradiated cells were intermediately cultured in the nutrient agar plates supplemented without and with 20µg/ml streptomycin for screening. The radiation effects on their viability and mutant frequency were studied with radiation dose. The results showed that its survival rate was reduced with the dose as biphasic function. The cells irradiated at dose higher than 1200 Gy do not form colony in the medium containing streptomycin though they could survive in nutrient agar. Therefore, potential streptomycin resistance mutations were collected as survivals from the cells irradiated with radiation dose ranging from 100 to 1000 Gy. Within this dose range, mutation frequency of Bacillus subtilis B5 increased with the rising dose. The greatest mutation frequency was determined as 1.61´10-3 obtained by irradiation at 1000 Gy, and the smallest as 3.09´10-6 at 100 Gy. The enzyme activities of 361 screened colonies from all irradiated samples were investigated in casein agar, and the results revealed 25 colonies having protease activity higher than parent strain.
Keywords: Bacillus subtilis, gamma irradiation, streptomycin, survival, mutation frequency, protease.References
[2] R.Gupta, Q.K.Beg, P. Lorenz, Bacterial alkaline proteases: molecular approaches and industrial applications, Appl Microbiol Biotechnol 59 (2002) 15.
[3] S. Li, X. Yang, S. Yang, M. Zhu, X. Wang, Technology prospecting on enzymes: Application, marketing and engineering, Comp struct biotech J 2(3) (2012) 1.
[4] W.H. Chu, Optimization of extracellular alkaline protease production from species of Bacillus, J Ind Microbiol Biotechnol 34 (2007) 241.
[5] R. Gupta, Q.K. Beg, P. Lorenz, Bacterial alkaline proteases: molecular approaches and industrial applications, Appl Microbiol Biotechnol 59 (2002) 15.
[6] A.Kodym, R. Afza, Physical and chemical mutagenesis, Methods in Molecular Biology 236 (2003) 189.
[7] C. Von Sonntag, The Chemical basic of radiation biology, Taylor & Francis (1987) New York, USA.
[8] A.Y.Kim, D.W.Thayer, Mechanism by which gamma irradiation increases the sensitivity of Samonella typhimurium ATCC 14028 to heat, Appl Environ Microbiol 62 (1966) 1759.
[9] S.D.Awan, N.Tabbasam, N.Ayub, M.E.Babar, M.Rahman, S.M.Ran, M.I. Rajoka, Gamma radiation induced mutagenesis in Aspergillus niger to enhance its microbial fermentation activity for industrial enzyme production, Mol Biol Rep 38 (2011) 1367.
[10] K.Ochi, T. Hosaka, New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters, Appl Microbiol Biotechnol 97 (2013) 87.
[11] K. Ochi, From microbial differentiation to ribosome engineering, Biosci Biotechnol Biochem 6 (2007) 1373.
[12] K.Kurosawa, T.Hosaka, N.Tamehiro, T.Inaoka,K. Ochi, Improvement of amylase production by modulating the ribosomal component S12 protein in Bacillus subtilis168, Appl Environ Microbiol 72 (2006) 71.
[13] Z.S.Tawfik, H.H.Swailam, M.A.Sayed, S.M. EL-Sonbaty, Effect of Gamma Irradiation and Culture Conditions and Media Composition on Metallothionein Production by Bacillus pantothenticus, 2nd International Conference on Radiation Sciences and Applications, Marsa Alam Egypt (2010).
[14] S.Yazdi, A.M. Ardekani, Bacterial aggregation and biofilm formation in a vortical flow, Biomicrofluidics 6 (2012) 044114.
[15] K.H.Yoon, S.In-Kyung, H.J.Kyung, P. Seung-Hwan, Hyper-CMCase-producing mutants of Bacillus sp. 79-23 induced by gamma-radiation, J Microbiol Biotechnol 9(4) (1999) 518.
[16] L.GuiJun, M.You-ting, Y.Su-ling, B.Fang, S.Hong-zhong, Study on γ-ray irradiation mutation of Bacillus subtilis NCD2, Agricultural Science & Technology 12(11) (2011) 1633.
H. Afsharmaesh, M.Ahmadzadeh, M.Javan-Nikkhah, K.Behboudi, Improvement in biocontrol activity of Bacillus subtilis MTB1 against Aspergillus flavus using gamma irradiation, Crop Protection 60 (2014) 83.