CN106191018A - A kind of UDP xylose synzyme deriving from Herba Phyllanthi Urinariae, its nucleotide sequence and application - Google Patents

Abstract

The present invention provides a uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger's eye, the amino acid sequence of which is shown in SEQ ID NO.1; The nucleotide sequence of the sugar synthase gene is shown in SEQ ID NO.3, as well as the vector and host cell containing the coding nucleotide sequence. The present invention also provides uridine-5'-diphosphate xylose synthase or a cell containing uridine-5'-diphosphate xylose synthase in catalytic substrate uridine-5'-diphosphate glucuronic acid reaction Applications.

Description

A uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, its nucleus Nucleotide sequence and application

technical field

The invention relates to a uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, its coding gene and its catalytic substrate uridine-5'-diphosphate glucuronic acid is uridine-5' -Application in xylose diphosphate reaction, which belongs to the field of genetic engineering.

Background technique

Uridine-5'-diphosphate xylose synthase (UXS) is the key enzyme to generate uridine-5'-diphosphate xylose (UDP-xylose) in organisms. UXS can decarboxylate uridine-5’-diphosphoglucuronic acid (UDP-glucuronic acid) to form UDP-xylose (Figure 1). UDP-xylose is an important glycosyl donor in the biosynthesis of glycoproteins, polysaccharides, and various glycosyl-modified secondary metabolites, such as flavonoid glycosides, triterpenoid saponins, and steroidal saponins. It exists widely in In animals, plants, fungi, bacteria. Many glycosylated compounds have better water solubility than their aglycones, which can increase the druggability of the compounds. However, the content of UDP-xylose is low and the synthesis is difficult, which leads to high price and is not conducive to the synthesis of xyloside, which brings great difficulties to the development of innovative drugs. Through the catalysis of UDP-glucuronic acid by UXS, one-step reaction can convert UDP-glucuronic acid into UDP-xylose, which can provide a stable and cheap glycosyl donor for the glycosylation of natural products, which can bring great economic benefit. Therefore, cloning and functional identification of the UXS gene is useful for enzymatically synthesizing the compound UDP-xylose or for large-scale preparation of xyloside natural products through synthetic biology techniques, reducing the need for traditional xyloside natural products due to chemical synthesis and plant extraction. It is of great significance to promote the sustainable development of society by reducing the pressure caused by the method on the environment and species.

So far, UDP-xylose synthase has been found and cloned in plants such as Arabidopsis thaliana, Hordeum vulgare, and bacteria such as Sinorhizobium meliloti gene, but there is no report on the isolation of this protein from the Asparagaceae plant Ornithogalum caudatum. The present invention obtains the sequence information of UDP-xylose synthase OsaUXS1 of Dieffenbachia tiger's eye by performing transcriptome sequencing on Dieffenbachia tiger's eye. The OsaUXS1 gene was cloned from the aseptic bulbs of Diofenbachia tiger's eye by extracting RNA and using RT-PCR, and its function was identified.

Contents of the invention

The technical problem solved by the present invention is to provide a uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger's eye, a nucleotide sequence encoding the enzyme, an expression vector containing the nucleotide sequence, and an expression vector containing the Nucleotide sequence or host cell of expression vector, and its application in catalyzing the reaction of substrate uridine-5'-diphosphate glucuronic acid.

In order to solve technical problem of the present invention, adopt following technical scheme:

The first aspect of the technical solution of the present invention provides a kind of uridine-5'-diphosphate xylose synthase derived from Dieffenbachia tiger eye, characterized in that:

a) the amino acid sequence shown in SEQ ID NO.1;

b) The amino acid sequence shown in SEQ ID NO.1 is replaced, deleted or added with 1-35 amino acids to form an amino acid sequence with equivalent functions.

The above-mentioned uridine-5'-diphosphate xylose synthase is characterized in that conventional modification can be carried out on uridine-5'-diphosphate xylose synthase; or on uridine-5'-diphosphate xylose synthase Labels for detection or purification are also attached to the xylose synthase.

Wherein, the conventional modification includes acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, immobilization modification; Tags include His ₆ , GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, Profinity eXact.

The second aspect of the technical solution of the present invention provides a nucleic acid molecule encoding the uridine-5'-diphosphate xylose synthase described in the first aspect, the nucleotide sequence of which is shown in SEQ ID NO.3.

The third aspect of the technical solution of the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

The fourth aspect of the technical solution of the present invention provides a host cell containing the expression vector described in the third aspect, wherein the host cell is preferably Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Streptomyces and plant cells, more preferably the host Cells were selected from Escherichia coli.

The fifth aspect of the technical solution of the present invention provides the uridine-5'-diphosphate xylose synthetase described in the first aspect or the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect or the expression vector described in the fourth aspect Application of host cells in catalytic substrate uridine-5'-diphosphate glucuronic acid reaction, wherein the catalytic substrate is uridine-5'-diphosphate glucuronic acid, and the product is uridine-5'-diphosphate xylose.

Beneficial technical effects:

UDP-xylose has a low content in organisms and is difficult to synthesize, resulting in high prices, which is not conducive to the synthesis of xylosides, and brings great difficulties to the development of innovative drugs. Through the catalysis of UDP-glucuronic acid by UXS1, one-step reaction can convert UDP-glucuronic acid into UDP-xylose, which can provide a stable and cheap sugar group donor for the glycosylation of natural products, and can also bring great economic benefit.

Description of drawings

Figure 1: Catalytic reactions involved in uridine-5'-diphosphate xylose synthase.

Figure 2: PCR analysis results of OsaUXS1 gene, where M is DNA molecular weight standard; 1 is PCR result of OsaUXS1 gene.

Figure 3: Schematic diagram of the recombinant plasmid pETDuet-OsaUXS1.

Figure 4: Silver staining results of OsaUXS1 recombinant protein SDS-PAGE analysis, where M is the protein molecular standard; 1 is OsaUXS1; 2 is the empty vector control.

Figure 5: Western blot results of OsaUXS1 recombinant protein, where CK is the control; 1 is OsaUXS1.

Figure 6: Silver staining results of OsaUXS1 protein SDS-PAGE analysis after imidazole elution and purification, in which 1-4 are the target proteins; M is the protein molecular standard.

Figure 7: OsaUXS1 recombinase catalyzed UDP-glucuronic acid HPLC-UV detection results, in which 1 is the experimental group; 2 is the control group (inactivated enzyme); 3 is the experimental group without NAD ⁺ ; 4 is without NAD ⁺ for the control group.

Figure 8: HPLC-MS results of UDP-glucuronic acid product catalyzed by OsaUXS1.

Figure 9: ¹ H NMR chart of UDP-glucuronic acid product catalyzed by OsaUXS1.

Figure 10: Effect of temperature on OsaUXS1 catalytic activity of UDP-glucuronic acid.

Figure 11: Effect of pH on OsaUXS1 catalytic activity of UDP-glucuronic acid.

Figure 12: OsaUXS1 catalyzed UDP-glucuronidase activity curve.

Figure 13: OsaUXS1 catalyzed UDP-glucuronic acid Lineweaver-Burk double reciprocal diagram.

detailed description

The present invention is further illustrated by the following examples, which are intended to be illustrative only and not to limit the scope of the claimed invention in any way.

Example 1 Transcriptome Sequencing and Sequence Analysis of Dieffenbachia tiger-eye

After extracting the total RNA from the sterile bulb of Dieffenbachia tiger eye, using the mRNA as a template, the first cDNA strand was synthesized with six base random primers (randomhexamers), and then the second cDNA was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I After purification by QiaQuick PCR kit and elution with EB buffer, end repair, poly(A) was added and sequencing adapters were connected, then fragment size selection was performed by agarose gel electrophoresis, and finally PCR amplification was carried out to construct A good sequencing library was sequenced with Illumina HiSeq ^™ 2000.

The original image data obtained by sequencing is converted into sequence data through base calling, that is, raw data or raw reads. Perform data filtering on rawreads, remove reads with adapters, duplicates, and low-quality sequencing, and obtain clean reads. Use the short reads assembly software Trinity to connect clean reads with a certain length of overlap into longer fragments, namely Contig. Then, compare the clean reads back to Contig, and determine the different Contigs from the same transcript and the distance between these Contigs through paired-end reads. Trinity connects these Contigs together to obtain sequences that cannot be extended at both ends. These The sequence is called Unigene. The Unigene sequence is compared to the protein database nr, Swiss-Prot, KEGG and COG (E value<0.00001) by blastx, and the Unigene is compared to the nucleic acid database nt by blastn (E value<0.00001), and the sequence of the given Unigene is obtained. The protein with the highest sequence similarity, so as to obtain the protein function annotation information of the Unigene.

Through functional annotation, a Contig with a length of 1543bp and a conserved sequence of UDP-xylose synthase gene was found from the transcriptome sequence of Diphtheria tiger eye, namely CL1039 (SEQ ID NO.2).

Example 2 Cloning of OsaUXS1 gene

Take 100mg of aseptic bulbs of Dieffenbachia tiger's eye, freeze them quickly in liquid nitrogen, grind them into fine powder with a mortar, and use Trizol extraction to extract total RNA from the aseptic bulbs of Dieffenbachia tiger's eye. Using RT-PCR Kit (ReverTra-Plus-, TOYOBO company) to reverse transcribe the total RNA of Diofenbachia sterile bulbs into cDNA. The reverse transcription system and procedure are as follows: Add 1 μg of total RNA, 4 μL of RNase Free H ₂ O, and 1 μL of Oligo(dT) ₂₀ to a 20 μL system, incubate at 65°C for 5 minutes, immediately place it on ice, and then re- Add 4 μL of 5×RT buffer, 1 μL of RNase Inhibitor (10 U/μL), 1 μL of dNTP Mixture (10 mM) and ReverTra Ace in sequence, incubate at 30°C for 10 minutes, incubate at 42°C for 60 minutes, denature at 85°C for 5 minutes, and place on ice for 5 minutes to complete cDNA synthesis synthesis. The cDNA was stored at -20°C for later use.

Two pairs of OsaUXS1-specific primers were designed using the cDNA of the sterile bulb of Diofenbachia tiger eye as a template, and the OsaUXS1 gene was amplified by nested PCR.

For the first round of PCR, the 50 μL system contained 5 μL of 10×PCR buffer, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO ₄ , and 10 μM of primers FCL1039-1 (5'-CAAATTACGAAGAGATCGGATC-3', SEQ ID NO.4) and RCL1039-1 ( 5'-TTAAGCGTGCAGACAATGATC-3', SEQ ID NO.5) 1.5 μL each, KOD-Plus-Neo 1 μL, cDNA 2 μL, supplemented with ddH ₂ O. PCR program: pre-denaturation at 94°C for 5 minutes; denaturation at 98°C for 30s, renaturation at 53°C for 45s, decreasing by 1°C for each cycle, extension at 68°C for 120s, a total of 15 cycles; denaturation at 98°C for 30s, renaturation at 38°C for 45s, extension at 68°C 120s, a total of 15 cycles; extension at 68°C for 7min, holding at 4°C. In the second round of PCR, the 50 μL system contained 5 μL of 10×PCR buffer, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO ₄ , and 10 μM of primers FCL1039-2 (5'-ATGGCGAAGGAGAGCTCCA-3', SEQ ID NO.6) and RCL1039-2 ( 5'-TTAACTGCTCTGTTTGCTGG-3', SEQ ID NO.7) 1.5 μL each, KOD-Plus-Neo 1 μL, first-round PCR product 2 μL, ddH ₂ O supplementation. PCR program: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 30 s, renaturation at 45°C for 45 s, extension at 68°C for 120 s, a total of 30 cycles; extension at 68°C for 7 min, and incubation at 4°C. The PCR product was analyzed by 0.8% agarose gel electrophoresis, and the results showed that a band about 1 kb in length was amplified ( FIG. 2 ).

The PCR product was connected to the pEASY ^TM -T1Simple (Transgen Company) vector, transformed into the Trans1-T1 (Transgen Company) strain, cultured on an LB plate containing 100 μg/mL carbenicillin, and single clones were picked for colony PCR to screen positive clones Sample delivery for sequencing. The results showed that the amplified PCR product was exactly the same as the transcriptome sequencing result, which was 1047bp in length (ie, SEQ ID NO.3), and the vector containing the gene was named pEASY-OsaUXS1.

Example 3 Construction of OsaUXS1 expression vector

According to the principle of homologous recombination of In-Fusion (Clontech Company), using the correctly sequenced plasmid pEASY-OsaUXS1 as a template, FETduetUXS1 (5'-gccaggatccgaattcgatggcgaaggagagctcc-3', SEQ ID NO.8) and RETduetUXS1 (5'-atgcggccgcaagcttttaactgctctgtttgctgg-3 ', SEQ ID NO.9) as primers, PCR was carried out through the following procedures and systems, 50 μL system containing 5 μL of 10×PCR buffer, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO ₄ , 1.5 μL of 10 μM primers FETduetUXS1 and RETduetUXS1, KOD- Plus-Neo 1 μL, plasmid 1 μL, supplemented with ddH ₂ O. PCR program: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 30 s, renaturation at 63°C for 45 s, extension at 68°C for 120 s, a total of 30 cycles; extension at 68°C for 7 min, and incubation at 4°C. A 1 kb OsaUXS1 gene was amplified. The gene was connected to pET-Duet1 treated with EcoRI and HindIII double enzymes by the In-Fusion method, and the recombinant product was transformed into Trans1-T1 strain, cultured on LB plates containing 50 μg/mL ampicillin, and single clones were picked Colony PCR was performed, and positive clones were screened for sequencing. The results showed that the vector was constructed correctly and named as pETDuet-OsaUXS1 (Fig. 3).

Example 4 Induced expression and detection of recombinant OsaUXS1 protein

The constructed plasmid pETDuet-OsaUXS1 was transformed into the expression host strain Transetta (DE3) by the heat shock method, and the transformation product was spread on LB solid medium (10 g Tryptone, 5g of yeast powder, and 10g of sodium chloride were dissolved in 1L of distilled water, 15g of agar powder was added, and then the pH was adjusted to 7.0), and incubated overnight at 37°C until a single colony grew out. Pick a single clone and transfer it to 20 mL LB liquid medium (10 g tryptone, 5 g yeast powder, 10 g sodium chloride dissolved in 1 L distilled water) containing ampicillin (50 μg/mL) and chloramphenicol (35 μg/mL), and then Adjust the pH to 7.0), cultivate at 37°C and 200rpm until the OD ₆₀₀ is about 1.0, and transfer to 50mL LB medium (10g Tryptone, 5g yeast powder, 10g sodium chloride dissolved in 1L distilled water, then adjust the pH to 7.0), when the _OD600 is 0.6, add IPTG (final concentration is 0.5mM), and induce Incubate for 12 hours. Take 1mL of the bacterial liquid, centrifuge at 12000rpm for 2min to collect the bacterial cells. Add 1 mL of pre-cooled lysis buffer (25 mM sodium phosphate buffer with pH 8.0), resuspend the cells, and disrupt the cells by ultrasonication (sonication for 5 s, stop for 10 s, total ultrasonication for 6 min). The crushed solution was passed through 12000rpm, centrifuged for 20min, and the supernatant was absorbed. Take 10 μL of supernatant for SDS-PAGE analysis. The results are shown in Figure 4, and it can be seen from the figure that, compared with the control, a specific band with a length of about 38 kDa appeared in the induced product of the experimental bacteria. It is consistent with the theoretical size of OsaUXS1, indicating that OsaUXS1 was successfully expressed in E. coli.

To further confirm the expression of OsaUXS1 gene, western blot analysis was carried out. The cells of E.coli[pETDuet-OsaUXS1] were collected by centrifugation, subjected to SDS-PAGE electrophoresis with sonicated solution, and detected by Western blot. The primary antibody used was anti-His-Tag mouse monoclonal antibody, and the secondary antibody was goat anti-mouse IgG (1:5000). The results showed that, compared with the control, E.coli[pETDuet-OsaUXS1] had a corresponding hybridization band at the corresponding position after immunoblotting (Fig. 5). Therefore, it was confirmed that the recombinant OsaUXS1 protein had specific immune activity, indicating that the OsaUXS1 gene was expressed in E.coli.

Example 5 Purification and quantification of recombinant OsaUXS1 protein

E.coli[pETDuet-OsaUXS1] was cultured at 25℃, 160rpm, induced by 0.5mM IPTG for 12 hours, then centrifuged at 12000rpm for 2min to collect the bacterial cells, a large number of bacterial cells were crushed with a cell disruptor (800MPa), centrifuged at 12000rpm for 2min , collect the supernatant of the crushed liquid, filter the supernatant through a 0.22 μm filter membrane, and then purify it by nickel gel affinity chromatography. The foreign protein was washed with rinse buffer (pH 8.0, 25 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole) until A280 was less than 0.010. Then, the recombinant OsaUXS1 bound to the nickel gel was eluted with 40 mL of elution buffer (pH 8.0, containing 25 mM sodium phosphate, 300 mM sodium chloride, and 100 mM imidazole), and the elution fraction was collected for SDS-PAGE analysis. The results are shown in Figure 6. It can be seen from the results that the purification effect of OsaUXS1 is better, and almost no impurity bands can be seen.

The purified recombinant OsaUXS1 protein was dialyzed with 2L buffer solution containing 25mM sodium phosphate (pH 8.0), and the medium was changed 4 times, then 10mL of glycerol was added to the dialysate, and the protein was stored at -20°C.

The purified OsaUXS1 protein was quantified using the Bio-rad protein assay kit with some modifications according to its instructions. First, the dye and ddH ₂ O were diluted and mixed at a ratio of 1:4, and then filtered through a 0.22 μM filter membrane for later use. The standard BSA protein and the protein to be tested were diluted in a certain gradient, vortexed and mixed, and then the protein was reacted with the dye according to the following system (100 μL: dry reagent dilution buffer; 10 μL: protein), and incubated at room temperature for at least 5 minutes with shaking. The A595 value of each protein was measured by a microplate reader, and the concentration equation of the standard BSA protein was obtained by linear regression analysis of different BSA standard protein concentrations and the corresponding A595 values. According to the obtained equation, the OsaUXS1 used in the catalytic UDP-glucuronic acid experiment was measured. The protein concentration was 0.409 mg/mL.

Example 6 OsaUXS1 protein function identification

In this experiment, UDP-glucuronic acid was used as the substrate to investigate the decarboxylation effect of OsaUXS1 on the substrate. The results showed that OsaUXS1 could catalyze the formation of UDP-xylose from UDP-glucuronic acid.

Product identification of UDP-glucuronic acid formation catalyzed by OsaUXS1

Using the purified OsaUXS1 protein as a catalyst, a 200 μL reaction system was established (50 μL of 200 mM phosphate buffer, 20 μL of 10 mM substrate UDP-glucuronic acid, 20 μL of purified protein, and 110 μL of sterilized water). After reacting at 30°C for 30 min, the reaction was terminated with 200 μL of chloroform. The reaction solution was filtered with a 0.22 μM nylon membrane, and 20 μL was injected for HPLC analysis. The chromatographic column is the WA-pak anion exchange column of Shodex Company. The HPLC conditions are shown in Table 1, wherein phase A is H ₂ O; phase B is 700 mM ammonium acetate-acetate buffer, pH 5.2. The detection wavelength is 261 nm.

Table 1

time Phase A% Phase B% Flow rate mL/min 0 98 2 1 20 50 50 1 32 0 100 1 33 98 2 1 40 98 2 1

HPLC result (Fig. 6) shows, OsaUXS1 can catalyze UDP-glucuronic acid, and experimental group substrate peak consumption is more, and a new product peak has appeared before substrate peak simultaneously, and the maximum absorption wavelength of this product is 261nm, is The characteristic absorption peak of uracil. The HPLC-MS analysis of the collected product (Fig. 8) showed that in the negative ion detection mode, the m/z of the [M-H]-ion formed by the product was 534.6219, which was consistent with the molecular weight of UDP-xylose.

In order to further confirm the structure of the new product, the sample was prepared by liquid phase, the sample was lyophilized and dissolved in deuterated water, and the structure was analyzed by ¹ HNMR (600M) (Figure 9). Attributed to each hydrogen signal, the results are as follows:

Xylosyl: H1"5.44(1H,dd)H2"3.40(1H,dt)H3"3.59(1H,t)H4"3.53(1H,dt)H5a"/H5b"3.64(2H,m)

Rib: H1'5.88(1H,d)H2'4.27(2H,m)H4'4.18(1H,m)H5a'(1H,m)H5b'(1H,m)Uridine:H5 5.87(1H,d)H6 7.85(1H,d)

Based on the above analysis, it can be confirmed that the new product catalyzed by OsaUXS1 to form UDP-glucuronic acid is UDP-xylose. Thus it was shown that OsaUXS1 has the function of UDP-xylose synthase.

At the same time, the experimental results also show that OsaUXS1 still has high activity without adding NAD ⁺ , and the conversion rate is 97.4% of that of adding NAD ⁺ .

Example 7 Determination of OsaUXS1 Catalyzed UDP-Glucuronid Enzyme Properties

Determination of the optimal temperature of UDP-glucuronic acid catalyzed by OsaUXS1

Take a 1.5mL EP tube, add 50μL of 200mM phosphate buffer, 20μL of purified enzyme, and 110μL of sterilized water, and incubate at different temperatures for 10min, then quickly add 20μL of 10mM UDP-glucuronic acid. After reacting for 30 min, 200 μL of chloroform was added to terminate the reaction, and the reaction solution was filtered through a 0.22 μM nylon membrane, and 20 μL was injected for HPLC analysis. The chromatographic column is the WA-pak anion exchange column of Shodex Company. The HPLC conditions are shown in Table 1, wherein phase A is H ₂ O; phase B is 700 mM ammonium acetate-acetate buffer, pH 5.2. The detection wavelength is 261 nm. Take the product with the largest peak area as 100%, and the rest take corresponding relative values.

The results show (Figure 10), the optimum temperature for OsaUXS1 to catalyze UDP-glucuronic acid is 30°C, and the activity can reach more than 50% in the range of 30-50°C. Too low or too high temperature is not conducive to the reaction. The activity at 4°C was 3.24% of the highest value, and the enzyme was inactivated at 60°C.

Determination of the optimal pH value of UDP-glucuronic acid catalyzed by OsaUXS1

Prepare 100 mM sodium acetate-acetic acid buffer solution with pH 4-6, 10 mM phosphate buffer solution with pH 6-9, and 100 mM Tris-HCl buffer solution with pH 7.5-9.2. Take 100 μL of buffer solutions with different pH, 20 μL of purified OsaUXS1 protein with a concentration of 0.409 mg/mL and 60 μL of sterilized water, incubate at 30°C for 10 min, and add 20 μL of UDP-glucuronic acid with a concentration of 10 mM. After reacting at 30°C for 30 minutes, add 200 μL of chloroform to stop the reaction, centrifuge at 12,000 rpm for 5 minutes, absorb the aqueous phase and filter it with a 0.22 μM nylon membrane, and take 20 μL of the filtrate for HPLC analysis. The HPLC conditions are shown in Table 1, and the detection wavelength is 261 nm. The product peak areas were detected by comparing HPLC and the catalytic efficiencies at different pHs were compared. The product with the largest peak area was taken as 100%, and the rest were taken as relative values.

The results of HPLC analysis ( FIG. 11 ) showed that the optimum pH for catalyzing UDP-glucuronic acid was 5.0, and its activity decreased as the pH increased or decreased.

Determination of Kinetic Parameters of UDP-Glucuronic Acid Catalyzed by OsaUXS1

UDP-xylose standard substances with different concentrations (pH 5.0, 50 mM phosphate) were prepared with reaction buffer, and detected by HPLC at a detection wavelength of 261 nm to obtain the peak area. The relationship between the peak area and the concentration of UDP-xylose is obtained by linear regression, and a fitting equation Y=111.9X+1.428 (0.1≤X≤1) is obtained, where X is the concentration of UDP-xylose (mM), and Y is the peak area change value. Establish a 100 μL reaction system with a pH of 5.0, 50 mM phosphate buffer, and a final concentration of OsaUXS1 of 40.9 μL/mL. Change the concentration of the substrate UDP-glucuronic acid, react at the optimum temperature of 30 ° C for 10 min, and immediately terminate each reaction with chloroform , Utilize HPLC to analyze the generation amount of UDP-xylose, utilize the peak area to obtain the concentration of UDP-xylose, and then obtain the reaction initial velocity (mM/min). According to the experimental results, the enzyme activity curve was made (Figure 12). Km and Vmax were automatically calculated using related programs in GraphPad Prism 5 (Fig. 13).

The enzymatic property data of OsaUXS1 are shown in Table 2

Table 2

Claims (10)

1. the UDP xylose synzyme deriving from Herba Phyllanthi Urinariae, it is characterised in that its aminoacid sequence is:

A) aminoacid sequence shown in SEQ ID NO.1；

B) 1-35 is amino acids formed has equal function through replacing, lack or adding for the aminoacid sequence shown in SEQ ID NO.1 Aminoacid sequence.

UDP xylose synzyme the most according to claim 1, it is characterised in that at UDP xylose synzyme On can carry out conventional modification；Or it is also associated with on UDP xylose synzyme for detection or the label of purification.

UDP xylose synzyme the most according to claim 2, it is characterised in that described conventional modify include acetylation, Amidatioon, cyclisation, glycosylation, phosphorylation, alkylation, biotinylation, fluorophor modify, Polyethylene Glycol PEG modify, Immobilization is modified；Described label includes His₆、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc、 Profinity eXact。

4. one kind encodes the nucleic acid molecules of UDP xylose synzyme described in claim 1.

Nucleic acid molecules the most according to claim 4, it is characterised in that the nucleotide sequence of described nucleic acid molecules such as SEQ ID NO.3 Shown in.

6. the expression vector containing the arbitrary described nucleic acid molecules of claim 4-5.

7. one kind contains the host cell of expression vector described in nucleic acid molecules described in claim 4 or claim 6.

Host cell the most according to claim 7, it is characterised in that its host cell includes escherichia coli, saccharomyces cerevisiae, finishes Red yeast, streptomycete, plant cell.

9. UDP xylose synzyme described in claim 1 or the arbitrary described nucleic acid molecules of claim 4-5 or right are wanted The expression vector described in 6 or the host cell described in claim 7 is asked to react at catalytic substrate UDP-Glc aldehydic acid In application.

Application the most according to claim 9, it is characterised in that described UDP-xylose synzyme is at catalytic substrate UDP After glucuronic acid, the product of formation is UDP xylose.