黑曲霉β-葡聚糖酶基因eglA6在乳酸克鲁维酵母中的表达及重组酶性质

Expression of β-Glucanase Gene eglA6 of Aspergillus niger in Kluyveromyces lactis and Characterization of Recombinant Enzyme

以黑曲霉(Aspergillus niger)高耐热葡聚糖酶编码基因eglA6在乳酸克鲁维酵母(Kluyveromyces lactis)中的表达及重组酶性质为研究目的。利用乳酸克鲁维酵母表达系统,构建表达eglA6基因的重组菌K.Lactis GG799/pKLAC1-eglA6 SL105,重组菌摇瓶发酵酶活达到23.0 U/mL。重组酶AnEglA6K表观相对分子质量为5.6×10^(4),明显高于同一基因在巴斯德毕赤酵母中的表达产物。重组酶最适pH为5.0,最适温度为75℃,在80℃时酶活半衰期为65.0 min。以微晶纤维素为底物时重组酶比酶活约为0.5 U/mg。基因eglA6在乳酸克鲁维酵母中的表达产物与其在巴斯德毕赤酵母中的表达产物有明显差异,前者相对分子质量明显高于后者,热稳定性和结晶纤维素降解活性也高于后者。以乳酸克鲁维酵母为宿主表达eglA6基因获得的重组葡聚糖酶具有很高的耐热性,适合在饲料工业中应用。

This study aimed to express gene eglA6 from Aspergillus niger,which encoding a highly thermal stable β-glucanase in Kluyveromyces lactis(K.Lactis),and investigated the properties of recombinant enzyme.The recombinant strain K.Lactis GG799/pKLAC1-eglA6 SL105 expressingβ-glucanase was constructed by using the K.Lactis expression system with an enzyme activity of 23.0 U/mL in shake flask.The apparent molecular weight of recombinant enzyme AnEglA6k was 5.6×10^(4),which was significantly higher than those of the same gene in Pichia pastoris.The optimum pH of AnEglA6K was 5.0 and the optimum temperature was 75℃.The half-life of AnEglA6K was 65.0 min at 80℃.The specific activity of AnEglA6K was about 0.5 U/mL with Avicel as substrate.The expression products of eglA6 in K.lactis and Pichia pastoris were significantly different.The molecular weight of product expressed by eglA6 in K.lactis was significantly higher than that in Pichia pastoris,and the thermal stability and crystalline cellulose degradation activity of product expressed by eglA6 in K.lactis were also higher than those in Pichia pastoris.The recombinant glucanase derived from the expression of eglA6 gene by K.lactis has high thermostability and is suitable for application in feed industry.