Abstract:
This research aimed at isolating and screening bacterial isolates associated with the gut of worker termites, Coptotermes formosanus for pullulanase production. The guts of the termites were mashed and the bacteria were isolated and identified by standard microbiological techniques. The identified bacterial isolates were initially screened for quantitative pullulanase production in mineral salt medium containing pullulan as the sole carbon source. Pullulanase activity and protein concentration in the enzyme production medium were determined by dinitrosalicylic acid and Bradford methods, respectively. The purification of the crude enzyme from the best pullulanase-producing bacterium was carried out by ammonium sulphate precipitation, ion-exchange chromatography and gel-filtration on DEAE Sephadex A-50 and Sephadex G-100, respectively and characterized. The molecular weight of the purified pullulanase was estimated by dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using a gel electrophoresis system. The bacterial isolates were tentatively identified and classified into genera Bacillus, Enterobacter, Escherichia and Staphylococcus. The identities of the best three pullulanase-producing bacterial strains were authenticated as Bacillus cereus, B. safensis and Psychrobacter pulmonis using 16S rRNA Gene Sequence Analysis. The purified pullulanase activities from B. safensis were optimal at pH 7.0 and 60 oC. The purified pullulanase was 100% relatively stable for 30 min at pH 11.0 and retained 93% of its residual activity at pH 5.0 for 18 hours of incubation. The enzyme was approximately 88% thermally stable for 30 min at 40 oC and retained 46% of its residual activity after 180 min of incubation. The molecular weight of the purified pullulanase was estimated to be 42.34 kDa. The apparent Km and Vmax of purified pullulanase were 6.85 mg/ml and 0.32 μmol/min/ml. The activities of the enzyme were stimulated by some of the metal ions and enzyme inhibitors such as SDS and urea. Novel chemical properties such as thermal, acid and alkaline stability, and the ability to withstand some enzyme inhibitors exhibited by B. safensis resident in the gut of worker termites suggest its industrial significance.
