The amylolytic enzyme plays a very important role in industrial applications. This study aimed to screen amylase producing Bacillus sp. and to promote its amylolytic activity by mutagenesis. Samples were collected from coastal mud samples and starch hydrolyzing isolates were screened. A single isolate having the highest enzyme activity was identified as Bacillus tequilensis by 16S rRNA analysis. A starch medium was optimized and fermentation period studies revealed that the mutant strain (after 60 sec of UV exposure) had higher activity (868 U/mL/min) than the parental strain (418 U/mL/min) after 36 hours of incubation at 37°C, pH 7.0. It was also found that amylase from intracellular mutant strain had maximum activity; on the other side parental strain had maximum activity with an extracellular enzyme. Optimized temperature, pH and salt concentration revealed that the intracellular amylase from mutant strain had the maximum activity of 978 U/mL/min, 985 U/mL/min, 960 U/mL/min respectively. Varying the source of carbon in the medium had a significant impact on enzyme activity. Metalloenzymes like amylases were reported to have strong activity towards metal ions, so amylase activity was analysed by adding different metal ions in the medium and found that calcium ions strongly promoted amylase activity and Fe2+, Zn2+, Cu2+, Mg2+ inhibited the activity. SDS-PAGE results showed that the molecular weight of isolated amylase to be approximately 55.0 kDa. Our study showed the capability of mutant B. tequilensis strain to produce double the amount of intracellular amylase than the parental strain.
Amylolytic, mutagenesis, metalloenzymes, metal ions, zymogram