Cloning Of Tea Polyphenol Oxidase Gene And Construction Of Enigeering Yeast For Its High Expression

Theaflavin, as a peculiar pigment material of tea, since it has the function of strongresistance to oxidation and free radicals, in recent years, got a lot of attention by researchersand became a research hotspot in health care and cancer cure fields. Now it is the hotcomposition in the research of tea depth development fields, and has a widely Applicationprospect in the fields of natural medicines, functional foods, daily chemical products,functional drinks etc. Its synthesis mechanism is tea flavin catechins are oxidativepolymerized into the o-quinones under the action of tea polyphenol oxidase (PPO), then theo-quinones occur a condensation reaction into theaflavins. At present the main productiontechnology of theaflavins is vitro enzyme oxidation method, thereinto polyphenol oxidase isthe key enzyme for catalytic oxidation forming into theaflavins. How to get a lot of highquality enzyme source is the key factoe to improve the production technology of theaflavins.This paper aims at constructing the expression vector of tea PPO, through geneticengineering means, with the fusion protein expression system, and further to transform it intothe advantage expression strains, in order to construct the tea PPO's genetic engineering strain,and its applied foundation research was studyed, so as to solve the key enzyme sourceproblem of the production of theaflavins, then to provide the theoretical basis and basicresearch for the industrial production of theaflavins. The main research results are as follows:Through synthesizing and optimizing the CTAB method, the highly purfied genomicDNA of Yingshuang tea plant was extracted and purified. Then the PPO's cds sequence wasamplified by PCR from the genome with the high fidelity polymerase LA-taq. The sequencingresults showed that the cds sequence's length of PPO was1800bp. The sequence had beensigned in the Genbank and its registered number is GQ214317.The bioinformatics analysis result showed that the Yingshuang tea PPO gene was highlyhomologous with the other tea PPO genes, the similarity was above99%; Its gene encoding atotal599amino acids, and its molecular formula was C3008H4677N813O900S18, molecular weightwas67207.2, the theoretical isoelectric point was6.24; PPO belongs to three superfamily(Tyrosinase. PPO1_DML and PPO1_KFDV), and the key Cu2+combined structure domain of PPO was highly conservative; It has no transmembrane area and no signal peptide,and most likely exists in the chloroplast with the chloroplasts transport peptides.The cloned PPO1gene was recombined to the pET-28vector to construct the expressionvector pET-PPO1, and transform it into the E.coli BL21(DE3). Then the prokaryoticexpression of PPO was successfully realized by being induced with IPTG. During theprokaryotic expression process, PPO easily formed to the inclusion body, and the enzymeactivity was lower; A71KD protein was screened by SDS-PAGE electrophoretic and the sizewas same with the prediction.The cloned PPO2gene was recombined to the pPICZα vector to construct the expressionvector pPICZα-PPO2, and transform it into the Pichia pastoris GS115. Many positivetransformants were successfully screened by PCR, and were induced by methanol. Thepurpose protein was successfully detected in the culture medium by using Western-Blottingmethod. Enzyme activity detection result showed that the induction product had high relativeenzyme activity, and could correctly play the biological function.The optimized strategy research of PPO's eukaryotic expression was made as follows:Firstly we modificated the gene of PPO, and constructed the fusion protein expression vectorof PPO (pPICZα-PPO3and pPICZα-PPO4), then transformed them (include pPICZα-PPO2)into the Pichia pastoris GS115, KM71and SMD1168to get9kinds of PPO recombinedtransformants. The corresponding positive transformants were screened by PCR, and wereinduced by methanol. Western-Blotting results show that: in the nine combinations, only thepPICZα-PPO2/GS115got PPO positive expression.Finally, the basic applied research of PPO genetic engineering strain showed that: theenzymatic activity of the crude enzyme liquid was63.6U; The genetic stability is good, thetarget protein was in expressed after3days induced, and still kept expression of the targetprotein in7days.