β-xylosidase (EC 3.2.1.37) is an important part of the xylanolytic enzymes system. In the present research, β-xylosidase gene Sxa derived from Selenomonas ruminantium was expressed in Pichia pastoris GS115. According to the codon bias and rare codons of P. pastoris, mRNA secondary structure and GC content, Sxa gene was optimized. The optimized full-length gene mSxa was obtained by gene synthesis technique and the recombinant yeast expression vector pPIC9K-mSxa was constructed. After being digested by restriction enzyme BglⅡ, the mSxa gene was transformed into P. pastoris GS115. Then, phenotype and geneticin G418 resistance screening, and PCR were adopted to identify the positive transformants. Finally, the recombinant P. pastoris GS115-pPIC9K-mSxa was obtained. Based on enzymatic activity assay, a high-level expression clone was picked up and then the enzymatic characteristics of the recombinant β-xylosidase were studied. The results showed that the molecular weight of the mSxa expressed in P. pastoris G115 was about 66 kDa. The maximum activity was achieved 287.61 IU/mL at fermenter level. Enzymatic characterization showed the β-xylosidase was stable between 40 ℃ and 60 ℃, and pH between 5.0 and 7.0. The optimal reaction temperature and pH were 55 ℃ and 6.0, and preferentially degrading the β-xylose glycosidic bond. The enzymatic activity was activated by Mn²⁺ and Ca²⁺, and inhibited by Fe³⁺, Cu²⁺, Co²⁺, Mg²⁺, EDTA and SDS. The study indicates that the modified β-xylosidase gene mSxa from Selenomonas ruminantium can express successfully with high activity in P. pastoris. The study lays a foundation for further industrial application of the β-xylosidase.
β-木糖苷酶 (β-xylosidase，酶编号EC 3.2.1.37)是木聚糖降解酶系中的重要组成部分。本研究以毕赤酵母Pichia pastoris GS115 为宿主菌尝试表达反刍兽月形单胞菌Selenomonas ruminantium 中的β-木糖苷酶基因Sxa。根据毕赤酵母对密码子的偏爱性、mRNA 二级结构、GC 含量和稀有密码子，对Sxa 基因进行优化；通过基因合成技术获得了全长基因mSxa 并构建重组酵母表达载体pPIC9K-mSxa；以Bgl Ⅱ酶切重组载体pPIC9K-mSxa，电击转化将mSxa 基因导入毕赤酵母GS115 中，获得的转化子经过表型和遗传霉素G418 抗性筛选、PCR鉴定，得到表达β-木糖苷酶基因的工程菌GS115-pPIC9K-mSxa；通过活性测定获得高效表达β-木糖苷酶的重组酵母，并对重组β-木糖苷酶的酶学性质进行了初步研究。结果表明，重组β-木糖苷酶的分子量约为66 kDa。在发酵罐水平表达的酶活性达到了287.61 IU/mL。对酶学性质研究显示，该酶在温度为40−60 ℃，pH 为5.0−7.0 时较稳定，其最适反应温度和pH分别为55℃和6.0，专一性地作用于β-木糖苷键。Mn²⁺和Ca²⁺对该酶具有激活作用，而Fe³⁺、Cu²⁺、Co²⁺、Mg²⁺、EDTA及SDS抑制其酶活性。本研究首次将反刍兽月形单胞菌的β-木糖苷酶基因转化到毕赤酵母中获得表达，并具有较高活性，为进一步工业化应用奠定了基础。.
Keywords: Pichia pastoris; Selenomonas ruminantium; enzymatic characteristics; heterologous expression; β-xylosidase.