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Journal of Tea Science ›› 2018, Vol. 38 ›› Issue (4): 396-405.doi: 10.13305/j.cnki.jts.2018.04.007

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

GAN Yudi, SUN Kang, LI Huijuan, DU Zhongying, ZHAO Zhen, PANG Xing, LI Xinghui, CHEN Xuan*   

  1. College of Horticulture, Nangjing Agricultural University, Nanjing 210095, China
  • Received:2018-04-04 Revised:2018-04-26 Online:2018-08-15 Published:2019-10-15

Abstract: 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, polyphenol oxidase, Prokaryotic expression, enzymatic activity

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