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。
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.
[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.
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