水牛瘤胃宏基因组的一个新的b-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性

Cloning and Expression of a b-Glucosidase Gene umcel3G from Metagenome of Buffalo Rumen and Characterization of the Translated Product

摘要:

以木质纤维素为原料、应用同步糖化共发酵工艺发酵生产酒精时需要酸性中低温高活力纤维素酶包括b-葡萄糖苷酶。本工作分6次构建了水牛瘤胃未培养微生物宏基因组文库, 获得1.26×105个克隆, 文库含外源DNA的总长度 约为4.8×106 kb。从文库中筛选到118个表达b-葡萄糖苷酶活性的独立克隆。发现其中8个克隆表达的b-葡萄糖苷酶在pH5.0、37oC条件下活性较强。对其中一个克隆进行了亚克隆, 序列分析发现一个2223 bp的潜在的编码b-葡萄糖苷酶基因(umcel3G)的开放阅读框(ORF), 其编码产物的氨基酸序列与来自于 Bacillus sp.的一个b-葡萄糖苷酶同源性最高, 具有60%的一致性和73%的相似性。该ORF在E.coli中的表达产物Umcel3G的分子量与预测大小相似, 酶谱分析表明该表达产物具有b-葡萄糖苷酶活性, 证实该基因为一个b-葡萄糖苷酶基因。测定了用Ni-NTA纯化的Umcel3G的酶学特性, 其最适pH和最适温度分别为6.0~6.5和45oC。一些金属离子如Ca2+、Zn2+能显著提高该酶的酶活, 而另外一些金属离子如Fe3+、Cu2+能抑制Umcel3G的活性。在pH4.5、35oC和5 mmol/L的 Ca2+存在的条件下, 用Ni-NTA纯化的重组酶的比活为22.8 IU/mg, 说明该酶在用SSCF工艺发酵生产酒精中有潜在的应用价值。

Abstract:

Metagenomic cosmid libraries containing 1.26×105 clones, covering about 4.8×106 kb metagenomic DNA of uncultured microorganisms from the contents of buffalo rumens were constructed, and 118 independent clones expressing b-glucosidase activity were isolated from the libraries. Screening of these clones showed that eight clones expressed relatively higher b-glucosidase activity at pH 5.0 and 37℃. One out of the eight clones was subcloned. Sequencing analysis showed that an open reading frame (ORF) of 2223 bp, termed umcel3G, potentially encodes a b-glucosidase. The encoded product shared highest homology with a b-glucosidase from Bacillus sp. at 60% identity and 73% similarity. The umcel3G was over-expressed in Escherichia coli and the size of the translated product Umcel3G on SDS-PAGE was in agreement with the predicted molecular mass. Zymogram analysis showed that Umcel3G exhibited b-glucosidase activity, confirming that this ORF encodes a b-glucosidase. The Umcel3G, purified with Ni-NTA column, exhibited optimal activity at pH 6.0~6.5 and 45oC. Certain ions such as Ca2+, Zn2+ had significant positive effect on the activity of Umcel3G. However, some ions such as Fe3+, Cu2+ gave significant inhibitory effect on the enzyme. The Ni-NTA purified recombinant b-glucosidase Umcel3G had a specific activity of 22.8 IU/mg at pH4.5, 35oC and at the presence of 5 mmol/L Ca2+, indicating that this enzyme has potential applications in the fermentative production of ethanol by simultaneous saccharification and cofermentation (SSCF) of lignocelluloses.