Tyrosine decarboxylase CtTyDC, encoding gene, kit and method for preparing tyramine

Abstract

This invention discloses a tyrosine decarboxylase CtTyDC, its encoding gene, a kit, and a method for preparing tyramine. The amino acid sequence of the tyrosine decarboxylase CtTyDC is shown in SEQ ID NO:1, or an amino acid sequence with equivalent function formed by substitution, deletion, or addition of one or more amino acids. The tyrosine decarboxylase CtTyDC provided by this invention catalyzes the decarboxylation of tyrosine to produce tyramine without the participation of PLP, greatly simplifying the reaction steps and saving reaction costs.

Description

Technical Field

[0001] This invention relates to a tyrosine decarboxylase CtTyDC, its encoding gene, a kit, and a method for preparing tyrosine. Background Technology

[0002] Tyrosine decarboxylase (TyDC) is an amino acid decarboxylase (AADC) that catalyzes the decarboxylation of tyrosine to produce tyramine. It serves as a bridge between primary metabolism and various secondary metabolites and plays a crucial regulatory role in controlling the biosynthesis of final products.

[0003] Pyridoxal 5'-phosphate (PLP) acts as a coenzyme in many enzymatic processes, including decarboxylation, deamination, transamination, and racemization. Enzymes that require PLP are generally called PLP-dependent enzymes, and they are widely involved in many key cellular metabolic pathways. Most of the tyrosine decarboxylases (TyDC) reported from plants (such as poppy, rice, Thalictrum lucidum, Arabidopsis thaliana, Rhodiola rosea, and Lycoris radiata) belong to the PLP-dependent enzyme family. They all require the cofactor PLP to catalyze the decarboxylation of tyrosine to tyramine, increasing the input of raw materials and production costs. The catalytic mechanism of PLP is as follows: the PLP cofactor binds to the protein by forming a Schiff base bond; the aldehyde group in the PLP molecule binds to the ε-amino group of the highly conserved active site lysine residue in the protein to generate an internal aldehyde imine. After the substrate enters the enzyme's active site, the unprotonated amino groups of the substrate attack the Schiff base bond of the inner aldehyde imine, leading to the decomposition of the inner aldehyde imine and the formation of a new Schiff base bond between the substrate and PLP, known as the outer aldehyde imine. This outer aldehyde imine loses its α-carboxyl group, forming a quinone intermediate. The quinone intermediate interacts with acidic tyrosine residues in the protein, causing protonation of the carbocyclic ring, which then leads to the regeneration of the imine complex and the release of aromatic amines and CO2 products. Researchers have found that the activity of the poppy tyrosine decarboxylase PsTyrDCII is regulated by the concentration of PLP in the cell. Based on this, the crystal structure of the PsTyrDCII-PLP bound form of tyrosine decarboxylase was determined. In the PsTyrDCII-PLP structure, the PLP molecule is coordinated with Asp287, Asn316, and Ser370 via hydrogen bonds. Furthermore, by comparing the structures of PsTyrDCII-PLP and PsTyrDCII, it was found that the binding of PLP did not induce significant conformational changes in the overall structure of PsTyrDCII. However, the binding pockets of PLP showed conformational changes at Phe124, His203, and Thr262, indicating that Phe124, His203, and Thr262 play important roles in binding and stabilizing the cofactor PLP.

[0004] It is evident that most of the known tyrosine decarboxylases (TyDC) are PLP-dependent enzymes. They all require the cofactor PLP to catalyze the decarboxylation of tyrosine to tyramine, which increases the input of raw materials, increases production costs, and reduces production efficiency. Summary of the Invention

[0005] In view of this, one object of the present invention is to provide a tyrosine decarboxylase CtTyDC. This tyrosine decarboxylase CtTyDC catalyzes the decarboxylation of tyrosine to produce tyramine without the participation of PLP, greatly simplifying the reaction steps and saving reaction costs, and has good application prospects in industrial production and practical applications.

[0006] Another object of the present invention is to provide a gene encoding the above-mentioned tyrosine decarboxylase CtTyDC.

[0007] Another object of the present invention is to provide a kit prepared using the above-mentioned tyrosine decarboxylase CtTyDC.

[0008] Another object of the present invention is to provide a method for preparing tyramine. This method utilizes the tyrosine decarboxylase CtTyDC to catalyze the decarboxylation of tyrosine to produce tyramine in vitro, and the reaction does not require the addition of the cofactor PLP.

[0009] The present invention achieves the above objectives using the following technical solutions.

[0010] The present invention provides a tyrosine decarboxylase CtTyDC, the amino acid sequence of which is shown in SEQ ID NO:1, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids.

[0011] The tyrosine decarboxylase CtTyDC according to the present invention is preferably derived from Cistanche tubulosa.

[0012] The present invention also provides a gene encoding the above-mentioned tyrosine decarboxylase CtTyDC, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0013] The present invention further provides a gene encoding the above-mentioned tyrosine decarboxylase CtTyDC, the full-length cDNA sequence of which is shown in SEQ ID NO:3.

[0014] The present invention also provides a kit prepared using the tyrosine decarboxylase CtTyDC as described above.

[0015] The present invention also provides a method for preparing tyramine, comprising reacting a reaction system containing the above-described tyrosine decarboxylase CtTyDC and a tyrosine substrate under suitable conditions to obtain tyramine.

[0016] According to the method of the present invention, preferably, the tyrosine substrate includes at least one of L-tyrosine and L-DOPA.

[0017] According to the method of the present invention, preferably, the reaction system further comprises a buffer solution.

[0018] According to the method of the present invention, preferably, the reaction system, in 150 μL, contains 1–8 mM tyrosine substrate, 10–70 μg tyrosine decarboxylase CtTyDC, and 80–120 mM sodium phosphate buffer solution, with the sodium phosphate buffer solution being brought to a final volume of 150 μL.

[0019] According to the method of the present invention, preferably, the suitable conditions include a reaction temperature of 20–40°C and a reaction time of 10–50 hours.

[0020] This invention cloned the tyrosine decarboxylase CtTyDC from *Cistanche tubulosa*. Enzymatic catalysis revealed that this enzyme catalyzes the decarboxylation of tyrosine to tyramine, and the reaction is independent of the cofactor PLP. Ultraviolet and proteomic analyses further ruled out the possibility of endogenous PLP involvement, thus further demonstrating the PLP-independent nature of the enzyme's tyrosine decarboxylation reaction. Structure-guided site-directed mutagenesis identified the key amino acid sites responsible for the PLP-independent characteristic of CtTyDC. Using the CtTyDC tyrosine decarboxylase of this invention to prepare tyramine reduces raw material input, effectively saves costs, simplifies reaction steps, and improves efficiency. Attached Figure Description

[0021] Figure 1 This is an agarose gel electrophoresis image of the coding region of the tyrosine decarboxylase CtTyDC gene of the present invention.

[0022] Figure 2 This is an SDS-PAGE electrophoresis image of the tyrosine decarboxylase CtTyDC obtained by heterologous expression in this invention.

[0023] Figure 3 This is an HPLC-HR-ESI-MS chromatogram of the L-tyrosine reaction catalyzed by the tyrosine decarboxylase CtTyDC of the present invention.

[0024] Figure 4 This is a high-performance liquid chromatogram of the reaction of L-tyrosine and D-tyrosine catalyzed by the tyrosine decarboxylase CtTyDC of the present invention.

[0025] Figures 5(A), 5(B), and 5(C) show the relative activities of the tyrosine decarboxylase CtTyDC at different reaction times, temperatures, and pH values ​​when catalyzing L-tyrosine.

[0026] Figure 6 The three-dimensional structure of the tyrosine decarboxylase CtTyDC of the present invention was obtained by homology modeling.

[0027] Figure 7 This is an activity diagram of the CtTyDC mutant protein of the tyrosine decarboxylase of the present invention. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0029] This invention marks the first time an enzyme capable of catalyzing tyrosine decarboxylation has been cloned from *Cistanche tubulosa*, a traditional Chinese medicine rich in phenylethanoid glycosides. Further screening, identification, and optimization yielded a highly active and stable tyrosine decarboxylase, CtTyDC. Based on this, the invention was completed.

[0030] The present invention provides a method for preparing tyramine, comprising reacting a reaction system containing tyrosine decarboxylase CtTyDC and a tyrosine substrate under suitable conditions to obtain tyramine. The "reaction system of tyrosine decarboxylase CtTyDC and tyrosine substrate" is sometimes simply referred to as the "reaction system".

[0031] <Tyrosine decarboxylase CtTyDC>

[0032] A tyrosine decarboxylase CtTyDC, wherein the amino acid sequence of the tyrosine decarboxylase CtTyDC is as shown in SEQ ID NO:1, or the sequence is modified by substitution, deletion or addition of one or more amino acids to form an amino acid sequence with equivalent function.

[0033] This invention is the first to screen and identify a tyrosine decarboxylase, named CtTyDC, from the plant *Cistanche tubulosa* (Schenk.) Wight. The tyrosine decarboxylase CtTyDC of this invention can be a natural protease or an enzyme containing mutations that still possesses catalytic tyrosine decarboxylation activity. Preferably, the tyrosine decarboxylase CtTyDC of this invention comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence with equivalent function formed by substitution, deletion, or addition of one or more amino acids. In some embodiments, the amino acid sequence of the tyrosine decarboxylase CtTyDC has more than 90%, preferably more than 92%, more preferably more than 95%, further preferably more than 98%, and even more preferably more than 99% homology with the sequence shown in SEQ ID NO:1 and originates from the same species, *Cistanche tubulosa*. In this application, "homology" refers to the similarity between two sequences, which can be determined by any algorithm known in the art. For example, the degree of identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm. Preferably, the tyrosine decarboxylase CtTyDC of the present invention has a molecular weight of 56.33 kDa; a theoretical isoelectric point of 6.68, indicating that it is an acidic protein; an instability index of 48.27, indicating that the protein may be unstable; and a grand average of hydrophobicity (GRAVY) of -0.041, indicating that it is a hydrophilic protein composed of 509 amino acids.

[0034] <Encoding gene>

[0035] A gene encoding the aforementioned tyrosine decarboxylase CtTyDC has a nucleotide sequence as shown in SEQ ID NO:2, which is 1,530 bp in length. The full-length cDNA sequence of this nucleotide sequence is shown in SEQ ID NO:3, which is 1,882 bp in length, with a 5′ untranslated region of 209 bp and a 3′ untranslated region of 143 bp.

[0036] This invention investigated key sites of the catalytic activity of CtTyDC protein through protein homology modeling, molecular docking, and structure-guided site-directed mutagenesis. It was found that mutations at sites F100, P101, S102, T166, T261, and L324 significantly reduced enzyme activity, but the activity recovered after the addition of coenzyme PLP to the reaction system. It is further speculated that these key sites lead to the characteristic of tyrosine decarboxylase CtTyDC having a catalytic reaction independent of exogenous cofactor PLP.

[0037] <Reagent Kit>

[0038] A kit prepared using the aforementioned tyrosine decarboxylase CtTyDC. Existing research indicates that tyrosine decarboxylases are closely related to the synthesis of various secondary metabolites, which have therapeutic effects on certain human diseases, such as Alzheimer's disease, tumors, and inflammation. Therefore, preparing tyrosine decarboxylase CtTyDC into a kit format allows for better clinical application and has greater practical value.

[0039] <Methods for preparing tyramine>

[0040] A method for preparing tyramine includes reacting a reaction system containing tyrosine decarboxylase CtTyDC and a tyrosine substrate under suitable conditions to obtain tyramine.

[0041] In this invention, the tyrosine substrate includes at least one of L-tyrosine and L-DOPA. Through in vitro catalytic activity studies of the tyrosine decarboxylase CtTyDC, this invention revealed that CtTyDC exhibits significant substrate selectivity and stereocatalytic specificity, demonstrating pronounced catalytic properties for L-tyrosine and L-DOPA, while failing to catalyze decarboxylation reactions when using L-phenylalanine, L-tryptophan, or L-histidine as substrates. This indicates that CtTyDC possesses strict conformational selectivity for its substrates, accepting only L-tyrosine and L-DOPA as substrates.

[0042] In some embodiments, the reaction system of the present invention may further include a buffer solution. Preferably, the pH value of the buffer solution is in the range of 6.5 to 8.5, and more preferably 8.0. Preferably, the buffer solution may be a phosphate buffer solution, such as potassium phosphate buffer solution or sodium phosphate buffer solution. The concentration of the salt in the buffer solution is not particularly limited, as long as it can provide the pH range described in the present invention. Preferably, the concentration of the salt is 50 to 150 mM, and more preferably 100 mM.

[0043] In some embodiments, the reaction system, in 150 μL, contains 1–8 mM tyrosine substrate, 10–70 μg tyrosine decarboxylase CtTyDC, and 50–150 mM sodium phosphate buffer solution, with the sodium phosphate buffer solution brought to 150 μL, and a reaction system scaled up proportionally.

[0044] According to a preferred embodiment of the present invention, the reaction system, in 150 μL, contains 4 mM L-tyrosine substrate, 50 μg tyrosine decarboxylase CtTyDC and 100 mM sodium phosphate buffer solution, with the sodium phosphate buffer solution being brought to a total of 150 μL.

[0045] The "suitable conditions" described in this invention refer to conditions that ensure the reaction between tyrosine decarboxylase CtTyDC and the tyrosine substrate. These conditions include a reaction temperature of 20–40°C, preferably 25–35°C, and more preferably 30°C. The conditions also include a reaction time of 10–50 hours, preferably 11–48 hours, and more preferably 12 hours.

[0046] Preferably, the reaction system of the present invention may further contain other substances required for the reaction.

[0047] Example 1 - Obtaining the gene encoding the tyrosine decarboxylase CtTyDC 1. Extraction of total RNA from fresh explants of Cistanche tubulosa and preparation of RACE template

[0048] Fresh fleshy stems of *Cistanche tubulosa* plants were selected, and total RNA was extracted using a liquid nitrogen quick-freezing and grinding method according to the specific procedures of the OMEGA RNA Plantkit. Agarose gel electrophoresis was used to assess quality, and RNA concentration was determined using a Nanodrop 2000 spectrophotometer. RNA with no residual genomic DNA and an A260 / 280 value below 2.0 was selected and reverse transcribed using the SMARTer RACE5′ / 3′ Kit (ClonTech) to obtain 5′-RACE-cDNA, 3′-RACE-cDNA, and total cDNA.

[0049] Obtaining 5′-RACE-cDNA: Take 0.2–2.0 μg of RNA, add 1.0 μL of 5′-CDS-primer A and ddH2O to a final volume of 3.75 μL, and perform PCR step 1 (72℃, 3 min; 42℃, 2 min). After the reaction, immediately place the mixture on ice, add 1.0 μL of SMARTer II A oligo and 4.25 μL of Mix (containing 2.0 μL of 5× First-strand buffer, 1.0 μL of 20 mM DTT, 1.0 μL of 10 mM dNTP, and 0.25 μL of RNase Inhibitor), mix well and briefly centrifuge, then add 1.0 μL of SMARTScribeReverse Transciptase and perform PCR step 2 (42℃, 90 min; 72℃, 10 min). After the reaction, dilute the product with 100 μL of RNase-free water.

[0050] Obtaining 3′-RACE-cDNA: Take 0.2–2.0 μg RNA, add 1.0 μL of 3′-CDS-primer A and ddH2O to a final volume of 4.75 μL, and perform PCR step 1 (72℃, 3 min; 42℃, 2 min). After the reaction, immediately place the sample on ice and add 4.25 μL of LMix (5× First-strand buffer 2.0 μL, 20 mM DTT 1.0 μL, 10 mM dNTP 1.0 μL, RNase Inhibitor 0.25 μL). Mix well and briefly centrifuge. Then add 1.0 μL of SMARTScribe Reverse Transciptase and perform PCR step 2 (42℃, 90 min; 72℃, 10 min). After the reaction, dilute the product with 100 μL of RNase-free water.

[0051] Obtaining RT-cDNA: Take 0.2–2.0 μg of RNA and mix it with 10 μM oligo dT... 20 Add 2.0 μL of ddH2O to a final volume of 5.0 μL and perform PCR step 1 (72℃, 3 min). Add 4.0 μL of Mix (2.0 μL of 5× First-strand buffer, 1.0 μL of 20 mM DTT, and 1.0 μL of 10 mM dNTP) to the reaction product, mix well, briefly centrifuge, and then add 1.0 μL of SMARTScribe Reverse Transciptase. Perform PCR step 2 (42℃, 90 min; 70℃, 15 min). After the reaction is complete, dilute the product with 10 μL of N-ase-free water.

[0052] 2. Rapid amplification of cDNA ends using RACE method

[0053] Cloning of the 5′ and 3′ sequences of the target gene was performed according to SMARTer. TMFollowing the requirements of the RACE cDNA Amplification Kit (Clontech), using approximately 40 ng of 5′ / 3′-RACE-cDNA obtained from RACE reverse transcription as a template, and based on the analysis of previously obtained Cistanche tubulosa transcriptome data, 5′ / 3′-RACE-specific primers for the CtTyDC sequence were designed. The 5′ RACE-specific primer sequence is shown in SEQ ID NO:4, and the 3′ RACE-specific primer sequence is shown in SEQ ID NO:5. Simultaneously, using the kit's universal primers UPM, the corresponding 5′ / 3′ end sequences were amplified with the high-fidelity DNA amplification enzyme KOD-Plus-Neo DNA Polymerase. The 5′ RACE PCR product was 1144 bp, and the 3′ RACE PCR product was 913 bp.

[0054] The reaction system for RACE amplification was as follows: 5′ / 3′-RACE-cDNA: 40 ng, UPM (10 μM): 1.0 μL, 5′ / 3′-RACE specific primer (10 μM): 0.5 μL, 10×KOD buffer: 1.0 μL, MgSO4 (25 mM): 0.6 μL, dNTPs (2 mM): 1.2 μL, KOD DNA Polymerase (1 U / μL): 0.2 μL.

[0055] The PCR reaction program was as follows: pre-denaturation at 94℃ for 2 min; followed by denaturation at 94℃ for 15 s, initial annealing at 65℃ for 30 s, decreasing the temperature by 0.5℃ per cycle, extension at 68℃ for 50 s, for a total of 30 cycles; then denaturation at 94℃ for 15 s, annealing at 54℃ for 30 s, extension at 68℃ for 50 s, for a total of 25 cycles; and finally extension at 72℃ for 1 min.

[0056] The 5′RACE and 3′RACE sequences obtained from sequencing were spliced ​​to obtain the full-length cDNA sequence as shown in SEQ ID NO:3. The cDNA fragment sequences obtained from 5′-RACE and 3′-RACE were analyzed, and a pair of specific primers with restriction enzyme sites were designed, as shown in SEQ ID NO:6 and 7. SEQ ID NO:6 introduced the EcoR I restriction site through primer design, and SEQ ID NO:7 introduced the Xho I restriction site through primer design. The full-length coding region of approximately 1530 bp was amplified using the pair of specific primers, as shown in SEQ ID NO:2. The encoding amino acid sequence is shown in SEQ ID NO:1. Agarose gel electrophoresis was performed as follows: Figure 1 As shown. Figure 1 In this context, M stands for DNA Marker, and the number to the left (e.g., 3000bp) indicates the size of the DNA in the Marker.

[0057] Example 2 - Bioinformatics Analysis of Tyrosine Decarboxylase CtTyDC

[0058] Based on the obtained full-length CtTyDC sequence, the open reading frames of the gene were analyzed using ORF Finder; homology analysis of the amino acid sequence encoded by CtTyDC was performed using BLASTX in NCBI; and the physicochemical properties of the encoded protein were predicted using the ExPASy online server (http: / / web.expasy.org / protparam / ). BLAST sequence analysis of the amino acid sequence encoded by CtTyDC showed that the two sequences had a similarity of up to 89% with reported aromatic amino acid decarboxylases. Predictions of the physicochemical properties of the protein encoded by CtTyDC using ExPASy showed a molecular weight of 56.33 kDa; a theoretical isoelectric point of 6.68, indicating an acidic protein; and a Grand average of hydrophobicity (GRAVY) of -0.041, indicating a hydrophilic protein.

[0059] Example 3 - Construction and prokaryotic expression of the prokaryotic expression vector for the tyrosine decarboxylase CtTyDC gene

[0060] To verify whether the obtained CtTyDC sequence possesses tyrosine decarboxylase function, suitable forward and reverse primers with restriction sites were designed based on the restriction site information of the pET28a expression vector and the obtained full-length CtTyDC sequence. Using RT-cDNA obtained by conventional reverse transcription as a template, PCR amplification was performed using KOD-Plus-Neo DNA Polymerase. PCR products with fragment sizes consistent with predicted values ​​were selected for gel recovery. The recovered products were then compared with... Simple cloning vectors were ligated and transformed into *E. coli* Trans1-T1 competent cells. Positive clones were selected using blue-white screening and colony PCR, and sequenced using M13F and SR primers to verify sequence accuracy. Plasmids were extracted from positive clones with correct sequencing results by shaking.

[0061] The extracted CtTyDC plasmid and pET28a vector were double-digested with the designed restriction enzyme sites. Digestion was performed at 37°C for 5 h, and the products were recovered by gel electrophoresis. The target gene (53 ng / 1.4 kb) was ligated into the pET-28a vector (66 ng / 5.3 kb) using 0.5 μL of NEB T4 DNA ligase, followed by the addition of 1.0 μL of 10×T4 buffer and ddH2O to a final volume of 10 μL. The mixture was incubated overnight at 16°C. 5 μL of the ligation product was transformed into competent *E. coli* Trans1-T1 cells and plated on LB agar plates containing Kana inhibitors. The cells were incubated overnight at 37°C. Positive clones were screened by colony PCR, and sequenced using T7F and T7R primers for sequence verification. Positive clones with correct sequencing results were cultured to extract plasmids. 5 μL of the recombinant plasmid pET-28a-CtTyDC was transformed into 50 μL of *E. coli* competent cells Transetta(DE3). The plasmids were plated on LB agar plates containing Kana and Chl antibodies and incubated overnight at 37°C. Positive clones were screened by colony PCR, and the sequences were verified by sequencing. Single colonies with correct sequencing results were inoculated into LB liquid medium (containing 50 μg / mL Kana and 40 μg / mL Chl) and incubated at 37°C, 200 rpm / min for 5 h. After incubation, the colonies were transferred to liquid medium at a 1:100 (V / V) ratio for scale-up. Incubation continued at 37°C, 200 rpm / min until the bacterial culture reached OD500. 600 When the concentration of the molecule is approximately 0.6, IPTG is added to a final concentration of 0.7 mM, and the cell induction is performed at 23°C and 180 rpm / min for 16 h. The cells are collected by high-speed centrifugation (7600 × g, 4°C, 6 min), 3 mL / g Lysis buffer is added, the cells are resuspended and mixed, and the mixture is placed in an ice-water mixture for ultrasonic disruption. The disruption is repeated for 2 s, paused for 4 s, and continued for 8 min. The lysate was centrifuged at high speed (8000×g, 4℃, 40min). The supernatant was filtered through a 0.45μm filter and loaded onto a Ni-NTAHis-Bind Resin affinity column pre-equilibrated with binding buffer (flow rate <1mL / min). After washing with 20 column volumes of binding buffer to remove contaminating proteins, the column was eluted with a gradient of imidazole concentrations (68mM, 164mM, 260mM, 404mM). The fractions containing the target protein were combined and concentrated to 1mL with 50kD millipore solution. After desalting twice with desalting buffer, the concentration was further concentrated to 1mL. Protein concentration was measured using an Easy Protein Quantitative Kit and a microplate reader for activity assay. SDS-PAGE results showed that CtTyDC protein was solublely expressed in E. coli. Figure 2 As shown, the size of the recombinant protein is basically consistent with the theoretical molecular weight of 56.33 kD. Figure 2In this context, M stands for protein marker, and the number on the left (e.g., 100kD) indicates the size of the protein band in the marker.

[0062] Example 4 - In vitro enzyme activity experiment and HPLC analysis of tyrosine decarboxylase CtTyDC protein.

[0063] HR-MS testing

[0064] The in vitro enzymatic reaction system for CtTyDC protein is as follows: 100 mM sodium phosphate buffer (pH 8.0), 4 mM L-tyrosine, 50 μg CtTyDC protein, reaction volume 150 μL. The reaction was carried out in a water bath at 28℃ for 12 h. The reaction was terminated by adding 2 volumes of methanol, vortexed, and centrifuged at 15000×g at 4℃ for 1 h. The supernatant was then analyzed using an Agilent 1260 HPLC system.

[0065] Chromatographic column: XBridge BEH Amide Column (4.6mm×150mm, 3.5μm), flow rate 1.0mL / min, column temperature 30℃, detection wavelength 280nm, mobile phase acetonitrile (A) and 0.1% formic acid water (B), elution program as follows: A:B (v / v): 0min (100:0)-10min (85:15)-22min (55:45).

[0066] High-resolution mass spectrometry analysis was performed using a Shimadzu LCMS-IT-TOF ion trap time-of-flight mass spectrometer. The chromatographic column and liquid chromatography conditions were the same as described above.

[0067] The mass spectrometry parameters are as follows: positive ion mode, negative ion mode, and automatic multi-stage MS. 1 MS 2 MS 3 Full scan; nebulizing gas: N2, flow rate: 1.5 mL / min; drying gas: N2, pressure: 100 MPa; interface voltage: 1.40 kV; ion trap vacuum: 1.9 × 10⁻⁶ -2 High-purity argon was used as both the cooling gas and the collision gas for collision-induced dissociation (CID). The ion accumulation time was set to 100 ms, and the CID collision energy was set to 50%.

[0068] By using high-performance liquid chromatography-ultraviolet characteristic absorption spectroscopy, combined with Figure 3The high-resolution mass spectrometry molecular weight analysis and molecular formula prediction shown indicate the formation of tyramine. The reaction formula of L-tyramine catalyzed by the tyrosine decarboxylase CtTyDC is shown below.

[0069]

[0070] Example 5 - Investigation of substrate selectivity and stereoselectivity of CtTyDC catalyst

[0071] To investigate the substrate selectivity of the obtained tyrosine decarboxylase CtTyDC, tyrosine structural analogs such as L-DOPA, L-phenylalanine, L-tryptophan, and L-histidine were selected as substrates for in vitro enzymatic reactions. The results showed that tyrosine decarboxylase CtTyDC also exhibited significant catalytic properties for L-DOPA, but could not catalyze the decarboxylation of other types of substrates. Furthermore, the stereoselectivity of tyrosine decarboxylase CtTyDC was examined. The results showed that when D-tyrosine was used as a substrate, tyrosine decarboxylase CtTyDC could not catalyze the decarboxylation reaction, while when L-tyrosine was used as a substrate, tyrosine decarboxylase CtTyDC could catalyze the decarboxylation reaction. Figure 4 As shown, this indicates that the tyrosine decarboxylase CtTyDC exhibits strict conformational selectivity for its substrates, a result consistent with the fact that all naturally occurring amino acids available to organisms in nature are levorotatory.

[0072] Example 6 - Optimal catalytic conditions for the in vitro enzymatic reaction of CtTyDC

[0073] Based on the identification of the function of tyrosine decarboxylase CtTyDC, the optimal conditions for CtTyDC-catalyzed in vitro enzymatic reactions were further explored. Using L-tyrosine as a substrate, the trend of CtTyDC-catalyzed reaction conversion over time and the effects of temperature and pH on enzyme catalytic activity were investigated.

[0074] To investigate the conversion rate over time, the following procedures were followed: In a sodium phosphate buffer system (pH 8.0), L-tyrosine was added first, followed by CtTyDC protein, and timing was started. Samples were taken at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and the reaction was terminated by adding twice the volume of methanol. The supernatant was collected by high-speed centrifugation and analyzed by HPLC. The results showed that the conversion rate increased linearly from 0 to 4 h; the increase slowed from 4 to 12 h; after 12 h, the conversion rate stabilized and approached 100% after 48 h of reaction.

[0075] The effect of temperature on the activity of tyrosine decarboxylase CtTyDC was investigated. The specific procedure was as follows: the reaction mixture was added to ice, and then the reaction was carried out at 0℃, 4℃, 16℃, 25℃, 30℃, 37℃, 42℃, 50℃, and 65℃, respectively. After 3 hours of reaction, twice the volume of methanol was added to terminate the reaction. The supernatant was collected by high-speed centrifugation and analyzed by HPLC. The effect of temperature on enzyme activity was as follows: between 0 and 30℃, enzyme activity increased with increasing temperature, reaching its peak at 30℃, and then decreased with further increases in temperature. Therefore, 30℃ was determined to be the optimal temperature for this reaction.

[0076] The effect of pH on the activity of tyrosine decarboxylase CtTyDC was investigated. The specific procedure was as follows: L-tyrosine and CtTyDC protein were added to buffers with different pH values: 0.1M acetate-sodium acetate buffer (pH 4.0–5.0), 0.2M phosphate buffer (pH 6.0–8.0), 0.2M Tris-HCl buffer (pH 8.0–9.0), and 0.1M sodium carbonate-sodium bicarbonate buffer (pH 9.0–11.0). The reaction was carried out at 30℃ for 3 hours, and then 2 volumes of methanol were added to terminate the reaction. The supernatant was collected by high-speed centrifugation and analyzed by HPLC. The effect of pH on enzyme activity was as follows: when the pH was below 6.5, enzyme activity was lost; between pH 6.5 and 8.0, enzyme activity increased with increasing pH, reaching its peak at pH 8.0, and then decreased with further increases in pH. Therefore, pH=8 was determined to be the optimal pH value for this reaction, and the results are shown in Figures 5(A), 5(B) and 5(C).

[0077] Example 7 - UV and mass spectrometry analysis of CtTyDC protein

[0078] Since PLPs are one of the most basic cofactors in organisms and exist in multiple organelles, and lysine residues in the protein sequence readily bind to PLPs, thus introducing endogenous PLPs, the addition of PLPs to the CtTyDC protein to catalyze the tyrosine decarboxylation reaction was not due to the introduction of endogenous PLPs. Therefore, to rule out the possibility that the CtTyDC protein's catalytic tyrosine decarboxylation reaction did not require the addition of PLPs due to the incorporation of endogenous PLPs, the CtTyDC protein was analyzed by ultraviolet and mass spectrometry.

[0079] The CtTyDC protein was detected by UV using NanoDrop 2000. The results showed that the CtTyDC protein only had an absorption peak at 280 nm, but no characteristic absorption peak of PLP, indicating that the CtTyDC protein does not have endogenous binding to PLP.

[0080] To further confirm the results, a certain amount of NaBH4 (158 mM, reducing agent; if endogenous PLP binding is present, the addition of NaBH4 can stabilize PLP binding, facilitating mass spectrometry detection) was added to CtTyDC protein (7.60 mM) and incubated at 25°C for 30 min. Protein purity was detected using SDS-PAGE. The protein gel was stained with Coomassie Brilliant Blue, the target band was excised, and the protein was reduced by adding 10 mM dithiothreitol (DTT), followed by 55 mM iodoacetic acid ammonium (IAM), and finally 1 μg of Trypsin enzyme was added. Enzymatic digestion was carried out overnight for 8–16 h. The resulting peptides were desalted using a C18 column, and the desalted peptides were dried and dissolved in 15 μL of Loading Buffer (0.1% formic acid, 3% acetonitrile). The peptide was analyzed using LC-MS / MS (ekspert™ nanoLC; AB Sciex TripleTOF 5600-plus). PLP is thought to bind to lysine residues in proteins via Schiff base bonds. If PLP binds to CtTyDC protein, K318 is considered the binding site. Therefore, proteomic analysis of the CtTyDC protein sequence showed that lysine at position 318 did not bind PLP, nor did any other lysine residues. This result confirms that CtTyDC protein does not endogenously bind PLP. This further confirms that the decarboxylation reaction catalyzed by the tyrosine decarboxylase CtTyDC is independent of PLP.

[0081] Example 8 - Homology modeling and molecular docking of CtTyDC protein

[0082] To elucidate the PLP-independent molecular mechanism of CtTyDC-catalyzed tyrosine decarboxylation, homology modeling and molecular docking studies were conducted on the CtTyDC protein. A tyrosine decarboxylase derived from *P. somniferum* (PDB ID: 6EEM) was selected as a template. The three-dimensional structure of the CtTyDC protein was constructed using Modeller 9.14 software. The constructed structures were evaluated using Ramachandran plot and PROFILE 3D scoring in the PROCHECK program. The structure with the lowest probability density function (PDF), lowest DOPE statistical potential, and GA341 score closest to 1.0 was selected as the modeling result and applied to subsequent molecular docking. The optimal structure is shown below. Figure 6As shown, its total PDF energy is 2542.99561 molpdf, DOPE score is -59838.97656, and GA341 score is 1.0000. The decarboxylation reaction catalyzed by tyrosine decarboxylase requires the participation of the substrate tyrosine and the coenzyme PLP; therefore, the binding sites of the two ligands need to be analyzed separately. Using NCBI BLAST alignment analysis, the protein crystal complex Dopa decarboxylase (PDB ID: 1JS3), which has high homology and contains both ligands, was selected as the alignment template. Molecular docking was performed using the AutoDock vina program with a grid size of 40×40×40 and a grid spacing of 0.375 nm. AutoDock Tools 1.5.6 was used to prepare for docking of PLP, tyrosine ligands, and the target protein receptor. The three-dimensional center coordinates of the PLP ligand binding pocket are x = 45.86, y = 35.322, z = 66.377; the three-dimensional center coordinates of the tyrosine ligand binding pocket are x = 40.974, y = 36.952, z = 67.83. Optimization was performed using the Lamarckian genetic algorithm (LGA), with docking parameters set to complete the docking process. The binding freedom (K...) was selected. cat The docking result with the lowest possible value ( / mol) was processed using Pymol software to search for... The amino acid residues within the specified range serve as active binding sites.

[0083] Example 9 - Construction of CtTyDC mutant vector and determination of enzyme activity

[0084] Based on the homology modeling and molecular docking results, this application selected 14 key sites in the CtTyDC amino acid sequence for mutation, including sites W91, Y99, F100, P101, S102, T165, T166, C167, H202, T261, H317, K318, L324, and R478, and performed alanine scanning mutations on the above sites.

[0085] Using pET28a-CtTyDC plasmid as a template, PCR amplification was performed with KOD-Plus-Neo high-fidelity enzyme and mutant primers. The PCR product was demethylated by adding 1 μL DMT enzyme and incubating at 37°C for 1 h. Afterwards, PCR purification was performed according to the BIOMIG Gel / PCR Extraction Kit instructions, and the resulting cells were transformed into *E. coli* DMT competent cells, plated on LB agar plates containing Kana inhibitors, and incubated overnight at 37°C. Positive clones were screened by colony PCR and sequenced using T7F and T7R primers. The successfully mutated plasmid was reintroduced into *Transetta* (DE3) strain for induced expression, and the expression products were purified. Results showed that the T165A mutant protein was not expressed, while the other mutant proteins were expressed normally.

[0086] To further investigate the effects of these amino acid site mutations on the catalytic activity of CtTyDC, we conducted an in vitro enzymatic reaction using L-tyrosine as a substrate. The reaction system was as follows: 4 mM L-tyrosine, 50 μg of purified CtTyDC mutant protein, and 100 mM sodium phosphate buffer (pH 8.0) to a final volume of 150 μL. The reaction was carried out at 30 °C for 12 hours. In addition, the effect of the presence of coenzyme PLP on the activity of the mutant protein was also investigated. The reaction system was as follows: 4 mM L-tyrosine, 50 μg of purified CtTyDC mutant protein, 1 mM PLP, and 100 mM sodium phosphate buffer (pH 8.0) to a final volume of 150 μL. The reaction was carried out at 30 °C for 12 hours. The results are as follows. Figure 7 As shown, mutations at W91, C167, H202, H317, K318, and R478 to alanine (Ala) resulted in complete loss of protein activity regardless of the addition of coenzyme PLP to the reaction system, indicating that these sites are key sites for the CtTyDC-catalyzed decarboxylation reaction. Mutations at F100, P101, S102, T166, T261, and L324 reduced enzyme activity, but the activity recovered after the addition of coenzyme PLP to the reaction system, suggesting that these sites are related to the PLP-independent catalytic characteristic of CtTyDC. Figure 7 In the diagram, the suffix "A" on the horizontal axis indicates an amino acid mutation. For example, W91A represents a mutation at site W91 into alanine (Ala).

[0087] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention. sequence list <110> Beijing University of Chinese Medicine <120> Tyrosine decarboxylase CtTyDC, encoding gene, kit and method for preparing tyramine <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 509 <212> PRT <213> Cistanche tubulosa <400> 1 Met Gly Ser Leu Gln Asn Lys Lys Pro Asp Gly Ala Ala Ser Ala Gly 1 5 10 15 Ile Ile Lys Pro Met Asp Pro Glu Glu Phe Arg Arg Gln Gly His Leu 20 25 30 Val Ile Asp Phe Ile Ala Asp Tyr Tyr Lys Asn Val Glu Lys Tyr Pro 35 40 45 Val Arg Ser Gln Val Glu Pro Gly Tyr Leu Lys Lys Arg Met Pro Asp 50 55 60 Ser Ala Pro His Gly Pro Glu Pro Ile Glu Glu Ile Leu Arg Asp Val 65 70 75 80 Gln Asn Asp Ile Val Pro Gly Ile Thr His Trp Gln Ser Pro Asn Tyr 85 90 95 Tyr Ala Tyr Phe Pro Ser Ser Gly Ser Ile Ala Gly Phe Leu Gly Glu 100 105 110 Met Leu Ser Thr Gly Phe Asn Ile Val Gly Phe Asn Trp Met Ser Ser 115 120 125 Pro Ala Ala Thr Glu Leu Glu Ser Ile Val Met Asp Trp Leu Gly Lys 130 135 140 Met Leu Lys Leu Pro Ser Glu Phe Leu Phe Ser Gly Gly Gly Gly Gly 145 150 155 160 Val Leu Gln Gly Thr Thr Cys Glu Ala Ile Leu Cys Thr Leu Val Ala 165 170 175 Ala Arg Asp Gln Met Leu Arg Lys Ile Gly Arg Glu Asn Ile Asn Lys 180 185 190 Leu Val Val Tyr Gly Ser Asp Gln Thr His Ser Ala Leu Gln Lys Ala 195 200 205 Ala Gln Ile Ala Gly Ile Asn Pro Asn Asn Phe Arg Ala Val Ser Thr 210 215 220 Thr Lys Glu Thr Ala Phe Gly Leu Thr Gly Lys Ala Leu Arg Ala Ala 225 230 235 240 Ile Glu Ser Asp Val Asp Leu Gly Leu Val Pro Leu Phe Leu Cys Ala 245 250 255 Thr Val Gly Thr Thr Ser Ser Thr Ala Val Asp Pro Leu Glu Pro Leu 260 265 270 Cys Asp Val Ala Glu Glu Tyr Gly Val Trp Val His Val Asp Ala Ala 275 280 285 Tyr Ala Gly Ser Val Cys Ile Cys Pro Glu Tyr Arg His Phe Ile Asp 290 295 300 Gly Val Glu Lys Ala His Ser Phe Ser Phe Asn Ala His Lys Trp Phe 305 310 315 320 Leu Thr Thr Leu Asp Cys Cys Cys Leu Trp Val Lys Asp Pro Gly Ala 325 330 335 Leu Val Lys Ala Leu Ser Thr Tyr Pro Glu Tyr Leu Arg Asn Lys Ala 340 345 350 Ser Glu Thr Lys Gln Val Val Asp Tyr Lys Asp Trp Gln Ile Thr Leu 355 360 365 Ser Arg Arg Phe Arg Ser Leu Lys Leu Trp Met Val Leu Arg Ser Tyr 370 375 380 Gly Val Ala Asn Leu Arg Lys Phe Leu Arg Ser His Val Lys Met Ala 385 390 395 400 Met Asn Phe Glu Gly Leu Val Gly Met Asp Lys Arg Phe Glu Val Val 405 410 415 Val Pro Arg Asn Phe Ala Thr Val Cys Phe Arg Ile Ser Pro Val Glu 420 425 430 Ile Ala Gly Asn His Gln Met Ile Ala Ser Arg Glu Glu Ala Ala Asn 435 440 445 Ser Phe Asn Ala Arg Leu Leu Glu Met Ile Asn Glu Ser Gly Glu Val 450 455 460 Tyr Met Thr His Ala Val Val Gly Gly Val Tyr Val Met Arg Phe Ala 465 470 475 480 Val Gly Ala Ser Leu Thr Glu Asn Arg His Val Ile Leu Ala Trp Lys 485,490,495 Val Val Gln Glu His Ala Asn Ala Leu Leu Ala Ser Ser Ser 500,505 <210> 2 <211> 1530 <212> DNA <213> Tubular chestnuts <400> 2 atgggcagcc ttcagaataa aaaaccagac ggtgccgcct ccgccggcat cattaaacct 60 atggaccccg agagttcag gcggcagggc catcttgtca tcgactcat cgccgattac 120 tacagaacg tagagaata ccctgtccga agccaagtcg aaccggcta tctaagaaa 180 cggatgccag attccgctcc acacggccca gaaccaatcg aggaatcct ccgcgacgtc 240 caaaacgaca tcgttccggg catcacacac tggcagagcc ccactatta tgcttactc 300 ccgtccagtg ggagtatagc aggatttctc ggagaatgc tgagcactgg ttttacatc 360 gttggattta actggatgtc gtctccggcc gccactgagc tggagcat cgtcatggac 420 tggctcggga aaatgctcaa gctcccgtcg gagttttgt tctccggcgg aggcggcgga 480 gtcttgcagg gaaccacctg cgaggccatt ctgtgcactc ttgtcgccgc ccgagaccag 540 atgctgagaa agatcggcag agagaatatt aacaagttgg tcgtgtacgg gtcggatcag 600 acccactccg cgttgcaaaa ggcggcccag atcgctggca tcaacccgaa taatttccgg 660 gccgtatcca ccacgaagga aacagcattc gggctgacgg ggaaggccct ccgcgccgcg 720 atcgagtccg atgtggatct cgggctggtg ccgctgttt tatgcgccac cgttgggacg 780 acatcgtcga ccgccgtgga cccgctggaa ccgctgtgcg acgtggcgga ggagtacggg 840 gtttgggtcc acgtggacgc ggcctacgcc ggcagcgttt gtatttgccc tgagtatcgc 900 catttcatcg atggggttga aaaagcgcat tcgttcagct ttaacgcgca taagtggttt 960 ttgacgactt tagattgctg ctgcctgtgg gttaaggacc caggcgccct ggtaaaagcg 1020 ctatcaactt atcccgagta tttgagaaac aaagcctcgg agacaaaaca ggtcgtcgat 1080 tacaaagact ggcaaatcac tctcagccga cgattccgat cactgaagct ctggatggtc 1140 ctccgtagct atggcgtggc caacctcagg aaattcctcc gcagccacgt gaaaatggcg 1200 atgaatttcg aaggtctggt cggaatggac aagcggttcg aggtggtggt gccgagaaac 1260 ttcgccaccg tctgctccg gatctcgccg gtggagatcg ccggaacca ccagatgatc 1320 gcttcgagggg aggaggcggc caatagtttt aacgcgagat tgctggagat gataaatgaa 1380 tcggtgagg tttacatgac gcacgcggtg gttggcggcg tttacgtgat gcgtttcgcc 1440 gtcggtgcga gcttgacgga aaacaggcac gttattgg cctggaagt tgtgcagga 1500 catgcaaacg ctctgttagc cagttcgtga 1530 <210> 3 <211> 1882 <212> DNA <213> Tubular chestnuts <400> 3 ctaatacgac tcactatagg gcaagcagtg gtatcaaccc agagtacatg ggggcaaacc 60 ccccccaaaa aaaaaggaaattattack acattattattattattattattattattack 120 tatcttcttt tctatttcct xaagctaa caagtgaag ataccacat aaagaataat 180 atttgcacca ggctcaat ttaacatcga tgggcagcct tcagataaa aaaccagacg 240 gtgccgccctc cgccggcatc attaaccta tggaccccga agagttcagg cggcaggcc 300 atcttgtcat cgacttcatc gccgattact acagaacgt agagaaatac cctgtccgaa 360 gccaagtcga accgggctat ctaaagaaac ggatgccaga ttccgctcca cacggcccag 420 aaccaatcga ggaaatcctc cgcgacgtcc aaaacgacat cgttccgggc atcacacact 480 ggcagagccc caactattat gcttacttcc cgtccagtgg gagtatagca ggatttctcg 540 gagaaatgct gagcactggt tttaacatcg ttggatttaa ctggatgtcg tctccggccg 600 ccactgagct ggagagcatc gtcatggact ggctcgggaa aatgctcaag ctcccgtcgg 660 agtttttgtt ctccggcgga ggcggcggag tcttgcaggg aaccacctgc gaggccattc 720 tgtgcactct tgtcgccgcc cgagaccaga tgctgagaaa gatcggcaga gagaatatta 780 acaagttggt cgtgtacggg tcggatcaga cccactccgc gttgcaaaag gcggcccaga 840 tcgctggcat caacccgaat aatttccggg ccgtatccac cacgaaggaa acagcattcg 900 ggctgacggg gaaggccctc cgcgccgcga tcgagtccga tgtggatctc gggctggtgc 960 cgctgttttt atgcgccacc gttgggacga catcgtcgac cgccgtggac ccgctggaac 1020 cgctgtgcga cgtggcggag gagtacgggg tttgggtcca cgtggacgcg gcctacgccg 1080 gcagcgtttg tatttgccct gagtatcgcc atttcatcga tggggttgaa aaagcgcatt 1140 cgttcagctt taacgcgcat aagtggtttt tgacgacttt agattgctgc tgcctgtggg 1200 ttaaggaccc aggcgccctg gtaaaagcgc tatcaactta tcccgagtat ttgagaaaca 1260 aagcctcgga gacaaaacag gtcgtcgatt acaaagactg gcaaatcact ctcagccgac 1320 gattccgatc actgaagctc tggatggtcc tccgtagcta tggcgtggcc aacctcagga 1380 aattcctccg cagccacgtg aaaatggcga tgaatttcga aggtctggtc ggaatggaca 1440 agcggttcga ggtggtggtg ccgagaaact tcgccaccgt ctgcttccgg atctcgccgg 1500 tggagatcgc cggaaaccac cagatgatcg cttcgaggga ggaggcggcc aatagtttta 1560 acgcgagatt gctggagatg ataaatgaat cgggtgaggt ttacatgacg cacgcggtgg 1620 ttggcggcgt ttacgtgatg cgtttcgccg tcggtgcgag cttgacggaa aacaggcacg 1680 ttattttggc ctggaaagtt gtgcaggaac atgcaaacgc tctgttagcc agttcgtgat 1740 ttgctttttg tcaacttttc agatttttgt tttaaatgtc ccttgttctg gaaaatatcc 1800 catgtttttg gtttgtgaaa ttattaaata attgaaatat tactattatg ggcaaaaaaa 1860 1882 <210> 4 <211> 28 <212> DNA <213> Artificial sequence <400> 4 gaacgaatgc gctttttcaa ccccatcg 28 <210> 5 <211> 26 <212> DNA <213> Artificial sequence <400> 5 atgcgccacc gttgggacga catcgt 26 <210> 6 <211> 31 <212> DNA <213> Artificial sequence <400> 6 gaattcatgg gcagccttca gaataaaaaa c 31 <210> 7 <211> 29 <212> DNA <213> Artificial sequence <400> 7 ctcgagcgaa ctggctaaca gagcgtttg 29

Claims

1. A tyrosine decarboxylase CtTyDC, characterized in that, The amino acid sequence of the tyrosine decarboxylase CtTyDC is shown in SEQ ID NO:

1.

2. The tyrosine decarboxylase CtTyDC according to claim 1, characterized in that, The tyrosine decarboxylase CtTyDC is derived from Cistanche tubulosa.

3. A gene encoding the tyrosine decarboxylase CtTyDC according to any one of claims 1 to 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

2.

4. A gene encoding the tyrosine decarboxylase CtTyDC according to any one of claims 1 to 2, characterized in that, Its full-length cDNA sequence is shown in SEQ ID NO:

3.

5. A kit prepared from the tyrosine decarboxylase CtTyDC according to any one of claims 1 to 2.

6. A method for preparing tyramine, characterized in that, The method includes reacting a reaction system comprising the tyrosine decarboxylase CtTyDC as described in any one of claims 1 to 2 and a tyrosine substrate under suitable conditions to obtain tyramine.

7. The method according to claim 6, characterized in that, The tyrosine substrate includes at least one of L-tyrosine and L-DOPA.

8. The method according to claim 6, characterized in that, The reaction system further includes a buffer solution.

9. The method according to claim 8, characterized in that, The reaction system, in 150 µL, contains 1–8 mM tyrosine substrate, 10–70 µg tyrosine decarboxylase CtTyDC, and 80–120 mM sodium phosphate buffer solution, with the sodium phosphate buffer solution brought to a final volume of 150 µL.

10. The method according to claim 6, characterized in that, The suitable conditions include a reaction temperature of 20–40°C and a reaction time of 10–50 hours.

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