Cloning&Microbial And Prokaryotic Expression Of Polyphenol Oxidase

Polyphenol oxidases(PPO) is the key enzyme in black tea processing. At the fermentation stage of black tea manufacture, tea polyphenols are oxidized by PPO and lead to the formation of two major pigments in black tea, theaflavins, thearubigins. They significantly contribute to the bright color and brisk taste of tea brews. Recently, theaflavins have attracted considerable interest because of their potential benefits for human health. However, the preparative separation of TFs is of significant challenge because of their low-level amount in black tea. Therefore, synthesizing theaflavins from tea catechins using biotechnology has an important theoretical and practical significance. The aim of this study is to select a better polyphenol oxidase gene, and to obtain its sequence by PCR, then to construct recombinant strain and to express PPO gene in Escherichia coli or yeast in vitro. The optimum medium ingredients and culture conditions were studied. The results were as follows:(1) Cloning and structure analysis of PPO genes from Fengshui PearPPO genes were cloned from the genomic DNA of Fengshui Pear by PCR. The full length of PPO gene was1782bp without introns, coding a precursor peptide. Precursor PPO consists of593amino acids with a molecular weight of about65.8KDa. It has a theoretical PI of8.4, and has a transit peptide consisting of47amino acids, but without signal peptide. The mature PPO without transit peptide consists of547amino acids with a molecular weight of about60.8KDa and a theoretical PI of6.69. Both PPOs are hydrophilic. Both of the PPO genes have an identity of99%both in the nucleartide sequence and the amino acid sequence compared with the Pyrus pyrifolia gene using the NCBI BLAST server.(2) The construction of prokaryotic expression vector,prokaryotic expression of PPO genes and its analysisBoth the precursor PPO gene and mature PPO gene were cloned into the prokaryotic expression vector (pET32a) successfully.The recombinant pET32a vectors were transformed into the competent E.Coli(DE3), and induced by IPTG. The protein accumulation got a peak in3-6hours at28℃. And if it was induced for longer time, the proein accumulation did not grow dramatically. The PPO had an activity of50U/(mL-min). PPOs can oxidize catechins into theaflavins in vitro, in which ester-type theaflavins had a proportion of98.8%and90%separately. (3) The construction of eukaryotic expression vectorBoth the precursor PPO gene and mature PPO gene were cloned into the eukaryotic expression vector(pPICZa-A) successfully.