分泌表达苹果酸乳酸酶的大肠杆菌系统的构建

苹果酸乳酸酶（简称苹乳酶）是果酒生产过程中苹果酸乳酸发酵（简称苹乳发酵）的关键酶。为了构建分泌表达苹乳酶的大肠杆菌表达系统，作者通过融合PCR将信号肽基因（487 bp）和苹乳酶基因（1 623 bp）的编码序列连接在一起，将融合片段（2110 bp）克隆到表达质粒pET28a（+）上并转化大肠杆菌，得到阳性克隆子。IPTG诱导阳性克隆子后可得到相对分子质量约为60 000左右的蛋白质，利用HPLC可在其发酵上清液中检测到240.7 mg/L的L-乳酸。这说明成功构建了分泌表达苹乳酶的大肠杆菌系统，该菌株的获得将为苹乳酶的研究和应用提供了技术平台。
Malolactic enzyme（MLE） is a key enzyme inmalactic fermentation（MLF）during fruit-wine producing. In order to construct the secretory Escherichia coli（E.coli）expression system of MLE. The signal peptide gene（487 bp） and the MLE gene（1 623 bp） were combined through fusion-PCR，then the fused fragment was inserted into pET28a（+） and transformed into E.coil. 60 000 protein was obtained when the positive strain was cultured and induced with IPTG. 240.7 mg/L L-lactic acids could be detected in the fermentation supernatant with HPLC. All the results indicated the secretory E.coli expression system of MLE was successfully constructed. This will provide a technical platform for the research and application of MLE