Cloning, expression and characterization of a thermostable pullulanase from newly isolated thermophilic Geobacillus sp. LM14–3

A novel strain LM14-3 producing thermostable pullulanase was separated from Lunma hot spring of Yunnan Province of China. According to colony morphology and 16S rRNA sequence, this strain was named as Geobacillus sp. LM14-3. The thermostable pullulanase with optimal pH 6.0 and optimal temperature 60°C was accumulated in the fermentation broth of Geobacillus sp. LM14-3. The corresponding pullulanase gene, designated as pul-3, was obtained through PCR. The domain structure of the pullulanase was consistent with that of pullulanase I according to sequence similarity. Pul-3 was expressed in E. coli. Maltotriose is the unique hydrolysis product of pullulan determined by HPLC, which confirmed its high specificity for alpha-1,6-glycosidic bond. The pullulanase reported previously usually lost their activity under high temperature, which was difficult to satisfy the demand of biotechnological applications, such as sugar making, detergent, etc. The thermostable pullulanase found in this study owned excellent thermal stability over a wide temperature span, and a half-life as long as 56 h at 60°C, which favors the potential industrial application.