链霉菌zxy19木聚糖酶酶学性质及酶基因克隆

The Xylanase Properties and the Xylanase Gene of Streptomyces sp. zxy19

摘要:

采用平板筛选法, 从红树林放线菌中筛选到一株有较强木聚糖酶活的菌株zxy19, 其16S rDNA序列与Streptomyces sampsonii 的同源性仅为96%。该菌株木聚糖酶活为852.41 IU/mL, 酶反应最适pH 值为7, 最适反应温度为60°C。用针对木聚糖酶基因保守结构域的一对简并引物扩增到该酶基因部分序列,进而通过反向PCR扩增到了完整的酶基因。对该基因序列分析结果表明此木聚糖酶基因属于糖基水解酶家族11的成员, 酶蛋白氨基酸序列与已报道序列同源性最高为79%(Streptomyces lividans xylanase B)。构建了该酶重组表达质粒pET-28a-xyl696, 经过IPTG诱导实现了该酶蛋白在大肠杆菌BL21(DE3)中的异源表达, 且通过镍柱纯化后的表达产物具有生物学活性。

Abstract:

A mangrove actinomycetes strain zxy19 that has shown strong xylanase activity was selected by plate screening. Sequencing analysis and Blast search of the 16S rDNA gene of the strain revealed that the highest identity in the database was 96% against Streptomyces sampsonii. Characterization of the xylanase enzymatic properties of zxy19 has shown that the optimal pH was 7 and the optimal temperature was 60°C. Degenerate primers were designed according to the conservative domains of actinomycetal xylanase to amplify the partial xylanase gene. The complete enzyme gene xyl696 was obtained by inverse PCR subsequently and confirmed by DNA sequence analysis. Blast search of the translated amino acid sequence suggested that the enzyme belongs to the glycosyl hydrolases family 11 with the highest similarity as 79% against the xylanase B produced by Streptomyces lividans. The xylanase gene was cloned into the expression vector to construct pET-28a-xyl696 and introduced into E. coli BL21(DE3). After IPTG inducing, the over expressed recombinant xylanase was purified by Ni2+-NTA affinity chromatography and proved to have catalytic activity.