Gene Cloning And Prokaryotic Expression Of Camellia Sinenesis Polyphenol Oxidase

Camellia sinensis polyphenol oxidase (PPO, EC.1.10.3.1) plays an essential role during the quality formation of preliminary tea products, particularly black tea. At the same time, the PPO is applied to the preparation of tea flavine and the treatment of industrial wastewater. So the PPO genes were cloned from the DNA genomics of Camellia sinensis Yihongzao and Anhuiyihao, and were expressed in Escherchia coli respectively. The homologous engineering protein with enzymic activity can be applied to tea processing, and tea flavine preparation. The results are as follows:1. PCR amplification and bioinformatics analysis of Camellia sinensis PPOPPO genes, amplified by PCR from the DNA genomic of Camellia sinensis Yihongzao and Anhuiyihao leaves respectively, were sequenced and analysed by Bio-softwares. The results showed that our PPO genes were highly homologous with those founded in other varieties, especially copper binding regions. Camellia sinensis PPO was one of cytoplasm-synthesized proteins, which was hydrophilic and non-transmembrane without signal peptide instead of chloroplast transfer peptide. The secondary structures of Camellia sinensis PPO were predicted and their three-dimensional structure models were builted successfully. The PPO genes of Anhuiyihao and Yihongzao were registered and published in GenBank, whose access numbers were EU787433 (Yihongzao 1800bp), FJ210643 (Anhuiyihao 1800 bp), FJ210644 (Anhuiyihao 1799 bp), FJ210645 (Anhuiyihao 1803 bp).2. Prokaryotic expression and activity determination of Camellia sinensis PPOAfter the expression vectors were constructed and screened, pET32a, with high expression level, was better than pET20bn to express Camellia sinensis PPO. However, pET20b and pET28a could not able to express. So the pET32a was chosen as the expression vector of Camellia sinensis PPO, despite of forming the inclusion bodies easily. For Anhuiyihao PPO was not expressed in different vectors, Yihongzao PPO was expressed in a great deal and purified by nickel ion column. Although the pureness of the purified Yihongzao PPO, examined by SDS-PAGE electrophoresis, was not high, the PPO, with a certain enzyme activity, could catalyse the catechol obviously and its maximum enzyme activity was 20.867U.3. Expression of Camellia sinensis sub-PPOThe sub-PPO genes (1532bp and 1053bp) were amplified by PCR, taking the full-lengh genes of Anhuiyihao PPO (FJ210643) and Yihongzao PPO (EU787433) as the template respectively. Compared with the full-length gene, the sub-PPO genes were more easily expressed in E. coli, indicated the chloroplast transfer peptide had an influence on PPO expression. The enzymic activities of the crude sub-PPOs (1532 bp) were higher than those of full-lengh PPOs, indicated the chloroplast transfer peptide had an influence on PPO activity.4. Denaturalization and renaturation of Camellia sinensis PPO engineering proteinFor the expressed proteins mostly were dissoluble, the inclusion bodies of Yihongzao PPO, Yihongzao PPO-1532 and Anhuiyihao PPO-1532 were purified through ultrafiltration renaturation after degeneration, respectively. Despite of the low rate of renaturation, the purified PPO engineering proteins were of electrophoresis pure, but lower rate of renaturation.