Isolation, characterization, and cloning of thermostable pullulanase from Geobacillus stearothermophilus ADM-11

Abstract

This study aimed to identify thermo-stable pullulanase-producing bacteria in soil samples of potato fields and food-producing companies. Pullulan agar medium was used to screen 17 bacterial strains, which were incubated at 65 °C. The isolate with the maximum activity (375U/ml) was selected and recognized as Geobacillus stearothermophilus ADM-11 by morphological, biochemical characterization, and 16S rRNA gene sequencing. The pullulanase production required optimum pH of 7 and temperature of 75 °C, respectively. The electrophoresis of purified pullulanase on SDS-polyacrylamide gel revealed 83 kDa of a molecular weight that is active at 70 °C and pH 7.0. It was also stable at 90 °C but its activity was decreased by 10 % at 100 °C. The action of pullulanase was increased and stabilized by Ca⁺² among the metal ions. Beta and gamma-cyclodextrins inhibited enzyme activity while ethylenediaminetetraacetate (EDTA) and phenylmethylsulfonyl fluoride (PMSF) have no significant effect on pullulanase activity. A full-length pullulanase gene was amplified from G. stearothermophilus ADM-11 using genomic DNA 2.1 kb of PCR product which was then purified and ligated in the cloning vector pTZ57R using the TA cloning technique. Colony PCR confirmed cloning on the positive clones after the pullulanase gene had been ligated and subjected to restriction digestion. It revealed 74 % similarity with the reported pullulanase gene from Geobacillus sp. 44C. The thermostability of pullulanase and its ability to degrade raw pullulan may therefore have wide-scale applications in starch processing, the detergent business, and new biotechnological applications.