Thermococcus siculi HJ21高温普鲁兰酶基因的克隆表达及其热稳定性保护剂研究

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

普鲁兰酶是水解α-1,6糖苷键的淀粉脱支酶,可提高淀粉制糖的糖化率。该研究将古菌Thermococcus siculi HJ21的超嗜热普鲁兰酶Pul-HJΔ782基因克隆在冷休克启动子cspA的质粒pColdⅠ,在宿主大肠杆菌(Escherichia coli)BL21中诱导表达,研究其酶学性质,通过单因素和响应面试验优化该酶的热稳定性保护剂,并考察保护剂对酶水解马铃薯淀粉的影响。结果表明,该酶的最适作用温度和pH分别为90℃和6.5,在温度80~100℃及pH 4.0~9.0范围稳定性良好。最佳热稳定性保护剂配方为Ca^(2+)添加量0.15 mol/L、甘油添加量5.4%、Tween 20添加量3.8 mL/L、明胶添加量4.0 g/L。在该优化条件下,90℃保温12 h后,酶活保留率为97.43%,比对照组提高了34.94%。该酶的糖化率显著提高,对马铃薯淀粉糖化40 h葡萄糖当量(DE)值为58.35%,比对照组比提高了11.78%。

Pullulanase is a starch debranching enzyme that hydrolyzesα-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 Ca^(2+) 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.