Expression Analysis And In Vitro Prokaryotic Expression Of Camellia Sinensis CsPPO

The quality of tea can be affected by changing the processing technology and regulation the content of the ingredients.As an important enzyme,PPO activity was regulated through fixation,withering and fermentation.It determined whether PPO was involved in the redox reaction and thus forms different tea qualities.In the processing of green tea,PPO activity was inhibited by the thermal denaturation which can guarante the quality of the green tea.In black tea processing,PPO catalyzed the reaction of catechins to form complex compounds such as theaflavins,thus forming the characteristics of black tea.However,the production efficiency of theaflavins was not very high.It was related to the insufficiency of PPO enzyme activity and the complexity of the synthesis mechanism.Therefore,it has become an urgent problem to solve the in vitro synthesis of theaflavins by finding stable,single,and highly active PPO enzyme sources..In recent years,many studies have investigated the activity of the enzyme by extracting the crude enzyme of PPO in vitro.Because many factors such as cultivar,picking method,and separation methods leaded to different PPO content and species.The obtained enzyme solution may be a mixture of several PPO isoenzymes,which was very difficult to separate and purify,and thus was not conducive to the study of the effect of a single enzyme source on the synthesis mechanism of theaflavins.Using genetic engineering techniques,qRT-PCR technology was used to explore differences in expression levels of CsPPO under different cultivares,picking standards,and different stress conditions;two prokaryotic expression vectors were selected to construct and express four engineering strains in order to obtain high-solubility,high-activity PPOs.Below are key research findings:(1)Bioinformatics analysis of PPO amino acids,CsPPO gene encoded 599 amino acids with a molecular weight of 67(kd);compared with other species,the 43th amino acid and the 70th amino acid were selected as the transfer peptide position.(2)The expression of CsPPO gene was different in different cultivares,different picking standards,and different stresses by qRT-PCR technique.The results showed that there was significant difference about CsPPO gene expression levels in different cultivares but no difference was found between ' Bai Ye 1th ' and 'Golden Bud '.The ' Fuding white ' and ' Zijuan ' also had no different expression in bud.The expression of CsPPO gene in different picking standard also appeared differences.In ' Bai Ye 1th ' and ' Golden Bud ',the expression of CsPPO gene was the same change trend.' Fuding white ' had the higher expression in a bud a leaf and a bud two leaves among cultivares.' Bai Ye 1th ' had the lower expression in a bud a leaf and a bud two leaves among cultivares.For the high temperature,drought,NaCl,cold stress by fluorescence quantitative detection of CsPPO gene expression resulted show that it was significant difference.The expression of CsPPO gene in drought stress was the same with the NaCl stress.CsPPO gene expression was presented differentially expressed trend,including drought and high treatment.(3)Four recombinant prokaryotic were construct with two prokaryotic expression vectors pET32a and pMALc5X.It was named 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 pMALc5X was easier extracted than by pET32a.The protein expressed by pMALc5X was found both in the supernatant and pellet,while by pET32a only found in the pellet.(4)The specific enzymatic activity of the four fusion proteins was determined in 1 h at pH 4,5.6,and 6,5 using catechol as a substrate.The specific activity of the four fusion proteins was measured at pH 5.6 when the catechol,EC,and ECG were used as substances.The results showed that the 'S' shaped curves graphed was showed during the specific activity measurement,and it indicated that all reactive speeds were not constant.The specific activity of the four fusion proteases with EC as substrate reaction was higher.The specific activity of pMALc5X-CsPPO43 was higher than that of the other,the highest activity reached 1.45×106 U·mg-1 when it reacted to EC.Therefore,pMALc5X-CsPPO43 can provide a enzyme source for the enzymatic oxidation preparation of the theaflavin in vitro.