Cloning And Expression Of Polyphenol Oxidase Isozymes From Camellia Sinensis Cv. Huangjinya And Cv. Jiukeng And Their Enzymatic Synthesis Of Theaflavins

Theaflavins(TFs)in black tea are characteristic components unique to fermented tea,and their content is directly related to the quality of black tea.In addition,TFs also have beneficial biological activities for the human body.At present,the method of directly extracting TFs from black tea has a high cost,while using chemical synthesis technology to obtain theaflavins has low efficiency and environmental pollution.Polyphenol oxidase(PPO)is a key rate limiting enzyme that promotes the synthesis of theaflavins from catechins.The publication of the entire genome of Camellia sinensis cv.Shuchazao tea varieties provides a rich data foundation for improving the content of TFs in tea from the perspective of genetic engineering.The use of heterologous expression systems to obtain recombinant tea PPO for efficient biosynthesis of TFs has become a potential feasible method to solve these problems.However,there are currently few research reports on the gene comparison and identification of tea PPO isozymes,as well as the directed synthesis of monomeric TFs.To investigate the enzymatic properties of tea PPO isozymes expressed in vitro and their ability to catalyze the synthesis of monomeric TFs,and to provide new ideas for enzymatic biosynthesis of TFs,this paper used prokaryotic in vitro cloning expression,enzymatic analysis characterization,and bioinformatics analysis methods to identify two tea strains suitable for producing black tea,Camellia sinensis cv.Huangjinya and Camellia sinensis cv.Jiukeng.The PPO isozymes were studied,and the main content is as follows:1.Analysis of the distribution and sequence characteristics of PPO isozymes in Huangjinya and Jiukeng.Four PPO isozyme genes were screened from the entire tea genome,including Cs PPO1(Gene ID: 114323507),Cs PPO2(Gene ID: 114267949),Cs PPO3(Gene ID: 114303710),and Cs PPO4(Gene ID: 114309907).Using the c DNA of Huangjinya and Jiukeng tender buds as templates for q RT-PCR analysis,it was found that both types of tea tender buds expressed PPO1 and PPO3,and the expression of PPO isozymes in Huangjinya is higher than that in Jiukeng,but there was no expression of PPO2 and PPO4.Through full length PCR amplification,electrophoresis,gel recovery,double enzyme digestion and plasmid linking,introduction of E.coli,sequencing and other steps,the CDS sequences of PPO1 and PPO3 from Huangjinya and Jiukeng were obtained for the first time,respectively named Hjy PPO1/3 and Jk PPO1/3.After sequencing analysis,it was found that the similarity of the same PPO isozyme sequence between Huangjinya and Jiukeng tea was as high as 99%,indicating high conservatism;The similarity between PPO1 and PPO3 is 74.2%,indicating a certain difference.Evolutionary tree analysis shows that the PPO1 and PPO3 of tea are more evolutionarily close to dicotyledonous fruit plants such as grapes,apples,and pears,while they are more distant in PPO affinity to monocotyledonous plants such as rice and wheat,as well as fungi.2.Enzymatic properties analysis of PPO1 and PPO3 in Huangjinya and Jiukeng.The enzymatic properties analysis of recombinant purified PPO1 and PPO3 showed that both the PPO isozymes of Huangjinya and Jiukeng had the ability to catalyze the synthesis of four monomer theaflavins(TF1,TFA,TFB,TF3)from four catechins(EC,EGC,ECG,EGCG),and PPO3 had a stronger catalytic ability than PPO1;However,there was no significant difference in catalytic efficiency between different varieties of the same PPO isozyme.Their affinity sequence with the substrate is EC>ECG>EGC>EGCG,with PPO1 having an optimal catalytic temperature of 35 ℃and an optimal p H of 5.5,PPO3 having an optimal catalytic temperature of 30 ℃ and an optimal p H of 6.0;After exceeding 65 ℃,both isozymes are inactivated.When the p H value is within the range of 4.5 to 6.5,both can maintain relative activity above40%.In addition,when the ratio of catechol-type catechin(EC,ECG)to pyrogallol-type catechin(EGC,EGCG)is 1:2,the synthesis ability of TFs is the strongest.3.Molecular docking analysis of PPO1 and PPO3 with catechin substrates.The molecular docking analysis results indicate that the gatekeeper amino acid residue Phe260 in PPO3 has stronger selectivity towards catechin series substrates and has aπ-π stacking system with His108.It is speculated that this is the reason why PPO3 has higher catalytic efficiency than PPO1.At the same time,the PPO3 active center can form more hydrogen bonds with the substrate and the required binding energy is lower,which also makes the efficiency of PPO3 binding substrate higher than PPO1.