Gene cloning and enzymatic characterization of alkali-tolerant type I pullulanase from Exiguobacterium acetylicum.

Abstract

A pullulanase gene (Pul3YH5) of 2568bp, which encodes a protein containing 855 amino acid residues, was cloned from the alkaliphilic bacterium Exiguobacterium acetylicum YH5. The pullulanase (Pul3YH5) contains the YNWGYDP motif of type I pullulanase as well as four conserved glycoside hydrolase sequences of the GH13 (α-amylase) family. When the pullulanase gene was cloned and expressed in Escherichia coli BL21 (DE3) plysS, the recombinant pullulanase had a molecular mass of ˜100·0kDa. It was optimally active at 50°C and pH 6·0, alkali-tolerant and displayed excellent stability (>93·0%) over a broad pH range (4·0-10·0) when incubated for 30min without substrate. The enzyme activity of Pul3YH5 was significantly enhanced in the presence of Co(2+) , Fe(2+) and Mn(2+) and was inhibited by Cu(2+) , SDS, β-mercaptoethanol and EDTA. The enzyme displayed the highest specificity forpullulan (Km =0·12±0·02mgml(-1) ), followed by soluble starch (Km = 0·69±0·04mgml(-1) ). Substrate hydrolysis demonstrated that pullulanase from E.acetylicum liberated maltotriose from pullulan, although hydrolytic activity was also detected with soluble starch, amylopectin, β-limited dextrin and glycogen. These enzymatic properties indicate that Pul3YH5 is alkali-tolerant pullulanase and that Pul3YH5 could be useful in the detergent industry. Pullulanases have great potential in various industries, ranging from food (high fructose and glucose syrups) to washing detergent industries. In this study, the gene encoding the novel pullulanase from E.acetylicum YH5 was cloned and sequenced, then expressed in E.coli. The properties of the recombinant enzyme in E.coli were also determined. The pullulanase from E.acetylicum YH5 is alkaline tolerant and has a high optimum temperature, making it a candidate for applications in the detergent industry.