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.

References 20

[1]	王曼玲, 胡中立, 周明全,等.植物多酚氧化酶的研究进展[J]. 植物学通报, 2005(2): 215-222.
[2]	宛晓春. 茶叶生物化学[M]. 北京:中国农业出版社, 2003.
[3]	刘仲华, 施兆鹏. 红茶制造中多献氧化酶同工酶谱与活性的变化[J]. 茶叶科学, 1989, 9(2): 141-150.
[4]	王坤波, 刘仲华, 赵淑娟, 等. 多醋氧化酶同工酶组成对茶黄素合成的影响[J]. 农业现代化研究, 2007, 28(5): 618-621.
[5]	谭淑宜, 朱尚同, 龚范武. 利用同工酶技术对江华苦茶等五个茶树品种亲缘关系的研究初报[J]. 湖南农学院学报. 1983(3): 63-69.
[6]	李桂琴. 鸭梨多酚氧化酶基因CDS区的克隆及表达[J]. 果树学报, 2008, 25(4): 577-580.
[7]	Newman S M, Eannetta N T, Yu H, et al.Organisation of the tomato polyphenol oxidase gene family[J]. Plant Molecular Biology, 1993, 21(6): 1035-1051.
[8]	Thygesen P W, Dry I B.Robinson. Polyphenol oxidase in potato[J]. Am Soc Plant Biol, 1995, 109(5): 525-531.
[9]	赵东, 刘祖生, 奚彪. 茶树多酚氧化酶基因的克隆及其序列比较[J]. 茶叶科学, 2001, 21(2): 94-98.
[10]	陈忠正, 李斌, 黎秋华, 等. 茶叶多酚氧化酶的基因克隆与序列分析[J]. 食品工业科技, 2006 (10): 109-111.
[11]	刘敬卫, 黄友谊, 丁建, 等. 茶树多酚氧化酶成熟蛋白的原核表达[J]. 茶叶科学, 2010, 30(2): 136-140.
[12]	吴贻良. 茶叶多酚氧化酶基因的克隆及微生物表达[D]. 广州: 华南师范大学, 2009.
[13]	王乃栋, 张丽霞, 黄晓琴, 等. 茶树PPO融合蛋白真核表达载体的构建与表达[J]. 茶叶科学, 2012, 32(3): 269-275.
[14]	陈娇, 孙长君, 杨昭, 等. 香蕉多酚氧化酶成熟蛋白的原核表达[J]. 热带作物学报, 2015, 36(12): 2198-2203.
[15]	刘敬卫. 茶树多酚氧化酶的基因克隆与原核表达[D]. 武汉: 华中农业大学, 2009.
[16]	陈炜烨, 刘冬冬, 徐建华, 等. ImageJ软件在重组质粒pET32a-CDK2中蛋白表达的应用[J]. 中国热带医学, 2014, 14(1): 23-25.
[17]	黄意欢. 茶学实验技术[M]. 北京: 中国农业出版社, 1997.
[18]	陈东生, 王坤波, 黄建安, 等. 茶树多酚氧化酶研究进展[J]. 茶叶通讯, 2012, 39(2): 17-21.
[19]	韩杰, 郑继平, 言普, 等. 原核表达番木瓜环斑病毒NIa-Pro及其双重酶活鉴定[J]. 热带作物学报, 2016, 37(4): 722-727.
[20]	Agarwal S, Jha S, Sanyal I.Expression and purification of recombinant human alpha1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity[J]. J Biotechnol, 2010, 147(1): 64-72.

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

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