Improvement of Bacillus strains by mutation for overproduction of exopolygalacturonases

Abstract

Pectinases, produced by microorganisms, have wide range application in food industry, textile processing, paper making, coffee and tea fermentation, etc. It accounts for 10% of the global industrial enzymes produced. The most important and widely used commercial pectinase polygalacturonase is produced by alkalophilic strains of Bacillus sp. and Streptomyces sp. Here, we explored 29 bacterial strains isolated from rotten mango samples for polygalacturonase production and selected 16 strains through preliminary screening by well-plate method for enzyme activity. The maximum zone of inhibition of pectin was observed up to 28 mm in diameter but one strain ZM11 was exhibiting no activity. Quantitative dinitrisalicylic acid (DNS) assay for polygalacturonase enzyme was also performed for the selected bacterial isolates. All the strains bestowed significant enzyme activity with the highest activity of 2.4 U/µL exhibited by strain ZM3 (P ≤0.05). Characterization of the isolates was performed using different biochemical tests which also confirmed the isolates as members of the genus Bacillus. Mutation was induced to the selected strains by UV light and acridine orange for different periods of time. Qualitative and quantitative assays of the mutant bacterial isolates showed that the enzyme activity increased to 4.62 U/µL which clearly indicated that induced mutation enhanced the ability of Bacillus strains to produce more polygalacturonase enzyme up to 3-fold as compared to the wild strains (P ≤0.05). Molecular characterization by 16S rRNA sequences further confirmed that the bacterial isolates belong to Bacillus subtilis and B. amyloliquefaciens.