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Gene cloning, functional expression and characterisation of a novel type I pullulanase from Paenibacillus
Jingjing Liu1, Yu Liu1, Feng Yan2
1College of Food Science and Nutritional Engineering, Beijing Advanced and Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing 100083, China.
Abstract:
A novel pullulanase gene (PbPulA) from Paenibacillus barengoltzii was cloned. PbPulA has an open reading frame of 2028 bp encoding 675 amino acids. It was heterologously expressed in Escherichia coli as an intracellular soluble protein. The recombinant pullulanase (PbPulA) was purified to homogeneity with a molecular mass of about 75 kDa on SDS-PAGE. PbPulA was optimally active at pH 5.5 and 50 °C. It was stable within pH 5.5-10.5. The enzyme exhibited strict substrate specificity towards pullulan, but showed relatively low activity towards amylopectin and no activity towards other tested polysaccharides. The Km and Vmax values of the enzyme on pullulan were 2.94 mg/mL and 280.5 μmol/min/mg, respectively. The addition of PbPulA in gelatinized rice and maize starches significantly increased the resistant starch type 3 (RS3) yields. Final yields from rice and maize starches were 10.82 g/100 g and 11.41 g/100 g, respectively. These properties make PbPulA useful in starch industries.
