Characterization of Thermostable and Acidophilic Type II Pullulanase from Geobacillus thermoleovorans NP1 and Its Industrial Applications

This research aimed to produce and characterize a thermophilic and an acidic pullulanase and determine the industrial areas of usage of this enzyme. Pullulanase producing Geobacillus thermoleovorans NP1 (NCBI accession number: MF355416) was isolated from soil samples and screened for pullulanase activity on agar medium containing pullulan (pH 7.0) at 55°C. The highest enzyme production occurred at 45°C and pH 7.0 for 12 h. The supernatant enzyme exhibited its maximal activity at 50°C and pH 6.0. It was highly stable between pH 3.0-12.0 for 24 h and 30-100°C for 60 min. The pullulanase was treated with two each concentration of various metal ions, chelators, detergents and inhibitors. The stability of the enzyme was saved at 5 and 10 mM concentrations of Ca2+, Mg2+, Cu2+, Zn2+, and Ba2+. It showed 145% activity with β-mercaptoethanol (%1). In the presence of 5 mM and 10 mM EDTA, NA1 pullulanase exhibited 92% and 101.3% activities, respectively. Each concentration of PMSF (5 and 10 mM) did not affect the enzyme stability. The enzyme activity was measured as 86.3% with 5% SDS. NP1 pullulanase maintained its initial activity with TritonX-100, Tween20 and Tween80. The native NP1 pullulanase is composed of two active subunits of 112 and 107 kDa. The hydrolysis products of pullulan and starch determined by thin layer chromatography analysis are glucose, maltose, maltotriose, and maltodextrin. This shows that NP1 pullulanase is a type II pullulanase (amylopullulanase). According to the wash performance tests, the mix of pullulanase and 1% commercial detergent removed nearly all of the stains (food gravy, chocolate, baby food). In conclusion, type II pullulanase producing by Geobacillus thermoleovorans NP1 is an acidic-neutral, a thermostable enzyme and resistant to some ions, chelators and detergents. Because of its thermo-acidic activity, it can be used in starch (liquefaction/saccharification), food and detergent industry