Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
Appl Biochem Biotechnol. 2019 Jan;187(1):424-437. doi: 10.1007/s12010-018-2832-1. Epub 2018 Jul 3.
A cosmid metagenomic library containing 1.3 × 10 clones was created from a soil sample. A novel gene (fae-xuan) encoding a feruloyl esterase was identified through functional screening. Primary sequence analysis showed that the gene consisted of 759 base pairs and encoded a protein of 252 amino acids. The gene was expressed in Escherichia coli BL21 (DE3) and the corresponding purified recombinant enzyme exhibited a molecular weight of 29 kDa. The FAE-Xuan showed high activity (40.0 U/mg) toward methyl ferulate with an optimal temperature and pH of 30 °C and 5.0, respectively. Besides methyl ferulate, FAE-Xuan can also hydrolyze methyl sinapate and methyl p-coumarate. The substrate utilization preferences and phylogenetic analysis indicated that FAE-Xuan belongs to type A FAE. FAE-Xuan was quite stable over a broad pH range from 3.0 to 10.0. The activity reduced remarkably in presence of Cu. FAE-Xuan can enhance the quantity of ferulic acid from de-starched wheat bran in presence of xylanase. The work presented here highlighted the effectiveness of metagenomic strategy in identifying novel FAEs with diverse properties for potential use in industrial production.
从土壤样本中创建了一个包含 1.3×10^6 个克隆的黏粒宏基因组文库。通过功能筛选，鉴定出一种新型基因（fae-xuan），其编码一种阿魏酸酯酶。初步序列分析表明，该基因由 759 个碱基对组成，编码 252 个氨基酸的蛋白质。该基因在大肠杆菌 BL21（DE3）中表达，相应的纯化重组酶显示出 29 kDa 的分子量。FAE-Xuan 对甲基阿魏酸具有很高的活性（40.0 U/mg），最适温度和 pH 分别为 30°C 和 5.0。除了甲基阿魏酸，FAE-Xuan 还可以水解甲基芥子酸和甲基对香豆酸。底物利用偏好性和系统发育分析表明，FAE-Xuan 属于 A 型 FAE。FAE-Xuan 在 pH 值为 3.0 至 10.0 的较宽范围内非常稳定。在存在铜的情况下，活性显著降低。FAE-Xuan 可以在木聚糖酶存在的情况下，增加去淀粉小麦麸皮中阿魏酸的含量。本研究突出了宏基因组策略在鉴定具有不同性质的新型 FAEs 方面的有效性，这些 FAEs 可用于工业生产。
