大肠杆菌多酚氧化酶的分子克隆及异源高效表达

Molecular cloning of the polyphenol oxidase from Escherichia coli and its heterologous high-efficiency expression

菜籽粕、棉籽粕等非粮饲料资源存在的酚类化合物芥子碱、棉酚等抗营养因子,严重影响了其饲喂价值。本课题组筛选出的一株大肠杆菌属芥子碱降解菌SDB2已被证实能通过分泌多酚氧化酶来降解芥子碱和棉酚,本试验旨在运用基因工程技术克隆SDB2多酚氧化酶基因,实现其高效表达,为SDB2多酚氧化酶在饲料工业上的规模化应用奠定基础。从大肠杆菌SDB2中克隆出多酚氧化酶基因,将其与质粒pET28a连接后转化至大肠杆菌BL21感受态细胞中培养,通过PCR鉴定及质粒酶切验证方法构建重组菌株,同时对重组克隆基因进行生物信息学分析。采用“2×2”交叉试验设计,以温度和诱导剂IPTG浓度两个影响因素诱导SDB2多酚氧化酶基因在重组菌株中高效表达,经SDS-PAGE和WB双重验证后,确立最佳诱导条件,最终纯化出目标酶蛋白。结果表明:(1)成功克隆SDB2多酚氧化酶基因,构建出异源表达重组菌株。(2)克隆出的SDB2重组多酚氧化酶基因含目标核苷酸741个,总编码氨基酸263个(含标签氨基酸20个),氨基酸组成的蛋白相对分子质量为28499.0,理论等电点为6.94,据此预测出SDB2重组多酚氧化酶三维结构模型。(3)确立SDB2多酚氧化酶异源高效表达的最佳诱导条件为温度37℃,IPTG浓度1 mmol,该条件下表达出大量可溶性酶蛋白,有利于工业化生产。本试验条件下,大肠杆菌SDB2多酚氧化酶成功实现了分子克隆及异源高效表达,为我国非粮饲料资源实现高效利用提供了技术参考。

Sinapine or gossypol(phenolic compounds)as anti-nutritional factor in non-grain feed resource rapeseed meal or cottonseed meal has seriously affected their application value in animal husbandry.A strain of Escherichia coli SDB2 screened by our research group has been proved to degrade sinapine and gossyrol by secreting polyphenol oxidase.This experiment aimed to clone the SDB2 polyphenol oxidase gene and achieve its high-efficiency expression by using genetic engineering technology,laying a foundation for the large-scale application of SDB2 polyphenol oxidase in feed industry.The polyphenol oxidase gene was cloned from Escherichia coli SDB2,which was connected with plasmid pET28a and transformed into Escherichia coli BL21 receptive cells for culture.The recombinant strain was verified by PCR and plasmid digestion,and the recombinant cloned gene was analyzed by bioinformatics.A“2×2”cross experiment design was used to induce the high-efficiency expression of SDB2 polyphenol oxidase gene in the recombinant strain by temperature and IPTG concentration.After SDS-PAGE and WB double verification,the optimal induction conditions were established,and the target enzyme protein was finally purified.The results showed that:(1)The polyphenol oxidase gene of SDB2 was cloned successfully and the recombinant strain was constructed.(2)The recombinant cloned SDB2 polyphenol oxidase gene contained 741 target nucleotides,263 encoded amino acids(including 20 tagged amino acids),the relative molecular weight of the protein composed of amino acids was 28499.0,and the theoretical isoelectric point was 6.94.Therefore,the three-dimensional structure model of SDB2 recombinant polyphenol oxidase was predicted.(3)The optimal induction conditions for heterogenic expression of SDB2 polyphenol oxidase were 37℃and 1 mmol IPTG.Under these conditions,a large number of soluble enzyme proteins were expressed,which was conducive to industrial production.The results indicated that the molecular cloning and heterologous expression of Escherichia coli SDB2 polyphenol oxidase were successfully achieved under the experimental conditions,which provided a technical reference for the efficient utilization of non-grain feed resources.