A pH-stable, detergent and chelator resistant type I pullulanase from Bacillus pseudofirmus 703 with high catalytic efficiency.

Pullulanase with high catalytic efficiency has attracted great attention from both the academic and industrial communities for its wide application in cold starch hydrolysis. A novel pullulanase gene pul703 was cloned from a mesophilic bacteria Bacillus pseudofirmus 703. Pul703 was characterized to be a type I pullulanase with maximal activity at 45°C and good low-temperature stability, more than 70% of activity was detected after incubation at 25-35°C for 72h. Pul703 obtained the maximal activity around pH 7.0-8.0, and was highly active and stable over a wide pH range of 5.5-9.5, more than 80% of activity was retained after 12h incubation in these pHs. Pul703 was EDTA-resistant and detergent-tolerant, with a relative activity of 100, 99, and 114.8% at the presence of 10mM EDTA, 10% of Triton X-100 and Tween 20, respectively. Pul703 can efficiently hydrolyze pullulan with a specific activity of 270U/mg, which was higher than all reported type I pullulanases. In addition, Pul703 can act synergistically with α-amylase BLA to efficiently hydrolyze amylopectin. These results suggested that Pul703 was a good candidate for cold starch hydrolysis.