Study On The Biocatalysis Of Tryptophan Decarboxylase(TDC2)in Ophiorrhiza Pumila

Ophiorrhiza pumila was a perennial herb which well known by people for the camptothecin?CPT?of its secondary metabolite.CPT was a cytotoxic quinoline alkaloid and a class of botanicals with unique anti-cancer mechanisms,which has been proved that can perform a good inhibitory effect on DNA topoisomerase I.Tryptophan decarboxylase?TDC?was a key enzyme that connected the primary metabolism and the secondary metabolism in CPT synthesis pathway.TDC was firstly isolated from Catharanthus roseus,which could catalyze the decarboxylation of tryptophan to form tryptamine,and its activity directly affected CPT biosynthesis and other related metabolic process.This research was based on the database of the transcriptome of the O.pumila,which had been established in our laboratory during the early stage,and a new TDC was isolated from the aseptic seedling of O.pumila by using molecular biology.The new TDC gene was named TDC2,based on which the sequence alignment analysis was performed,and the prokaryotic expression,protein purification and enzymatic properties were studied.The main research results of this paper were as follows:1.Cloning and sequence analysis of TDC2 geneThe sterile seedlings of O.pumila was served as raw materials,which was used to extract the total tissue RNA and then reverse transcribed into genomic c DNA.Using it as a template for PCR amplification,and then the TDC2 of O.pumila was cloned.Its sequence was 1536 bp,contained no introns,and encoded by 511 amino acids.Phylogenetic tree analysis showed that TDC2 from O.pumila was more closely related to Mitragyna speciosa and Catharanthus roseus than to Camptotheca acuminata and Rauvolfia verticillate.This gene encoded a tryptophan decarboxylase protein with a molecular weight of 57.01 k Da and a theoretical isoelectric point of6.39,which was an unstable hydrophobic protein.It distributed widely in the cytoplasm,nucleus,Golgi,mitochondria and vacuoles,of which up to 52.2%in the cytoplasm with the most distribution.The software predicted that the protein had four possible phosphorylation sites,no signal peptide,and no transmembrane structure.Secondary structure analysis showed that the protein had 22?-helices and 10?-sheets,where 33 random coils connected?-sheets.2.Specific expression of tissue about TDC2 geneIn order to study the spatial expression characteristics of TDC2 gene from O.pumila,and further explore the relationship between the expression of TDC2 and secondary metabolism of CPT,this study takes Actin as the internal reference gene,analyzing the relative expression of TDC2 gene in the roots,stems and leaves by using Real-time PCR technology.Tissue expression profile revealed that the TDC2gene was highly expressed in the roots,which was consistent with the fact that the site of CPT synthesis was mainly in the root.These results suggested that the TDC2 gene might also be involved in the CPT metabolism and synthesis in O.pumila.3.Optimization of expressional conditions about recombinant protein TDC2The TDC2 gene was constructed into prokaryotic expression vectors p ET28a and p Cold TF by double digestion reaction and one-step cloning and recombination,respectively.The identified recombinant plasmids p ET28a-TDC2 and p Cold TF-TDC2 were transformed into protein-expressing strain BL21?DE3?for prokaryotic expression.The expression of the two recombinant proteins could be identified by 10%sodium lauryl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?.The results showed that the recombinant protein p Cold TF-TDC2mainly existed in the form of soluble protein while the recombinant protein p ET28a-TDC2 mainly existed in the form of inclusion body.A specific protein band,namely the expressed recombinant protein TDC2,could be detected at the relative molecular weight of 115 k Da.The p Cold TF-TDC2 strain with soluble expression of target protein was optimized for culture conditions.The optimal expression conditions were as follows:after the culture to the middle and late logarithmic stage?3h,OD600=0.8-1.0?,0.1mmol·L^-1 isopropyl?-D-1-thiogalactopyranoside?IPTG?was added to fermented liquid,and then the culture was induced under the conditions of20?,220r for 24h.4.Purification of recombinant protein TDC2 and study on its enzymaticpropertiesThe bacterial cells induced under the optimal expression conditions were collected,broken and then purified by Ni-NTA affinity chromatography to obtain the target protein at a concentration of 0.25 mg·m L^-1in the end.In order to study the enzymatic properties of the target protein,the recombinant protein TDC2 was catalyzed in vitro.The related enzymatic properties were as follows:the K_mwas 1.08 mmol·L^-1,the maximum reaction rate of V_max was 0.20 umol·min^-1,the conversion number k_catwas0.78 S^-1,and the value of k_cat/K_m was 774.5 S^-1·mol^-1.The optimum reaction temperature was 45?,the optimum p H was 8.0,and the optimum cofactor concentration was 0.1mmol·L^-1.The influence of metal ions on the enzymatic reaction was detected with 10 mmol·L^-1 of metal ions,showing that Mn²⁺had a certain promotion effect on the catalytic activity of TDC2,and Cu²⁺,Fe²⁺as well as Zn²⁺had a strong inhibition effect on the catalytic activity of TDC2.