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Paper   IPM / Biological / 13286
School of Biological Sciences
  Title:   Improving chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis
1.  M.Vahed.
2.  E.Motalebi.
3.  G. Rigi.
4.  K. Akbari Noghabi.
5.  M. Soudi.
6.  M. Sadeghi.
7.  G. Ahmadian.
  Status:   Published
  Journal: Journal of Microbiol Biotechnol
  No.:  11
  Vol.:  23
  Year:  2013
  Pages:   1519-1528
  Supported by:  IPM
Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30

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