A Pyruvate Decarboxylase Mutant with Improved Catalytic Efficiency and Its Application

Abstract

The present invention discloses a pyruvate decarboxylase mutant with improved catalytic efficiency and its application. The present invention constructs a pyruvate decarboxylase mutant derived from Candida tropicalis for catalytic production of 4-hydroxyphenylacetaldehyde. The catalytic half-life (5.7 h) and TTN (24,600) of the mutant of the present invention are increased by 2.85 times and 2.07 times respectively compared with the control, and the mutant exhibits high decarboxylation activity. When the obtained mutant of the present invention uses 4-hydroxyphenylpyruvic acid as a substrate and reacts for 12 h at 25 °C and pH 6.5, the yield of 4-hydroxyphenylacetaldehyde can reach 5.08 g / L, and the molar conversion rate of the substrate reaches 67.28%. By adopting the method of the present invention, the production capacity of the unit catalyst and the reaction efficiency of the catalyst are improved, and the industrialization process of producing 4-hydroxyphenylacetaldehyde by the enzyme conversion method is accelerated.

Description

Technical Field

[0001] The present invention relates to a pyruvate decarboxylase mutant with improved catalytic efficiency and its application, belonging to the technical field of enzyme engineering. Background Art

[0002] 4-Hydroxyphenylacetaldehyde (4-HPAA) is a natural aromatic aldehyde, which can be used as a precursor for the production of important pharmaceutical intermediates in industry (such as benzylisoquinoline alkaloids and tyrosol), and has very considerable economic value.

[0003] Currently, the industrial production of 4-HPAA mainly relies on chemical conversion technologies, such as hydrolyzing tyrosine or synephrine in the presence of strong acids. However, the chemical process requires harsh reaction conditions, high process complexity and is not environmentally friendly. Based on these considerations, biocatalysis is an attractive alternative. The enzyme conversion method has the advantages of high yield, high conversion rate and short conversion cycle, etc., and thus has more industrial application value.

[0004] The enzyme involved in the biocatalytic production of 4-HPAA - pyruvate decarboxylase (PDC, pyruvate decarboxylase, EC 4.1.1.1) is an intracellular enzyme, a non-oxidative enzyme dependent on thiamine diphosphate (ThDP), and is a holoenzyme composed of coenzyme ThDP, Mg 2+ and protein. It acts on the substrate 4-hydroxyphenylpyruvic acid (4-HPPA) in the presence of cofactor thiamine diphosphate and Mg 2+ to produce 4-HPAA and CO2. It widely exists in various organisms such as yeast, mold, bacteria and plants. The structures, relative molecular weights, enzymatic properties, etc. of pyruvate decarboxylases from different sources are not the same. The PDC mentioned in the present invention is derived from Candida tropicalis, that is, CtPDC. Since the decarboxylation conversion rate of CtPDC catalyzing 4-HPPA to produce 4-HPAA is not high, its enzymatic properties and structure have been studied. The data show that low enzyme activity is the main reason limiting the decarboxylation efficiency of CtPDC, so it has also become the rate-limiting enzyme in the multi-enzyme cascade tyrosol synthesis pathway existing in this laboratory.

[0005] In recent decades, protein engineering has become an effective strategy for improving the properties of enzymes at the molecular level, such as the most effective method for expanding the substrate range, enhancing enzyme activity, and improving enzyme stability. Therefore, designing CtPDC through protein engineering may solve the problem of its low catalytic activity. Protein engineering modifications can be mainly classified into four categories: traditional directed evolution (i.e., irrational design), semi-rational design, rational design (based on structure and computer technology), and the combined application of multiple strategies. Currently, there has been no remarkable progress in the research on modifying PDC using protein engineering. In most studies, the mutants only verified the role of key residues or studied the catalytic promiscuity of the enzyme, and there was no significant effect on enhancing the decarboxylation activity of the enzyme. Therefore, the work of modifying PDC using protein engineering still has a long way to go. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a recombinant pyruvate decarboxylase CtPDC mutant capable of preparing 4-HPAA. The amino acid sequence of the pyruvate decarboxylase is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2. The enzyme activity of the CtPDC mutant provided by the present invention is improved compared with that of the wild type. The strain constructed using this pyruvate decarboxylase mutant has improved the decarboxylation conversion rate during the preparation of 4-HPAA and, to a certain extent, relieved the rate-limiting bottleneck in the cascade step of producing tyrosol.

[0007] The first object of the present invention is to provide a pyruvate decarboxylase mutant with improved catalytic efficiency. The mutant is a mutation of phenylalanine at position 395, glycine at position 415, and tyrosine at position 477 in the pyruvate decarboxylase (CtPDC) parent having the amino acid sequence shown in SEQ ID NO.1, or a combination of multiple mutations.

[0008] Further, the mutant is obtained by mutating phenylalanine at position 395 of the pyruvate decarboxylase parent to serine.

[0009] Further, the mutant is obtained by mutating glycine at position 415 of the pyruvate decarboxylase parent to serine.

[0010] Further, the mutant is obtained by mutating phenylalanine at position 395 of the pyruvate decarboxylase parent to serine and simultaneously mutating the amino acid at position 415 to serine.

[0011] Further, the mutant is obtained by mutating glycine at position 415 of the pyruvate decarboxylase parent to serine and simultaneously mutating the amino acid at position 477 to phenylalanine.

[0012] Further, the mutant is obtained by mutating the phenylalanine at the 395th position of the pyruvate decarboxylase parent into serine, mutating the amino acid at the 415th position into serine, and simultaneously mutating the amino acid at the 477th position into phenylalanine.

[0013] The second object of the present invention is to provide a gene encoding the mutant.

[0014] The third object of the present invention is to provide an expression vector carrying the gene.

[0015] The fourth object of the present invention is to provide a strain expressing the mutant.

[0016] Further, the strain uses Escherichia coli as the host.

[0017] The fifth object of the present invention is to provide the application of the strain in the preparation of 4-hydroxyphenylacetaldehyde. The application is to use 4-hydroxyphenylpyruvic acid as the substrate, use the strain as the catalyst, and react for 6 - 12 h at pH 6.0 - 6.5 and 25 - 30 °C to prepare 4-hydroxyphenylacetaldehyde.

[0018] Further, the addition amount of the strain in the reaction system is a final concentration of 15 - 25 g / L.

[0019] Further, the final concentration of 4-hydroxyphenylpyruvic acid in the reaction system is 5 - 10 g / L.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention constructs a pyruvate decarboxylase mutant derived from Candida tropicalis for catalytic production of 4-hydroxyphenylacetaldehyde. The catalytic half-life (5.7 h) and TTN (24600) of the mutant of the present invention are respectively increased by 2.85 times and 2.07 times compared with the control, and the mutant shows higher decarboxylation activity. When the obtained mutant uses 4-hydroxyphenylpyruvic acid as the substrate and reacts for 12 h at 25 °C and pH 6.5, the yield of 4-hydroxyphenylacetaldehyde can reach 5.08 g / L, and the molar conversion rate of the substrate reaches 67.28%. By using the method of the present invention, the production capacity of the unit catalyst and the reaction efficiency of the catalyst are improved, and the industrialization process of producing 4-hydroxyphenylacetaldehyde by the enzyme conversion method is accelerated. Description of the Drawings

[0022] Figure 1 The SDS-PAGE diagram showing the expression of CtPDC protein by SDS-PAGE: Lane M is the low molecular weight protein Marker; Lane 1 is the protein expression in the supernatant; Lane 2 is the protein expression in the precipitate.

[0023] Figure 2Generate the molar conversion rate of 4-HPAA for each mutant.

[0024] Figure 3 HPLC or GC detection spectra of the decarboxylation reaction substrates and products; among them, Figure 3 A is the HPLC chart of the substrate 4-HPPA standard; Figure 3 B is the GC chart of the product 4-HPAA standard. Specific implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0026] Gene source: The biological enzyme CtPDC gene involved in this patent is derived from Candida tropicalis. The pET28a(+) plasmid is purchased from Novagen (Madison, WI, U.S.A.). Restriction endonucleases, PrimeSTAR, etc. are purchased from TaKaRa (Dalian, China). The one-step homologous recombination enzyme is purchased from Vazyme (Nanjing, China). All CtPDC mutants are obtained by molecular modification, and the rest of the reagents are purchased from the market.

[0027] Prepare LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and sterilize at 121 °C for 20 min.

[0028] Prepare TB medium: tryptone 12 g / L, yeast extract (Angel yeast powder FM802) 24 g / L, glycerol 4 mL / L, KH2PO4 2.31 g / L, and K2HPO4 12.31 g / L.

[0029] Prepare sodium phosphate buffer at pH 6.5: a mixed buffer of 0.2 mol / L disodium hydrogen phosphate and sodium dihydrogen phosphate. For the specific formula, please refer to "Industrial Microbiology Experiment Technology Manual" (China Light Industry Press, edited by Zhuge Jian).

[0030] The detection methods involved in the following examples are as follows:

[0031] Detection of the contents of 4-hydroxyphenylpyruvic acid (4-HPPA) and 4-hydroxyphenylacetaldehyde (4-HPAA):

[0032] The system uses a sodium phosphate solution as the buffer, 5 mM MgSO4 and 2 mM ThDP as cofactors, the concentration of the converted substrate is 50 g / L, the concentration of whole cells is 20 g / L, and the reaction is carried out in a constant temperature shaker at 25 °C and 220 rpm for 12 h. 500 μL of the converted reaction solution sample is extracted with an equal volume of ethyl acetate and shaken, centrifuged at 12000 rpm for 5 - 10 min, the upper organic phase is aspirated and diluted by a certain multiple, and then passed through a 0.22 μm organic membrane for gas chromatography GC detection of the product 4-HPAA; another 500 μL is taken, the reaction is terminated with an equal volume of NaOH, centrifuged at 12000 rpm for 5 - 10 min, the supernatant is taken, diluted by a certain multiple, and then passed through a 0.22 μm water membrane for high performance liquid chromatography HPLC detection of the substrate 4-HPPA.

[0033] The specific GC analysis method is as follows:

[0034] The instrument is a Shimadzu gas chromatograph, and the chromatographic column is RTX-5 (inner diameter 0.25 mm, film thickness 1.00 μm); split injection is used, the split ratio is 10:1, and the injection volume is 1 μL; the total flow rate of nitrogen is 14 mL / min, and the flow rate of hydrogen is 32 mL / min; the temperature programming of the column oven is: the initial temperature of the chromatographic column is 80 °C, heated to 250 °C at a rate of 10 °C / min, and then held for 2 min. Under these detection conditions, the retention time of the product 4-HPAA is 12.442 min.

[0035] The specific HPLC analysis method is as follows:

[0036] The instrument is a Dionex UltiMate 3000 high performance liquid chromatograph, and the chromatographic column is an Aminex HPX-87H Ion Exclusion Column (300×7.8 mm); the mobile phase is 0.03% dilute sulfuric acid, the flow rate is 0.6 ml / min, the injection volume is 10 μL, the wavelength of the ultraviolet detector is 210 nm, and the analysis program for one sample is 20 min. Under these detection conditions, the retention time of the substrate 4-HPPA is 14.088 min.

[0037]

[0038] Among them, m(4-HPAA) represents the mass of 4-HPAA, g; m(4-HPPA) represents the initial mass of 4-HPAA, g; 136.15 and 180.16 represent the relative molecular masses of 4-HPAA and 4-HPAA respectively.

[0039] Example 1: Heterologous expression of CtPDC

[0040] Construction of genetically engineered bacteria and expression of proteins:

[0041] Using the nucleotide sequence of the target protein-encoding gene in Candida tropicalis (shown as SEQ ID NO. 2) as a template, PCR amplification was performed using F1 and R1 as primers (the underlined parts are the Nco I and Xho I restriction enzyme cleavage sites respectively. To avoid frameshift mutations, the last base 'g' of the sequence 'ccatgg' of this restriction enzyme cleavage site was deleted). The amplification conditions were: 95°C for 5 min, 29 cycles (98°C for 10 s, 55°C for 30 s, 72°C for 1 min 50 s), and 72°C for 5 min.

[0042] F1: ctttaagaaggagatata ccatg ATGAGTGAGATTACGCT;

[0043] R1: gtggtggtggtggtg ctcgag TTCTTGGGCGGCATTGGT.

[0044] The cDNA sequence of the coding region of the CtPDC gene was obtained. After recovering the PCR product, it was ligated to the pET-28a(+) plasmid vector digested with the same double enzymes by homologous recombination to obtain the recombinant expression plasmid pET-28a(+)-CtPDC. The recombinant plasmid pET-28a(+)-CtPDC was transformed into E. coli BL21(DE3). After PCR identification, the positive engineering bacteria were named E. coli BL21 / pET-28a(+)-CtPDC.

[0045] The engineering bacteria E. coli BL21 / pET-28a(+)-CtPDC were inoculated into LB liquid medium. After culturing for 8 - 12 h, a seed solution was obtained. The seed solution was inoculated into fresh TB liquid medium at an inoculation amount of 2% (v / v). After culturing for 2 - 3 h (OD = 0.6 - 0.8), IPTG with a final concentration of 0.42 mM was added, and the culture was continued at 25°C for 14 h to induce the expression of the recombinant target protein. 150 mL of the induced fermentation broth was centrifuged at 6000 r / min to collect the bacteria. After the bacteria were lysed, SDS-PAGE was used to detect the protein expression.

[0046] The results were as Figure 1 shown: Lanes 1 and 2 were respectively the band sizes of the proteins contained in the supernatant and the precipitate. It can be seen that CtPDC can be heterologously expressed in E. coli BL21.

[0047] Example 2: Preparation of 4-hydroxyphenylacetaldehyde by whole cells

[0048] In a 10 mL reaction bottle, 0.08 g of whole-cell E. coli BL21 / pET-28a(+)-CtPDC expressing CtPDC protein after induced culture, 0.04 g of 4-hydroxyphenylpyruvate, 3720 μL of 0.2 M sodium phosphate buffer, 200 μL of 100 mM magnesium sulfate solution, and 80 μL of 50 mM ThDP coenzyme solution were added respectively, and the reaction was carried out in a constant temperature shaker at 25°C and 200 rpm for 12 hours, and the reaction pH was 6.5.

[0049] Take 500 μL of the reaction liquid sample after conversion, extract it with an equal amount of ethyl acetate, shake it, centrifuge it at 12000 rpm for 5 to 10 minutes, take the upper organic phase, dilute it a certain number of times, pass it through a 0.22 μm organic membrane, and use gas phase GC to detect the yield of the product 4-HPAA; take another 500 μL, terminate the reaction with an equal amount of NaOH, centrifuge it at 12000 rpm for 5 to 10 minutes, take the supernatant, dilute it a certain number of times, pass it through a 0.22 μm water membrane, and use high performance liquid chromatography HPLC to detect the remaining amount of substrate 4-HPPA.

[0050] The detection results of gas chromatography and high performance liquid chromatography showed that the yield of 4-hydroxyphenylacetaldehyde prepared by whole cell transformation using E. coli BL21 / pET-28a(+)-CtPDC was 2.31 g / L, and the conversion rate was 30.57%.

[0051] Example 3: Construction and screening of single mutants

[0052] Single mutant construction: Design of CtPDC F395S , CtPDC G415S , CtPDC Y477F , CtPDC H116A , CtPDC D28A The primers for the mutation sites are shown in Table 1 , and mutants were constructed by whole plasmid PCR.

[0053] Table 1 Sequences of primers for single mutant mutation

[0054]

[0055] Construct the PCR amplification system: KOD Plus Neo enzyme 0.5 μL, Buffer 2.5 μL, dNTP 2.5 μL, Mg 2+ 1.5 μL, 0.2 μL of each primer for each mutation site, template (CtPDC WT )0.5μL, water 17.1μL; reaction conditions are: ①94℃5min; ②98℃10s; ③55℃30s; ④72℃4min; ⑤recycle ②~④3 steps 29 times; ⑥72℃5min; ⑦keep warm at 12℃.

[0056] Incubate the above reaction system at 37 °C for 3 h to digest the plasmid template (the digestion system is: 0.5 μL of DpnI, 45 μL of the above reaction PCR product, and 5 μL of 10×T Buffer). After digestion, the digested product is introduced into Escherichia coli BL21 competent cells by chemical transformation method. The specific steps of the chemical transformation method are as follows:

[0057] (1) Introduce 10 μl of the homologous recombination product into 100 μl of Escherichia coli BL21 competent cells;

[0058] (2) Incubate on ice for 15 - 20 min;

[0059] (3) Heat shock in a 42 °C water bath for 90 s, and then quickly place it on ice and let it stand in the ice bath for 3 - 5 min;

[0060] (4) Add 800 μl of antibiotic - free LB medium, mix well, and culture at 37 °C and 200 rpm for 1 h;

[0061] (5) Centrifuge at 5000 rpm for 2 min to collect the bacteria;

[0062] (6) Remove the supernatant, and resuspend the remaining 100 - 200 μL by pipetting and spreading it onto a kanamycin - resistant plate containing 0.05 mg / mL kanamycin, and incubate at 37 °C for about 12 h.

[0063] (7) Pick a single colony and culture it in kanamycin - resistant LB containing 0.05 mg / mL kanamycin at 200 rpm and 37 °C for 12 h, and then send it to the company for sequencing. The one with correct sequencing is the positive transformant.

[0064] Genetically engineered bacteria E. coli BL21 / pET - 28a(+)-CtPDC were respectively prepared F395S 、E. coli BL21 / pET - 28a(+)-CtPDC G415S 、E. coli BL21 / pET - 28a(+)-CtPDC Y477F 、E. coli BL21 / pET - 28a(+)-CtPDC H116A 、E. coli BL21 / pET - 28a(+)-CtPDC D28A 。

[0065] Subsequently, the above - obtained genetically engineered bacteria were induced to express, and then according to the method of Example 2, whole - cell transformation was carried out to prepare 4 - hydroxyphenylacetaldehyde, and the better mutants were screened out. The results are shown in Table 2. The genetically engineered bacteria E. coli BL21 / pET - 28a(+)-CtPDC F395S 、E. coli BL21 / pET - 28a(+)-CtPDCG415S 、E.coli BL21 / pET-28a(+)-CtPDC Y477F showed good effect in catalyzing 4-hydroxyphenylpyruvic acid to generate 4-hydroxyphenylacetaldehyde.

[0066] Table 2 Conversion rate of single mutants in catalyzing the generation of 4-HPAA

[0067]

[0068] Example 4: Construction and screening of double mutants and triple mutants

[0069] (1) Construction of double mutants

[0070] The double mutants in this example were constructed by whole plasmid PCR according to the primers in Table 3 on the basis of the corresponding single mutants. For example, on the basis of the mutant CtPDC G415S , using the mutant primers F395S-F and F395S-R (Table 1), the double mutant CtPDC G415S / F395S was constructed by whole plasmid PCR.

[0071] The primers used are shown in Table 3. For the specific implementation method, refer to the steps in Example 3 to prepare the genetically engineered bacteria containing double mutants: E.coli BL21 / pET-28a(+)-CtPDC F395S / G415S 、E.coli BL21 / pET-28a(+)-CtPDC G415S / Y477F 、E.coli BL21 / pET-28a(+)-CtPDC G415S / K231A 、E.coli BL21 / pET-28a(+)-CtPDC G415S / T390A .

[0072] Table 3 Mutant primer sequences of double mutants

[0073]

[0074] (2) Screening of double mutants

[0075] Inoculate the mutant strains with correct sequencing into LB seed medium, culture at 200 rpm and 37 °C for 10 h. Respectively, inoculate the seed liquid according to the inoculation amount of 2% (v / v) into fresh TB liquid medium, culture at 200 rpm and 37 °C until OD600 = about 0.8, add IPTG with a final concentration of 0.42 mM for induction, and the induction conditions are 200 rpm and 25 °C for 14 h.

[0076] Subsequently, the obtained genetically engineered bacteria were subjected to whole-cell transformation to prepare 4-hydroxyphenylacetaldehyde according to the method of Example 2, and the superior double mutants were screened out. The results are shown in Table 4. The engineered bacteria E. coli BL21 / pET-28a(+)-CtPDC F395S / G415S and E. coli BL21 / pET-28a(+)-CtPDC G415S / Y477F showed good effects in catalyzing 4-hydroxyphenylpyruvic acid to generate 4-hydroxyphenylacetaldehyde.

[0077] Table 4 Conversion rate of double mutants in catalyzing the formation of 4-HPAA

[0078]

[0079] (3) Construction of triple mutants

[0080] Based on the mutant CtPDC F395S / G415S , mutant primers Y477F-F and Y477F-R (Table 1) were used to construct triple mutants by whole-plasmid PCR. The specific implementation method refers to the steps in Example 3 to prepare the genetically engineered bacteria E. coli BL21 / pET-28a(+)-CtPDC F395S / G415S / Y477F containing the triple mutant CtPDC F395S / G415S / Y477F . According to the method in Example 2, whole-cell transformation was carried out to prepare 4-hydroxyphenylacetaldehyde. The detection result was that the genetically engineered bacteria E. coli BL21 / pET-28a(+)-CtPDC F395S / G415S / Y477F containing the triple mutant had a conversion rate of 67.28% in catalyzing 4-hydroxyphenylpyruvic acid to generate 4-hydroxyphenylacetaldehyde, and the corresponding yield was 5.08 g / L.

[0081] Example 5: Expression and purification method of mutant enzymes

[0082] The positive transformants of the mutant recombinant strains prepared in Examples 3 and 4 were inoculated into LB medium and cultured at 37 °C until the OD 600 reached 0.6 - 1.0, then IPTG with a final concentration of 0.42 mM was added to induce the expression of the enzyme. The induction temperature was 25 °C and the induction time was 14 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4 °C and 6000 rpm for 10 min to collect the cells. 10 mL of binding solution A (20 mM sodium phosphate, 0.5 mM NaCl, 20 mM imidazole, 1% glycerol, pH adjusted to 8.5 with HCl) was added to fully resuspend the cells, and then the centrifuge tube was placed in an ice bath and put into an ultrasonic cell disruptor. The conditions for ultrasonic disruption were: working time 4 s, interval time 4 s, for a total of 10 min. The obtained disrupted solution was centrifuged at low temperature and high speed at 4 °C and 8000 rpm for 30 min to obtain the crude enzyme solution. It was filtered through a 0.22 μm microporous filter membrane and reserved.

[0083] Prepare a nickel ion affinity chromatography column. First, use a constant flow pump to pump ultrapure water into the column to wash the column (about 6 - 12 times the column volume) at 4°C, and then balance the column environment with 10 mL of binding solution A. When the pH value of the effluent at the lower end of the column is consistent with that of the low-salt concentration buffer solution pumped into the column (about 5 times the column volume of buffer solution is required), add the obtained crude enzyme solution after passing through the membrane to the column. First, wash the miscellaneous proteins with binding solution A until baseline equilibrium, and then elute with elution solution B (20 mM sodium phosphate, 0.5 mM NaCl, 500 mM imidazole). Collect the eluate of the absorption peak, measure the enzyme activity, and obtain the target protein that reaches electrophoresis purity.

[0084] Example 6: Enzyme activity determination of the parental enzyme and mutants

[0085] Pure enzyme solutions of the parental enzyme Q0 and the beneficial mutants Q1 - Q6 obtained in Examples 3 and 4 were obtained by the method of Example 5, and then the decarboxylase activity was verified. The specific implementation method is as follows:

[0086] Using 4-hydroxyphenylpyruvic acid as the substrate, the enzyme activity of pyruvate decarboxylase and its mutants was determined by the yield of 4-hydroxyphenylacetaldehyde (4-HPAA) generated in 2 min. Reaction system (500 μL): 20 mM 4-hydroxyphenylpyruvic acid (0.1 M MOPS buffer solution at pH 6.5), cofactor 5 mM MgSO4 and 2 mM ThDP, an appropriate amount of pure enzyme solution of pyruvate decarboxylase or its mutants, react at 200 rpm and 25°C for 2 min; immediately add an equal volume of ethyl acetate after the reaction, shake and then centrifuge at 12,000 rpm for 10 min, take the upper layer (organic phase), pass through a 0.22 μm organic membrane and then perform GC detection. The enzyme activity of pyruvate decarboxylase is defined as: the amount of enzyme required to generate 1 μmol of 4-hydroxyphenylacetaldehyde within 1 min at 25°C and pH 6.5.

[0087] Table 5 Enzyme activity of beneficial mutants

[0088]

[0089] Example 7: Performance determination of the parental enzyme and mutants

[0090] (1) Determination of kinetic parameters

[0091] To evaluate beneficial mutants, the present invention determined the kinetic parameters of mutant parent Q0 and mutants Q1 to Q6 at 37°C. kcat / Km was calculated by measuring the initial rate of 4-hydroxyphenylacetaldehyde produced from different concentrations of 4-hydroxyphenylpyruvate under the condition of 37°C. For the specific implementation steps, please refer to the literature On the effects of site-specific mutations on activity and expression of the Streptomyces PMF phospholipase D, J. Mol. Catal. B. Enzym. 41(2006)1-7 and Biochemistry (Higher Education Press, edited by Wang Jingyan). The results are shown in Table 6.

[0092] (2) Determination of catalytic half-life

[0093] Since the transformation reaction is catalyzed by whole cells in vivo, the catalytic half-life of the in vivo reaction (referring to the time required for the remaining enzyme activity to be half during the catalytic reaction with 4-hydroxyphenylpyruvate as the substrate) was further verified. The catalytic half-lives of the parental enzyme and mutants were determined through the residual enzyme activity experiment during the transformation process.

[0094] Genetically engineered bacteria containing the parental enzyme of CtPDC and genetically engineered bacteria containing mutants were each added to the reaction solution with a final concentration of 20 g / L of wet cells. Using 4-hydroxyphenylpyruvate as the substrate, their residual enzyme activities were measured every 1 h during the entire transformation process (the initial enzyme activity at 0 h of the reaction was set as 100%), and the measurement was carried out for a total of 12 h; the results are shown in Table 6.

[0095] The reaction system involved is as follows: 0.08 g of the prepared genetically engineered bacteria, 0.04 g of 4-hydroxyphenylpyruvate, 3720 μL of 0.2 M sodium phosphate buffer, 200 μL of 100 mM magnesium sulfate solution, and 80 μL of 50 mM ThDP coenzyme solution were respectively added to a 10 mL reaction flask, and the reaction was carried out in a constant temperature shaker at 25°C and 200 rpm for 12 h, and the reaction pH was 6.5.

[0096] (3) Determination of specific enzyme activity

[0097] According to the detection method of specific enzyme activity, the specific enzyme activity data of the parental enzyme of CtPDC and its mutants were respectively detected. The results are shown in Table 6.

[0098] Specific enzyme activity is defined as the number of enzyme activity units per milligram of protein (U / mg protein).

[0099]

[0100] (4) Calculation of TTN

[0101] The TTN is determined by the amount of product generated and the amount of catalyst consumed. This relationship is particularly evident in homogeneous catalysis and is called the turnover number. Using the GC method, the TTN can be calculated by measuring the number of moles of 4-HPAA and the number of moles of pure enzyme consumed. The reaction system involved is as follows: The purified parental CtPDC enzyme protein and the protein containing the mutant are added to the reaction solution at a final concentration of 10 μmol, respectively. Using 4-HPPA as the substrate, the reaction is carried out in a constant temperature shaker at 25 °C and 200 rpm for 2 min. After the reaction is completed, the number of moles of 4-HPAA generated is measured by GC. The TTN is calculated according to the following formula, and the results are shown in Table 6.

[0102]

[0103] Table 6 Kinetic parameters of parental CtPDC enzyme and its mutants

[0104]

[0105] The results show that the Km values of the mutants are lower than that of the WT, and the Km of the triple mutant is the lowest, indicating that the triple mutant has an increased affinity for the substrate; compared with Q0, the catalytic half-lives of all mutants are prolonged. Consistent with this, the total turnover number (TTN) of each mutant is increased compared with Q0, and the TTN of Q6 is 2.07 times that of Q0.

[0106] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. A pyruvate decarboxylase mutant with improved catalytic efficiency, characterized in that, The mutant is a mutation of one or a combination of mutations in phenylalanine at position 395, glycine at position 415, and tyrosine at position 477 after translation of the pyruvate decarboxylase parental coding gene with a nucleotide sequence as shown in SEQ ID NO.

2. The mutation is any one of the following: Mutating phenylalanine at position 395 of the pyruvate decarboxylase parent to serine; Mutating glycine at position 415 of the pyruvate decarboxylase parent to serine; Mutating phenylalanine at position 395 of the pyruvate decarboxylase parent to serine and simultaneously mutating the amino acid at position 415 to serine; Mutating glycine at position 415 of the pyruvate decarboxylase parent to serine and simultaneously mutating the amino acid at position 477 to phenylalanine; Mutating phenylalanine at position 395 of the pyruvate decarboxylase parent to serine, mutating the amino acid at position 415 to serine, and simultaneously mutating the amino acid at position 477 to phenylalanine.

2. A gene encoding the mutant according to claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A strain expressing the mutant according to claim 1.

5. Use of the strain according to claim 4 in the preparation of 4-hydroxyphenylacetaldehyde, characterized in that, The application is to use 4-hydroxyphenylpyruvic acid as a substrate, the strain as a catalyst, and react under the conditions of pH 6.0 - 6.5 and 25 - 30 °C for 6 - 12 h to prepare 4-hydroxyphenylacetaldehyde.

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