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.
Key words
Camellia sinensis /
enzymatic activity /
polyphenol oxidase /
Prokaryotic expression
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