两种原核表达载体对CsPPO蛋白表达活性的影响

doi:10.13305/j.cnki.jts.2018.04.007

Abstract

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 1.45×10⁶βU·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

CLC Number:

TS272.2
Q52

GAN Yudi, SUN Kang, LI Huijuan, DU Zhongying, ZHAO Zhen, PANG Xing, LI Xinghui, CHEN Xuan. Effect of Two Prokaryotic Expressed Vectors on the Activity of PPO from Camellia sinensis[J]. Journal of Tea Science, 2018, 38(4): 396-405.

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

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