灰色链霉菌中乙酰木聚糖酯酶基因的克隆、表达及酶学性质研究

摘要

根据GenBank数据库中已报道的灰色链霉菌（Streptomyces griseus）全基因组序列，分析得到假定的乙酰木聚糖酯酶基因序列并设计引物；利用分子克隆的方法得到该菌株基因组中乙酰木聚糖酯酶基因，并构建原核表达载体pET28a-Sgraxe，经IPTG诱导表达重组SgrAxe，Ni-NTA亲和层析法纯化该蛋白。结果显示，克隆得到乙酰木聚糖酯酶基因axe，其序列全长1008 bp，编码336个氨基酸。SDS-PAGE检测带有pET28a-Sgraxe转化菌株诱导表达产物相对分子量约为37 kD，与理论值相符。纯化的重组SgrAxe酶学性质表明，该酶最适反应温度为50℃，最适pH8.0，热稳定性较强，pH作用范围广；金属离子对酶均表现为抑制作用，尤其是Zn2+严重抑制酶活力；重组酶特征的分析揭示了其在工业中潜在的应用价值。%Primers were designed by putative axe annotated from the whole genome sequence of Streptomyces griseus. The axe gene was firstly cloned and linked to the prokaryotic expression vector, expressed recombinant protein induced with IPTG and purified with the Ni-NTA affinity chromatography. The results showed the cloned gene axe contained an open reading frame of 1 008 bp encoding 336 amino acid residues. The transformant harboring pET28a-Sgraxe was expressed with a protein molecular weight of 37 kD by SDS-PAGE analysis. The results indicated that the purified recombinant enzyme exhibited optimum activity at pH8.0 and 50℃, high thermal stability and strong alkali resistance range. Metal ions on recombinant enzyme activity was inhibited especially Zn2+. The analysis of recombinant enzymatic properties reveals potential application in industry.