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

研究旨在开发耐热纤维素酶,以提高畜禽对纤维素饲料的利用率和养殖经济效益。从蛋白质信息数据库UniProt中获取热纤梭菌(Clostridium thermocellum)葡聚糖外切酶CelS的序列信息,经信号肽删除和密码子优化,利用大肠杆菌表达系统,实现了CelS的过量表达。通过自诱导表达条件优化,有效提高了CelS可溶表达的比例。将胞内上清液通过镍离子亲和层析和热处理分离纯化获得可溶CelS,将胞内沉淀通过洗涤、变性和复性后获得包涵体复性CelS。酶学特性检测发现,两种表达形式CelS的酶活力无显著性差异,最适反应温度均为70℃,最适反应pH值为5.5~6.0,对无定形纤维素(PASC)的活性最高,对结晶纤维素(Avicel)和玉米秸秆(CS)次之。CelS与海栖热袍菌(Thermotoga maritima)葡聚糖内切酶EG12B协同作用水解CS,比单独水解酶活力提高了81.8%。试验结果表明,重组CelS表现出优越的耐热性能和纤维素水解活性,为进一步开发酶制剂提供了有力支持,有利于提高纤维素饲料的利用率。

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.