Cloning and expression of thermostable pullulanase gene from Thermococcus siculi HJ21 and its thermostability protectant

Pullulanase is a starch debranching enzyme that hydrolyzes α-1, 6-glucoside bond, which can improve the glycosylation rate of starch. In this study, the super thermophilic pullulanase Pul-HJΔ782 gene of Thermococcus siculi HJ21 was cloned into the plasmid pColdⅠwith cold shock promoter cspA and induced to express in host Escherichia coli BL21, and its enzymatic properties were investigated. The formula of thermal stability protectant of the enzyme was optimized by single factor and response surface tests, and the effect of the protectant on hydrolysis of potato starch was studied. The results showed that the optimum temperature and pH of the enzyme were 90 ℃ and 6.5 ℃, respectively, and the stability was good in the range of 80-100 ℃ and pH 4.0-9.0. The optimal formula of the thermal stability protectant was Ca²⁺ addition 0.15 mol/L, glycerol addition 5.4%, Tween 20 addition 3.8 ml/L, and gelatin addition 4.0 g/L. Under the optimized conditions, the enzyme activity could be retained 97.43% after holding at 90 ℃ for 12 h, which was 34.94% higher than that of the control group. The glycosylation rate of the enzyme was significantly improved, and the glucose-equivalent (DE) value of potato starch for 40 h was 58.35%, which was 11.78% higher than that of control group.