title: Improving the chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.
abstract: 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% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Vahed
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Motalebi
authors.forename: Garshasb
authors.initials: G
authors.lastname: Rigi
authors.forename: Kambiz
authors.initials: K
authors.lastname: Akbari Noghabi
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Soudi
authors.forename: Mehdi
authors.initials: M
authors.lastname: Sadeghi
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Ahmadian
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D060847:Metabolic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009608:Nitrous Acid / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014466:Ultraviolet Rays
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases; D009608:Nitrous Acid