Isolation and molecular identification of α-amylase producing bacteria from hot water spring by 16S rRNA gene sequencing

ABSTRACT

Among different types of microbial enzymes, amylases are the most widely used in industries as they are produced in large quantity and in an economic way as compared to plants and animals. Moreover, thermostable amylase has significance as compared to the amylase from mesophiles. Therefore, hot water springs are explored to dig into its bacterial diversity. In the current study, we tried to isolate amylase producing bacteria from the soil and water samples collected from the hot water spring in Rampur, Himachal Pradesh, India. The samples were serially diluted before plating on the Luria Bertani agar plates. A total of 42 bacterial morphotypes were isolated and were screened for amylolytic activity by starch agar plate method. Among the 42 bacterial isolates 25 showed amylolytic activity. Production of amylase was carried out at different temperatures and pH to optimize the temperature and pH conditions for each isolate. All the 25 isolates were characterized based on morphology, biochemical tests and molecular analysis. Sequencing of 16S rRNA gene for the 25 isolates followed by BLAST search revealed a majority of them (19) identified as Bacillus licheniformis. Other isolates were identified as B. subtilis, B. safensis, B. halodurans, B. stratosphericus, Caldimonas hydrothermale and Exiguobacterium mexicanum. An attempt was made to amplify amyN gene which codes for α-amylase but successful amplification was achieved only from Bacillus licheniformis alone.