角毛壳菌木聚糖酶基因xyn24在毕赤酵母中的高效表达及产酶的固定化研究

Xynalase Gene Overexpression of Chaetomium cupreum in Pichia pastoris and Immobilization of Enzyme

以生防真菌角毛壳菌(Chaetomium cupreum)菌丝体时期的基因文库为基础,筛选得到编码木聚糖酶基因的片段,经拼接及RT-PCR扩增得到全长690bp的编码β-1,4-木聚糖酶基因序列,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达的木聚糖酶重组载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,表达蛋白分泌到培养基中,分子质量约为19.4 ku;以水溶性壳聚糖为底物测得酶活为2.72 U/mL。SDS及金属离子Mn2+对酶活性具有较强的激活作用;转化子经10代传代培养后酶活性稳定。通过制备的壳聚糖微球对木聚糖酶进行了固定化研究,并与游离酶的性质进行了比较。结果表明:固定化酶的最适pH范围与游离酶相比向酸性方向偏移;固定化酶的最适反应温度、酸碱稳定性及热稳定性均有所提高。

Based on the gene library of the Chaetomium cupreum mycelium, gene fragments encoding xylanase were obtained after screening, total length of 690bp encoding matureβ-1 ,4-xylanase was got through splicing and RT- PCR amplification. The recombinant methods were used to build the expression vector of xylanase in Pichia pastoris. Recombinant protein was highly expressed under the control of the AOX1 gene promoter and secreted into the medium with molecular weight of about 19.4 ku. The activity was 2.72U/mL in the measurement with chitosan as the sub- strate. SDS and metal ions Mn2＋ activated the enzyme. Activity of transformants was stable after 10 generations. Xyla- nase immobilization of the preparation of chitosan micro-spherical and the nature of the free enzyme were compared. The results showed that the optimum pH of the immobilized enzyme shifted to the acidic direction, and the optimum temperature of immobilized enzyme, pH stability and thermal stability were improved.