Abstract: A pullulanase (named PulBa) from Bacillus amyloliquefaciens HxP-21 was isolated and purified, and its enzymatic properties were studied to provide a theoretical basis for the application of pullulanase in starch processing. The pullulanase was isolated and purified from the fermentation broth of strain HxP-21 by sequential ammonium sulfate precipitation, anion exchange chromatography and dextran gel filtration chromatography. The yield of PulBa was 53.2%, and the procedure resulted in a 20.8-fold purification and a specific enzyme activity of 176.5 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) measured PulBa to be electrophoretically pure, with a molecular mass of 51.2 kDa. PulBa had high enzyme activity in the range of 45–70 ℃ and pH 3–6, with optimum temperature of 55 ℃ and optimum pH of 4.5. PulBa also exhibited good pH stability and thermostability. More than 80% of its initial activity was retained after being incubated at 40–70 ℃ for 120 min. PulBa was highly stable over an acidic pH range of 3-7, and more than 60 U/mL of its activity was retained after 6 h incubation under these pH conditions. PulBa showed different sensitivities to various metal ions and chemical reagents, and Mg2+ and Ca2+ were able to significantly enhance its enzyme activity. The most suitable substrate for PulBa was pullulan, and it also showed hydrolytic activity on potato amylopectin, corn amylopectin, soluble starch and glycogen, but no effect on α-cyclodextrin and β-cyclodextrin and amylose. When pullulan was used as a substrate, its Km and Vmax were 1.34 mg/mL and 24.6 μmol/(min·mg), respectively. Results indicated that PulBa was a typical type I pullulanase. Thin layer chromatography (TLC) further demonstrated that PulBa specifically cleave the α-1,6-glycosidic bonds of amylopectin to produce maltotriose. Therefore, PulBa had high thermostability and acidic pH tolerance, making it a promising candidate for application in biotechnological industries such as starch processing.

Key words: pullulanase; Bacillus amyloliquefaciens; purification; enzymatic characterization