Cloning, overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27

A gene encoding a special type of pullulanase from Thermus thermophilus HB27 (TTHpu) was cloned. It has an open reading frame of 1428 bp encoding a mature protein with a molecular mass of 52 kDa. The gene was expressed in Escherichia coli using pHsh and pET28a vectors. The pHsh expression system produced a 3.6-fold higher recombinant pullulanase than pET28a. The recombinant TTHpu was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The purified TTHpu exhibited highest activity at pH 6.5 and 70 degrees C. More than 90% activity was retained after incubation at 60-70 degrees C for 2 h and the half-life was 2 h at 80 degrees C. The stability of the enzyme was in a pH range from 6.0 to 8.0. Manganese at 5 mM enhanced its activity up to 298%. The K-m and V-max, for the enzyme activity on pullulan were 0.0031 mg mL(-1) and 23.8 mu mol min(-1), respectively. Unlike the most of pullulan-hydrolyzing enzymes described to date, this enzyme can attack alpha-1,6- and alpha-1,4-glycosidic linkages in pullulan, and produce a mixture of maltotriose, maltose and glucose. The enzyme could be further employed for industrial saccharification of starch. (C) 2013 Elsevier Inc. All rights reserved.