低温木糖苷/阿拉伯呋喃糖苷酶 AX543的基因克隆及性质研究

摘要

旨在获得在低温条件下具有高催化能力的低温木糖苷酶，并对其进行异源表达研究和酶学性质分析。利用 Touch down PCR 和 TAIL PCR 方法，从枝顶孢菌中克隆得到一个序列新颖的 GH43家族双功能木糖苷酶/阿拉伯呋喃糖苷酶基因 ax543，该酶基因在毕赤酵母中成功表达。酶学性质分析发现重组酶 AX543的最适温度为25℃，在15℃和4℃仍有54%和21%的相对酶活；具有木糖苷酶和阿拉伯呋喃糖苷酶活性，并可以降解木二糖、木三糖、桦木木聚糖、榉木木聚糖和小麦阿拉伯木聚糖；可以与木聚糖酶 Xyn11-1协同作用，协同度达1.46；具有高木糖/阿拉伯糖耐受性，抑制常数 Ki 分别为84.78 mmol/L 和54.01 mmol/L。%This work aims to obtain the cold-active xylosidase with high catalytic activity at low temperature,and to study its heterologous expression,as well as analyze its enzymatic characteristics. A novel psychrophilic bifunctional xylosidase/arabinofuranosidase gene ax543 in GH43 family was cloned using Touch down PCR and TAIL PCR,and was successfully expressed in Pichia pastoris. Analysis of biochemical characterization suggested that recombinant enzyme AX543 exhibited optimal activity at 25℃,and remained 54% and 21%relative activities at 15℃ and 4℃. Moreover,AX543 with xylosidase and arabinofuranosidase activity hydrolyzed xylobiose,xylotriose, birchwood xylan,beechwood xylan,and wheat arabinoxylan,and showed a 1.46 fold synergic effect with xylanase Xyn11-1. AX543 also presented xylose and arabinose tolerance with Ki 84.78 mmol/L and 54.01 mmol/L.