Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Appl Environ Microbiol. 2012 Jun;78(12):4294-300. doi: 10.1128/AEM.07473-11. Epub 2012 Apr 6.
Four types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designated bglA13, bglA16, bglA51, and bglM2, were found in the cDNA library of Neocallimastix patriciarum J11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias to Streptococcus equinus. The presence of expansion and several predicted secondary structures in the 3' untranslated regions (3'UTRs) of bglA16 and bglM2 suggest that these two genes were duplicated recently, whereas bglA13 and bglA16, which contain very short 3'UTRs, were replicated earlier. These findings indicate that the β-glucanase genes from N. patriciarum J11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes of Streptococcus equinus, Ruminococcus albus 7, and N. patriciarum J11 were cloned and expressed by Escherichia coli. The recombinant β-glucanases cloned from S. equinus, R. albus 7, and N. patriciarum J11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higher k(cat) values than others. These results support the notion that duplicated β-glucanase genes, namely, bglA16 and bglM2, increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms in N. patriciarum J11.
四种类型的β-1,3-1,4 葡聚糖酶（β-葡聚糖酶，EC 3.2.1.73）基因，分别命名为 bglA13、bglA16、bglA51 和 bglM2，在 Neocallimastix patriciarum J11 的 cDNA 文库中被发现。它们彼此之间高度同源，与链球菌具有密切的系统发育关系，并表现出相似的密码子偏好性。bglA16 和 bglM2 的 3'非翻译区（3'UTR）中存在扩展和几个预测的二级结构，表明这两个基因最近发生了复制，而 bglA13 和 bglA16 则包含非常短的 3'UTR，它们更早被复制。这些发现表明，来自 N. patriciarum J11 的β-葡聚糖酶基因可能是通过来自细菌的水平转移和随后在瘤胃真菌中的复制而产生的。链球菌、白色瘤胃球菌 7 和 N. patriciarum J11 的β-葡聚糖酶基因通过大肠杆菌进行克隆和表达。从链球菌、白色瘤胃球菌 7 和 N. patriciarum J11 克隆的重组β-葡聚糖酶是内切酶，具有相似的底物特异性，但在其他测试中表现出不同的性质。细菌β-葡聚糖酶的比活性和催化效率也明显低于真菌β-葡聚糖酶。我们的结果还表明，在水平基因转移事件中，酶的活性和某些特性发生了变化。当使用大麦来源的β-葡聚糖作为底物时，真菌β-葡聚糖酶的比活性范围为 26529 至 41209 U/mg 蛋白。它们还表现出相似的 pH 和温度最适值、底物特异性、底物亲和力和水解模式。然而，最近复制的两个β-葡聚糖酶 BglA16 和 BglM2 显示出比其他酶更高的 k(cat) 值。这些结果支持了这样的观点，即复制的β-葡聚糖酶基因，即 bglA16 和 bglM2，提高了β-葡聚糖酶的反应效率，并表明β-葡聚糖酶的催化效率可能是决定 N. patriciarum J11 中复制形式进化命运的一个标准。
