两种原核表达载体对CsPPO蛋白表达活性的影响

为了选择合适载体,获得稳定表达且具有高活性的可溶性茶树多酚氧化酶,本研究以茶树叶片为材料,克隆茶树多酚氧化酶基因（CsPPO）,切除含43个氨基酸和70个氨基酸的肽段后分别与pET32a载体、pMAL-c5X载体进行重组构建原核表达载体,分别命名为pET32a-CsPPO₄₃、pET32a-CsPPO₇₀、pMALc5X-Cs PPO₄₃、pMALc5X-CsPPO₇₀,利用E. coil Transetta（DE3）菌株进行表达,经SDS-PAGE电泳检验,pMAL-c5X载体表达的目的蛋白易于提取,大量出现在上清液中;而pET32a载体表达后蛋白则基本存在于沉淀中。利用邻苯二酚、ECG、EC 3种底物在410βnm处检测CsPPO蛋白活性发现,pMALc5X-CsPPO对不同底物反应时酶活性随时间增加均呈现明显“S”型变化,表明茶树PPO氧化儿茶素类的反应并不是匀速过程。pMALc5X-CsPPO₄₃比活力比pMALc5X-CsPPO₇₀的高,其中pMALc5X-CsPPO₄₃与EC反应时酶比活力最高,最大比活力为1.45×10⁶β U·mg^-1。因此,可利用pMALc5X-CsPPO₄₃合成工业用PPO,为进一步研究其与儿茶素的反应机理,利用体外酶法高效获得茶黄素奠定基础。

Effect of Two Prokaryotic Expressed Vectors on the Activity of PPO from Camellia sinensis

To get stably soluble tea polyphenol oxidase with high activity, two vectors were selected to express CsPPO. A tea polyphenol oxidase gene was cloned from tea leaves. After trimming two peptide fragments with 43 and 70 amino acids, the sequences were connected into pET32a and pMAL-c5X vectors with BamH I/xho I and Sal I/ BamH I and named as pET32a-CsPPO₄₃, pET32a-CsPPO₇₀, pMALc5X-CsPPO₄₃ and pMALc5X-CsPPO₇₀ respectively. After expressed of recombinant plasmids in the E.coil Transetta (DE3) strain, SDS-PAGE results showed that the protein expressed by pMAL-c5X was easier extracted than that by pET32a. The proteins expressed by pMAL-c5X were found in both the supernatant and pellet, while those by pET32a were only found in the pellet. The activity of CsPPO was detected by 1,2-benzenediol, ECG and EC as substrates in 410 nm with micro-plate reader. The ‘S’ shaped curves graphed with three substrates oxidized by the pMALc5X-CsPPO indicated that all reaction speeds were not constant. The specific activity of pMALc5X-CsPPO₄₃ was higher than that of pMALc5X-CsPPO₇₀, with the highest activity reaching 1.45×10⁶ βU·mg^-1 when reacted to EC. Therefore, pMALc5X-CsPPO₄₃ could be used to synthesize industrial PPO, but its role in catechin mechanism still needs further research.