嗜热木聚糖酶的克隆表达及其在酶解生产低聚木糖中的应用

Cloning and Expression of Thermophilic Xylanase and Its Application in Xylooligosaccharides Production

目的:为了获得一种耐高温的木聚糖酶,并对其生物特性进行表征,使其能够更有效地应用于低聚木糖的制备。方法:首先通过生物信息学分析,确定来自厌氧孢子菌Caldicellulosiruptor saccharolyticus DSM 8903的木聚糖酶(Cs Xyn B)基因序列,然后扩增Cs Xyn B基因,以p ET-20b为载体,构建重组质粒,将其转入大肠杆菌(Escherichia coli)BL21(DE3)中表达,重组酶通过热处理和镍柱亲和层析纯化获得纯酶,将纯酶用于后续酶学性质和降解木聚糖的研究。结果:通过生物信息学方法研究表明,Cs Xyn B属于糖苷水解酶第10家族。Cs Xyn B的最适反应温度为80℃,最适反应p H为6.5。在65℃以内,p H为5.5~7.5的条件下重组木聚糖酶的稳定性较高。以榉木木聚糖为底物时,Cs Xyn B的K_m,V_(max)和K_(cat)值分别为1.8 mg/m L,46.0 U/mg和60.7 s^(-1)。在65℃、p H6.5条件下,以榉木木聚糖为底物,木聚糖酶Cs Xyn B反应1 h,生成的低聚木糖的主要成分为木二糖和木三糖。结论:Cs Xyn B是一种极具特点的耐高温木聚糖酶,可以催化水解木聚糖生成高含量的木二糖和木三糖,在生产低聚木糖的应用中具有潜在价值。

Objective：A thermostable xylanase was obtained and characterized, the results showed that it could be used for preparation of xylo- oligosaccharides （ XOSs ）. Methods ： The xylanase （ CsXynB ） gene from Caldicellulosiruptor saccharolyticus DSM 8903 was analyzed by bioinformatics methods.Then amplified CsXynB gene was cloned into pET-20b.The recombinant plasmid was transformed into Escherichia coli BI21 （ DE3 ） .The recombinant CsXynB was purified by heat treatment and the Ni-NTA chromatography. The purified CsXynB was applied to determinate the enzymatic properties and was used in the production of XOSs. Results： CsXynB was classified into the glycoside hydrolase family 10 （GH10）by the bioinformatics analyses.CsXynB exhibited optimal activity at pH6.5 and 80 ℃.Further, CsXynB was stable up to 65 ℃ and pH values of 5.5 to 7.5.When beechwood xylan was used as substrate, the Km,VmaxandKcat values were 1.8 mg/mL,46.0 U/mg, and 60.7 s-L respectively.Productian of XOSs from beechwood using CsXynB was investigated. Upon incubation with substrate recombinant CsXynB at pH6.5 and 65 ℃ for 1 h, beechwood xylan was hydrolyzed to XOSs with chain length of 2 and 3.Conclusion ：CsXynB was a new and thermostable enzyme which could be used to produce xylooligosaccharides.