Fu Tingting, Hu Wei, Chen Yong, Wei Huan, Yang Guang
College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China.
Sheng Wu Gong Cheng Xue Bao. 2017 May 25;33(5):785-795. doi: 10.13345/j.cjb.160433.
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（EC 3.2.1.37）是木聚糖分解酶系统的重要组成部分。在本研究中，源自反刍月形单胞菌的β-木糖苷酶基因Sxa在毕赤酵母GS115中表达。根据毕赤酵母的密码子偏好性和稀有密码子、mRNA二级结构以及GC含量，对Sxa基因进行了优化。通过基因合成技术获得了优化后的全长基因mSxa，并构建了重组酵母表达载体pPIC9K-mSxa。用限制性内切酶BglⅡ消化后，将mSxa基因转化到毕赤酵母GS115中。然后，采用表型和遗传霉素G418抗性筛选以及PCR来鉴定阳性转化子。最终，获得了重组毕赤酵母GS115-pPIC9K-mSxa。基于酶活性测定，挑选出一个高水平表达克隆，然后研究了重组β-木糖苷酶的酶学特性。结果表明，在毕赤酵母G115中表达的mSxa的分子量约为66 kDa。在发酵罐水平下，最大活性达到287.61 IU/mL。酶学特性表明，β-木糖苷酶在40℃至60℃以及pH值5.0至7.0之间稳定。最佳反应温度和pH分别为55℃和6.0，且优先降解β-木糖苷键。酶活性被Mn²⁺和Ca²⁺激活，被Fe³⁺、Cu²⁺、Co²⁺、Mg²⁺、EDTA和SDS抑制。该研究表明，来自反刍月形单胞菌的修饰后的β-木糖苷酶基因mSxa能够在毕赤酵母中成功表达且具有高活性。该研究为β-木糖苷酶的进一步工业应用奠定了基础。
