Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp SH3

A pullulanase-encoding gene from psychrotrophic Exiguobacterium sp. SH3 was cloned and expressed in both E. coli and Bacillus subtilis. The functional recombinant enzyme (Pul-SH3) was purified as a His-tagged protein. Pul-SH3 was characterized to be a cold-adapted type I pullulanase with maximum activity at 45 A degrees C. Using fluorescence spectroscopy, the melting temperature of Pul-SH3 was determined to be about 52 A degrees C. The enzyme was able to hydrolyze pullulan, soluble starch, potato starch, and rice flour. It was exceptionally tolerant in the pH range of 4-11, exhibiting maximum activity at pH 8.5 and more than 60 % of the activity in the pH range of 5-11. Being a detergent-tolerant pullulanase, Pul-SH3 retained 99, 89, and 54 % of its activity at 10 % concentration of Triton-X100, Tween 20, and SDS, respectively. The enzyme also exhibited an activity of 80.4 and 93.7 % in the presence of two commercial detergents, Rika (7.5 % v/v) and Fadisheh (2.5 % w/v), respectively. The enzyme was even able to remain active by 54.5 and 85 % after 10-day holding with the commercial detergents. Thermal stability of the enzyme could w on silica. Pul-SH3 with several industrially important characteristics seems desirable for cold hydrolysis of starch.