饲料研究2024，Vol.47Issue(5)：82-88,7.DOI:10.13557/j.cnki.issn1002-2813.2024.05.016
热纤梭菌耐热葡聚糖外切酶的克隆表达及酶学特性分析
Cloning,expression and enzymatic properties of thermostable exoglucanase from Clostridium thermocellum
摘要
Abstract
The aim of this study was to develop heat-tolerant cellulase enzymes to improve the utilization rate of cellulosic feed and the economic benefits of livestock and poultry.The sequence information of an exonuclease CelS from Clostridium thermocellum was obtained from the protein information database UniProt,and the overexpression of CelS was achieved by signal peptide deletion and codon optimization,and the expression system of Escherichia coli was used.Through the optimization of self-induced expression conditions,the proportion of soluble expression of CelS was effectively increased.The intracellular supernatant was separated and purified by nickel ion affinity chromatography and heat treatment to obtain soluble CelS,and the intracellular precipitate was washed,denatured and renatured to obtain inclusion body renatured CelS.The enzymatic characteristics showed that there was no significant difference in the enzyme activities of the two forms of CelS,the optimal reaction temperature was 70 ℃,the optimal reaction pH was 5.5～6.0,and the activity of amorphous cellulose(PASC)was the highest,followed by crystalline cellulose(Avicel)and corn stover(CS).CelS and EG12B synergistically hydrolyzed CS with Thermotoga maritima,which increased the activity of hydrolase by 81.8％compared with hydrolase alone.The results showed that recombinant CelS exhibited superior heat tolerance and cellulose hydrolysis activity,which provided strong support for the further development of enzyme preparations,and was conducive to improving the utilization rate of cellulose feed.关键词
热纤梭菌/葡聚糖外切酶/可溶表达/包涵体/酶学特性Key words
Clostridium thermocellum/exoglucanase/soluble expression/inclusion body/enzymatic properties分类
农业科技引用本文复制引用
王珊珊,刘阳,任艳艳,王令,路宏朝,张涛..热纤梭菌耐热葡聚糖外切酶的克隆表达及酶学特性分析[J].饲料研究,2024,47(5):82-88,7.基金项目
陕西省教育厅重点科学研究计划项目(项目编号:20JY006) （项目编号:20JY006）
