Position: Home > Articles > Efficient Secretory Expression of Thermoacidiphilic Type Ⅲ Pullulan Hydrolase and Its Enzymatic Properties
Science and Technology of Food Industry
2020
(3)
98-103+109
嗜热酸性普鲁兰水解酶Ⅲ的高效分泌表达及其酶学性质
作 者：
曾静;郭建军;袁林
单 位：
江西省科学院微生物研究所
关键词：
普鲁兰水解酶Ⅲ;双功能酶;枯草芽孢杆菌表达系统;分泌表达;酶学性质
摘 要：
本文探索了嗜热酸性普鲁兰水解酶ⅢTk-PUL在枯草芽孢杆菌表达系统中的高效分泌表达条件,并对重组Tk-PUL的酶学性质进行了初步研究。通过构建Tk-PUL分泌表达信号肽筛选库,并结合高通量筛选方法,确定引导Tk-PUL在枯草芽孢杆菌中高效分泌表达的信号肽。结果表明,在信号肽AmyE的引导下,重组Tk-PUL在枯草芽孢杆菌表达系统中高效分泌表达。Tk-PUL属于单结构域双功能酶,同时具有α-淀粉酶活性和普鲁兰酶活性。重组Tk-PUL的α-淀粉酶活性的最适反应pH为4.5,最适反应温度为100℃,对应的绝对酶活为54.08 U/mg,于100℃的半衰期约为2 h。重组Tk-PUL的普鲁兰酶活性的最适反应pH为4.5,最适反应温度为100℃,对应的绝对酶活为110.39 U/mg,于100℃的半衰期约为2 h。本研究为Tk-PUL在淀粉酶法制糖工业中的应用奠定了基础。
译 名：
Efficient Secretory Expression of Thermoacidiphilic Type Ⅲ Pullulan Hydrolase and Its Enzymatic Properties
作 者：
ZENG Jing;GUO Jian-jun;YUAN Lin;Institute of Microbiology,Jiangxi Academy of Sciences;
关键词：
type Ⅲ pullulan hydrolase;;bifunctional enzyme;;Bacillus subtilis expression system;;secretory expression;;enzymatic properties
摘 要：
The conditions for the efficient secretory expression of thermoacidophilic type Ⅲ pullulan hydrolase Tk-PUL in Bacillus subtilis were explored,and the enzymatic properties of recombinant Tk-PUL were preliminarily studied.To identify the signal peptide that guide Tk-PUL efficiently secretory expressed in Bacillus subtilis,a screening library of signal peptides for Tk-PUL was constructed and high-throughput screening method was used. Results showed that,under the guidance of signal peptide AmyE,recombinant Tk-PUL was efficiently secretory expressed in Bacillus subtilis.Tk-PUL was a bifunctional enzyme with a single active site,Tk-PUL possessed both α-amylase and pullulanase activities. The highest α-amylase activity was observed at pH4.5 and 100 ℃,the corresponding specific activity was 54.08 U/mg,and its half-life at 100 ℃ was 2 h. The highest pullulanase activity was observed at pH4.5 and 100 ℃,the corresponding specific activity of 110.39 U/mg,and its half-life at 100 ℃ was 2 h. This study establishes the foundation for the application of Tk-PUL in starch processing industry.
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