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
倪豪 1郝悦 1于轶群 1薛陆州 1郝青芳 1亢新鑫 1王淑军1
作者信息
- 1. 江苏海洋大学海洋科学与水产学院江苏省海洋生物资源与环境重点实验室,江苏连云港 222005;江苏海洋大学海洋科学与水产学院江苏省海洋生物技术重点实验室,江苏连云港 222005
- 折叠
摘要
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-HJ△782基因克隆在冷休克启动子cspA的质粒pCold Ⅰ,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Ca2+添加量0.15mol/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％.
Abstract
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-HJ△782 gene of Thermococcus siculi HJ21 was cloned into the plasmid pCold Ⅰ with cold shock pro-moter 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.
关键词
Thermococcus/siculi/HJ21/普鲁兰酶/基因克隆表达/热稳定性/保护剂Key words
Thermophilic pullulanase HJ21/pullulanase/gene cloning and expression/thermostability/protectant引用本文复制引用
基金项目
国家重点研发计划项目(2022YFC2805101)
出版年
2024
NETL
NSTL
万方数据