生物质降解菌系中GH11家族耐热木聚糖酶基因的克隆与鉴定

Cloning and identification of GH11 family thermostable xylanase gene from biomass degradation strains

以玉米芯粉为基质富集培养牛粪中生物质降解菌系,提取该降解菌系的宏基因组DNA,并以其为模板,通过简并引物PCR扩增出200bp左右的木聚糖酶基因片段,选择与耐热木聚糖酶相似性最高且丰度最高的基因序列,应用mTAIL-PCR扩增得到1 059bp的基因全长序列(xynHAD4),编码352个氨基酸,经序列比对分析,该木聚糖酶(XynHAD4)属于典型的GH11家族木聚糖酶,与来源于Dictyoglomus thermophilum的xylanase229B相似度为93%。将xynHAD4连接于表达载体pET22b(+),在E.coli BL21中成功得到表达,表达产物经纯化后,测定其分子质量约为36kDa,最适作用温度和pH分别为80℃和6.0,该酶在90℃保温1h,残余酶活为72%,pH作用范围较广,在pH4.0保存1h,残余酶活为82%,可见,XynHAD4属于耐热木聚糖酶,并且在酸性条件下可保持较高稳定性,在食品、饲料等领域具有潜在的应用价值。

The biomass degradation strains in cow dung were enrichment cultured using corncob powder as substrates. And then the metagenome DNA of the biomass degradation strains were extracted and purified. Various DNA segments encoding partial of xylanases,which were about 200bp,were obtained through degenerate primer and the metagenome DNA model. In the PCR segments,the most abundant DNA sequence with the highest similarity with thermostable xylanases was used to amplify the whole gene sequence by mTAIL-PCR. The new gene（ xynHAD4） was1 059bp encoding 352 amino acids. The new xylanase（ XynHAD4） was belonged to Family GH11 of xylanases and showed the highest similarity by 93% with the thermostable xylanase 229B from Dictyoglomus thermophilum. The new gene was cloned into the expression vector pET-22b and expressed in E. coli BL21（ DE3）. The recombinant xylanase was purified by Ni2 ＋-NTA affinity chromatography. The molecular weight of the purified XynHAD4 was estimated to be 36kDa by SDS-PAGE. It revealed optimal activity at 80° C and pH 6. 0 and remained high activity over a large range of pH（ 4. 0 ~ 10. 0）. It remained at least 82% residual activity at pH 4. 0 for 1 h. Therefore,the thermostable xylanase（ XynHAD4） with high stability at acidic condition is a promising candidate for food and feed industry.