Isolation, Screening and Characterization of Pullulanase producing Bacillus subtilis

Abstract

Agro-waste is produced in large quantities in a country like India and contains starch as one of its main ingredients. This waste material can be recycled to create new commodities that are marketable. Currently, the amylase enzyme is mostly used for saccharification of starch, although this process takes time. Pullulanase enzyme plays an economically significant role in the solution because it quickly saccharifies starch with a high yield of glucose or maltose. In the current investigation, bacteria, fungi and actinomycetes were isolated from different soil samples and food waste and Organisms were grown in enriched media supplemented with 1% starch, 1% glucose, and 1% sucrose added, respectively. A total of 57 bacteria, 10 fungi, and 5 actinomycetes were isolated and qualitatively screened using the agar plate screening method on pullulan containing media for the pullulanase enzyme. Eight bacterial isolates were selected based on maximum pullulan consumption zone, which were then quantitatively screened for the pullulanase enzyme in liquid medium using pullulan as a substrate. After 24 hours of incubation, bacterial culture (NP 7) produced the maximum pullulanase activity (33.33 U/mL). Furthermore, the strain that produced the highest amount of activity was characterized using biochemical and molecular techniques. The isolates were identified as Bacillus subtilis. Keywords: agro waste, amylase, Bacillus subtilis, enriched media, Pullulan, Pullulanase, Saccharification