Synthetic Biology and Biofuel Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India.
Appl Environ Microbiol. 2011 Jul;77(14):4859-66. doi: 10.1128/AEM.02808-10. Epub 2011 Jun 3.
Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a Paenibacillus strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of Paenibacillus. Endo5A and Xyl11D were overexpressed in Escherichia coli, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.
以降解生物质为目的，寄居于植物和木材中的昆虫，其肠道中栖息着大量的细菌菌群。我们根据其分泌多种植物水解酶的能力，从棉铃虫肠道中分离到一株名为 ICGEB2008 的巨大芽孢杆菌。在本研究中，我们克隆、表达并鉴定了来自 ICGEB2008 菌株的两种酶，β-1,4-内切葡聚糖酶（Endo5A）和β-1,4-内切木聚糖酶（Xyl11D），并基于 Endo5A 和 Xyl11D 合成了重组双功能酶。通过使用基因组步移法从 ICGEB2008 菌株的基因组中获得编码 Endo5A 的基因。使用针对保守木聚糖酶序列的引物获得编码 Xyl11D 的基因，这些引物是通过比对其他巨大芽孢杆菌物种的木聚糖酶序列鉴定出来的。Endo5A 和 Xyl11D 在大肠杆菌中过表达，并对其最适活性进行了表征。Endo5A 和 Xyl11D 的最大比活性均在 50°C 和 pH6 至 7 时达到。为了利用这一特性，我们通过将编码基因首尾相连或通过甘氨酸-丝氨酸（GS）接头融合，构建了 Endo5A 和 Xyl11D 的四种嵌合模型。我们使用 I-TASSER 服务器预测了这四种模型的三维结构，并使用圆二色性（CD）光谱分析了它们的二级结构。嵌合模型 Endo5A-GS-Xyl11D 中，通过接头将两种酶分隔开，在 I-TASSER 服务器上获得的 C 分数最高，表现出与天然酶最接近的二级结构特性，酶活性分别比 Endo5A 和 Xyl11D 高 1.6 倍和 2.3 倍。由于其广泛的底物范围，这种双功能酶可能对水解植物生物质有效。
