Application of oxygenase in catalyzing 4-ethene guaiacol to synthesize vanillin

Abstract

The invention relates to an application of oxygenase in catalyzing 4-ethene guaiacol to synthesize vanillin. The amino acid sequence of the oxygenase comprises a sequence as shown in SEQ ID NO.1. The invention also relates to an application of the oxygenase in catalyzing 4-ethene guaiacol to synthesize vanillin. The invention provides an application of oxygenase in catalyzing synthesis of vanillin from 4-ethene guaiacol. The 4-ethene guaiacol oxygenase is in soluble expression in host bacteria. Crude enzyme liquid expressed by the strain stably catalyzes conversion of 4-ethylene guaiacol into vanillin, the conversion rate within 2 hours is 80%, the conversion rate within 24 hours exceeds 98%, and 11.5 g / L of vanillin can be accumulatively produced within 6 hours.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to an oxygenase capable of catalyzing 4-vinylguaiacol to synthesize vanillin and application thereof. Background Art

[0002] Vanillin, also known as vanillin, has a strong aroma and is widely used in food. It is known as the king of food flavors. The natural extraction method for producing vanillin has problems such as a long cycle, occupation of land, great environmental impact, and high prices, making it difficult to meet market demand. Chemical synthesis is currently the main synthesis method, but the vanillin obtained by this route is classified as synthetic vanillin, not natural vanillin, and is therefore prohibited from use in the food and pharmaceutical fields. Biotransformation refers to a method of using microorganisms to convert natural substrates into vanillin, which is classified as natural vanillin and can be used as an alternative to natural extraction methods. It has a very broad application prospect.

[0003] In the pathway of ferulic acid to vanillin, the rate-limiting step is the conversion of 4-vinylguaiacol to vanillin. The researchers screened out a phenol oxygenase Cso2 that can catalyze the conversion of 4-vinylguaiacol to vanillin. By heterologously expressing phenolic acid decarboxylase and Cso2 in microorganisms, a non-coenzyme-dependent pathway for the synthesis of vanillin with ferulic acid as a substrate was successfully constructed, but Cso2 had poor stability. Later, the researchers further discovered phenol oxygenase (Ado) from Thermothelomyces thermophila, which can catalyze the conversion of 4-vinylguaiacol to vanillin. Phenolic acid decarboxylase (PAD) and Ado from Bacillus coagulants DSM1 were heterologously expressed in Escherichia coli BL21, and the expression of Ado contained some insoluble inclusion bodies.

[0004] At present, the research on enzymes catalyzing this step is limited to Cso2 and Ado. Therefore, it has become an urgent problem to explore more efficient, stable and easy-to-express vanillin synthases and seek a cheap and efficient method for producing natural vanillin, which will help realize the biosynthesis of natural vanillin. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an application of an oxygenase in catalyzing the synthesis of vanillin from 4-vinylguaiacol, wherein the 4-vinylguaiacol oxygenase is soluble and expressed in a host bacterium. The crude enzyme solution expressed by the oxygenase stably catalyzes the conversion of 4-vinylguaiacol into vanillin, with a conversion rate of 80%-98%, and can produce 11.5 g / L of vanillin in 6 hours.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of an oxygenase in catalyzing the synthesis of vanillin from 4-vinylguaiacol, wherein the amino acid sequence of the oxygenase comprises the sequence shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] .

[0010] The invention discloses a highly active, high-temperature resistant 4-vinylguaiacol oxygenase derived from Thermothielavioides terrestris NRRL 8126, and provides a genetic engineering strain capable of efficiently and solublely expressing the 4-vinylguaiacol oxygenase. The crude enzyme solution of the genetic engineering strain can stably catalyze the conversion of 4-vinylguaiacol into vanillin at 50°C, with a conversion rate of 80%-98%, and can cumulatively produce 11.5 g / L of vanillin in 6 hours, and has good industrial application prospects.

[0011] Preferably, the nucleic acid sequence of the oxygenase comprises the sequence shown in SEQ ID NO.2.

[0012] SEQ ID NO.2:

[0013]

[0014] In a second aspect, the present invention provides a method for synthesizing vanillin, the method comprising: utilizing the oxygenase described in the first aspect to catalyze 4-vinylguaiacol to synthesize vanillin.

[0015] The method for synthesizing vanillin specifically comprises: constructing a recombinant plasmid containing the nucleic acid sequence of the oxygenase described in the first aspect, transferring the recombinant plasmid into a host cell to obtain a crude enzyme solution, and adding 4-vinylguaiacol and a buffer to synthesize vanillin.

[0016] The method for synthesizing vanillin comprises the following steps:

[0017] (1) connecting the nucleic acid sequence of the oxygenase described in the first aspect to a plasmid vector to construct a recombinant plasmid;

[0018] (2) transferring the recombinant plasmid constructed in step (1) into a host bacterium to obtain a genetically engineered strain;

[0019] (3) activating the genetically engineered strain, performing antibiotic screening, and performing seed culture to obtain seed liquid;

[0020] (4) inducing expression in the seed solution;

[0021] (5) collecting the genetically engineered strain by the first centrifugation, resuspending the cells and breaking them, and collecting the supernatant by the second centrifugation to obtain the genetically engineered crude enzyme solution;

[0022] (6) 4-vinylguaiacol is dissolved in a buffer solution, and a crude enzyme solution of a genetically engineered strain is added to the 4-vinylguaiacol solution to react and synthesize vanillin.

[0023] Preferably, the plasmid vector in step (1) includes pETDuet1-Vgo.

[0024] Preferably, the host bacteria in step (2) include any one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris or Saccharomyces cerevisiae.

[0025] Preferably, the temperature for activating the bacteria in step (3) is 25-35°C, and the time is 0.5-2h. The 25-35°C may be, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, etc. The 0.5-2h may be, for example, 0.5h, 1h, 1.5h or 2h, etc.

[0026] Preferably, the shaking speed of the bacterial cell activation in step (3) is 150-300 rpm, for example, it can be 150 rpm, 200 rpm, 220 rpm, 250 rpm or 300 rpm.

[0027] Preferably, the inoculum amount for bacterial activation in step (3) is 0.5-3%, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5% or 3%.

[0028] Preferably, the antibiotics used in the antibiotic screening in step (3) include any one of ampicillin, kanamycin, streptomycin or penicillin, or a combination of at least two of them.

[0029] Preferably, the concentration of the antibiotic is 50-150 μg / mL, for example, it can be 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL or 150 μg / mL, etc.

[0030] Preferably, the temperature of the seed culture in step (3) is 25-35°C, and the time is 8-16h. The 25-35°C may be, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, etc. The 8-16h may be, for example, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h or 16h, etc.

[0031] Preferably, the shaking speed of the bacterial cell activation in step (3) is 150-300 rpm, for example, it can be 150 rpm, 200 rpm, 220 rpm, 250 rpm or 300 rpm.

[0032] Preferably, the step of inducing differentiation in step (4) comprises: inoculating the seed solution into a differentiation induction medium.

[0033] Preferably, the inoculum amount of the seed solution inoculation is 0.5-3%, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3%.

[0034] Preferably, the differentiation induction medium is an induction composition and a basal medium.

[0035] Preferably, the basal culture medium comprises LB medium.

[0036] Preferably, the inducing composition comprises 0.05-0.5 mM IPTG and / or 0.5-5 mM Fe2Cl. The 0.05-0.5 mM may be, for example, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM or 0.5 mM, etc. The 0.5-5 mM may be, for example, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM or 5 mM, etc.

[0037] Preferably, the OD of the seed solution is 600 It can be 0.1-5, for example, it can be 0.1, 0.5, 0.6, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0038] Preferably, the speed of the first centrifugation in step (5) is 6000-10000 rpm, and the time is 2-8 min. The 6000-10000 rpm may be, for example, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm or 10000 rpm, etc. The 2-8 min may be, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min or 8 min, etc.

[0039] Preferably, the speed of the second centrifugation in step (5) is 6000-10000 rpm, and the time is 20-35 min. The 6000-10000 rpm may be, for example, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm or 10000 rpm, etc. The 20-35 min may be, for example, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min or 35 min, etc.

[0040] Preferably, the step (5) further includes a purification operation.

[0041] Preferably, the purification method comprises nickel column purification.

[0042] Preferably, the nickel column purification step comprises: adding the protein-containing supernatant to the nickel column, adding a cleaning solution and then adding an eluent to obtain a crude enzyme solution.

[0043] Preferably, the eluent comprises tris(hydroxymethylaminomethane) hydrochloride, sodium chloride and imidazole.

[0044] Preferably, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride is 30-70 mM, for example, 30 mM, 40 mM, 50 mM, 60 mM or 70 mM.

[0045] Preferably, the concentration of sodium chloride is 300-800 mM, for example, it can be 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM or 800 mM, etc.

[0046] Preferably, the concentration of imidazole is 100-500 mM, for example, it can be 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM or 500 mM, etc.

[0047] In the present invention, the concentration of the eluent required for purification of the crude enzyme solution has a great influence on protein purification and subsequent vanillin yield.

[0048] Preferably, the buffer in step (6) comprises any one or a combination of at least two of sodium citrate-citric acid, Tris-HCl, Na2HPO4-NaH2PO4 or Na2CO3-NaHCO3.

[0049] Preferably, the final concentration of the buffer in step (6) is 1.5-2.5 M, for example, it can be 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M or 2.5 M, etc.

[0050] Preferably, the pH of the buffer solution in step (6) is 5.0-11.0, for example, 5, 6, 7, 8, 9, 10 or 11.

[0051] In the present invention, the pH of the buffer has a great influence on the activity of 4-ethyleneguaiacol oxygenase, thereby affecting the yield.

[0052] Preferably, the concentration of the 4-vinylguaiacol solution in step (6) is 0.5-5 g / L, for example, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L.

[0053] Preferably, in step (6), the volume ratio of the crude enzyme solution to 4-vinylguaiacol is 1:(6-12), wherein (6-12) may be, for example, 6, 7, 8, 9, 10, 11 or 12.

[0054] Preferably, the reaction time in step (6) is 10-720 min, and the temperature is 20-70° C., wherein the 20-70° C. may be, for example, 20° C., 30° C., 40° C., 50° C., 60° C. or 70° C., etc. The 10-720 min. may be, for example, 10 min., 50 min., 100 min., 150 min., 200 min., 250 min., 300 min., 350 min., 400 min., 450 min., 500 min., 550 min., 600 min., 650 min., 700 min. or 720 min.

[0055] In the present invention, the reaction temperature and pH have a great influence on the yield of vanillin, and the specific mechanism is that different temperatures and solution pH have a great influence on the activity of 4-ethyleneguaiacol oxygenase, thereby affecting the yield.

[0056] Compared with the prior art, the present invention has at least the following beneficial effects:

[0057] 1. The present invention provides an oxygenase that can catalyze the synthesis of vanillin from 4-vinylguaiacol. The nucleic acid sequence of the oxygenase is derived from the oxygenase Vgo of Thermothielavioides terrestris, which belongs to the carotenoid oxygenase family. The present invention creatively finds that the oxygenase can promote the conversion of 4-vinylguaiacol into vanillin, and constructs a recombinant plasmid and a recombinant strain having the nucleic acid sequence for realizing the biological synthesis of natural vanillin.

[0058] 2. The present invention optimizes the method for producing natural vanillin based on the above-mentioned recombinant strain, and makes adjustments in terms of temperature, purification eluent and pH, thereby significantly improving the yield of vanillin. The conversion rate is 80% in 2h, and the conversion rate exceeds 98% in 24h. The cumulative production of vanillin is 11.5 g / L in 6h. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is the protein expression diagram of three recombinant plasmids.

[0060] Figure 2 It is a protein expression diagram of a recombinant plasmid.

[0061] Figure 3 It is the HPLC detection spectrum of the synthesis of vanillin and vanillin from 4-vinylguaiacol.

[0062] Figure 4 This is the standard curve of 4-vinylguaiacol.

[0063] Figure 5 This is the standard curve of vanillin.

[0064] Figure 6It is a line graph of vanillin production at different temperatures.

[0065] Figure 7 It is a line graph of vanillin production at different pH values.

[0066] Figure 8 This is a graph showing the conversion rate of 4-vinylguaiacol to vanillin at different times.

[0067] Fig. 9 is the cumulative amount of vanillin at different times.

[0068] Fig.10 This is the electrophoresis diagram of proteins eluted with different imidazole concentrations. DETAILED DESCRIPTION

[0069] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0070] Example 1

[0071] This example explores the potential of 4-ethyleneguaiacol oxygenase

[0072] Using computer simulation screening, three proteins, KAI0202932.1, KAF4443388.1, and XP_003653923.1, were screened by comparing catalysis and protein structure. Proteins with a similarity greater than 50% to Ado (Uniprot registration number G2QIL8) were further searched in the Uniprot database; protein sequence analysis was performed using MEGA.11 to screen XP_003653923.1, A0A446BT67, and B2AMA0. Since the amino acid sequence of A0A446BT67 was the same as that of XP_003653923.1, it was no longer synthesized. Finally, XP_003653923.1, KAI0202932.1, KAF4443388.1, and B2AMA0 were used for subsequent experiments.

[0073] (1) Construction of pETDuet1-XP_003653923.1 recombinant plasmid

[0074] The oxygenase Vgo from Thermothielavioides terrestris belongs to the carotenoid oxygenase family, and its NCBI registration number is XP_003653923.1. It has the amino acid sequence shown in SEQ ID NO.1. The codon-optimized sequence of the oxygenase was sent to Zhonghe Gene Biology Co., Ltd. for synthesis, and a histidine tag was added to facilitate protein purification. The linear fragment of Vgo is 1806 bp and has the nucleotide sequence shown in SEQ ID NO.1. The plasmid vector is pET_duet1, and has the nucleotide sequence shown in SEQ ID NO.3.

[0075] (2) Construction of pETDuet1-KAI0202932.1 recombinant plasmid

[0076] The protein KAI0202932.1 from the fungus Astrocystis sublimbata belongs to the carotenoid oxygenase family, has the NCBI accession number KAI0202932.1, and has the amino acid sequence shown in SEQ ID NO.4. The codon-optimized sequence of the oxygenase was sent to Zhonghe Gene Biology Co., Ltd. for synthesis, and a histidine tag was added to facilitate protein purification. The linear fragment of KAI0202932.1 is 1710 bp and has the nucleotide sequence shown in SEQ ID NO.5. The plasmid vector is pET_duet1, which has the nucleotide sequence shown in SEQ ID NO.3.

[0077] (3) Construction of pETDuet1-KAF4443388.1 recombinant plasmid

[0078] The hypothetical protein KAF4443388.1 from Fusarium austroafricanum, NCBI accession number is KAF4443388.1. It has the amino acid sequence shown in SEQ ID NO.6. The codon-optimized sequence of the oxygenase was sent to Zhonghe Gene Biology Co., Ltd. for synthesis, and a histidine tag was added to facilitate protein purification. The linear fragment of KAF4443388.1 is 1605 bp and has the nucleotide sequence shown in SEQ ID NO.7. The plasmid vector is pET_duet1, which has the nucleotide sequence shown in SEQ ID NO.3.

[0079] (4) Construction of pETDuet1-B2AMA0 recombinant plasmid

[0080] The protein B2AMA0 from NCBI has the accession number B2AMA0. It has the amino acid sequence shown in SEQ ID NO.8. The codon-optimized sequence of the oxygenase was sent to Zhonghe Gene Biology Co., Ltd. for synthesis, and a histidine tag was added to facilitate protein purification. The linear fragment of B2AMA0 is 1821 bp and has the nucleotide sequence shown in SEQ ID NO.9. The plasmid vector is pET_duet1, which has the nucleotide sequence shown in SEQ ID NO.3.

[0081] Example 2

[0082] This example screens 4-ethyleneguaiacol oxygenase

[0083] (1) Cultivation of recombinant engineering strains

[0084] Pipette 5 μL of the recombinant plasmid prepared in Example 1 into 50 μL of BL21 competent cells, gently blow to suspend and mix, place on ice for 30 minutes, heat shock at 42°C for 30 seconds, and place on ice for 2 minutes. Add 550mL of liquid LB medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L), and culture at 30°C, 220rpm for 1h. Pipette 50 μL of bacterial solution and spread it on an LB solid plate containing 100 μg / mL ampicillin, and culture it upside down at 30°C for 12h. Pick a single well-grown strain and culture it in a liquid LB medium containing 100 μg / mL ampicillin, 30°C, 220rpm for 12h to obtain a seed solution.

[0085] (2) Induced expression of target protein

[0086] The seed solution was inoculated into fresh liquid LB medium at 1% inoculation rate and cultured at 30°C and 220 rpm until OD 600 =0.6, add IPTG and Fe2Cl, the final concentrations are 0.2mM and 1mM respectively, and induce at 16℃ for 16h.

[0087] Collect the bacterial solution and centrifuge at 8000 rpm for 5 min to remove the supernatant; wash the cells with 5 mL of 50 mM pH = 7.4 phosphate buffer, centrifuge and remove the supernatant; suspend the cells with Tri-HCl buffer; measure OD with a UV spectrophotometer 600 The cells were broken on ice using a cell disruptor at 40 kHz, ultrasonicated for 3 seconds, paused for 5 seconds, and ultrasonicated for a total of 30 minutes. The cells were centrifuged at 8000 rpm for 25 minutes, the supernatant was collected, and the precipitate was suspended with an equal volume of Tri-HCl buffer to obtain a crude enzyme solution. Protein samples were subjected to SDS-PAGE gel electrophoresis to verify protein expression.

[0088] Specific results such as Figure 1 As shown in the figure, M stands for Maker; 1 represents lane 1, which is the supernatant of E.coil_BL21(DE3) protein; 2 represents lane 2, which is the supernatant of E.coil(DE3)-pETDuet1-KAF4443388.1 protein; 3 represents lane 3, which is the precipitate of E.coil(DE3)-pETDuet1-KAF4443388.1 protein; 4 represents lane 4, which is the precipitate of E.coil(DE3)-pETDuet1-KAF4443388.1 protein; t1-KAI0202932.1 protein supernatant; 5 represents lane 5 is E.coil(DE3)-pETDuet1-KAI0202932.1 protein precipitate; 6 represents lane 6 is E.coil(DE3)-pETDuet1XP_003653923.1 protein supernatant; 7 represents lane 7 is E.coil(DE3)-pETDuet1-XP_003653923.1 protein precipitate;

[0089] Specific results such as Figure 2 As shown in the figure, M stands for Maker; 1 represents lane 1, which is the supernatant of E.coil_BL21(DE3) protein; 2 represents lane 2, which is the supernatant of E.coil(DE3)-pETDuet1-B2AMA0 protein; 3 represents lane 3, which is the precipitate of E.coil(DE3)-pETDuet1-B2AMA0 protein.

[0090] Example 3

[0091] This example is used to synthesize vanillin

[0092] A 1 g / L 4-vinylguaiacol solution was prepared, the solvent was a phosphate buffer solution (pH = 7.4) containing 20% ​​glycerol; 900 μL of the 4-vinylguaiacol solution was taken, 100 μL of the crude enzyme solution was added, and the reaction was carried out at 30°C for 10 min; after the reaction was completed, 1 mL of methanol was added to terminate the reaction, and the sample was detected by HPLC.

[0093] The 4-vinylguaiacol and vanillin standard samples were dissolved in 100% methanol solution, and the contents of vanillin and 4-vinylguaiacol were detected by high performance liquid chromatography (HPLC).

[0094] The HPLC detector was Agilent 2006, the chromatographic column was C18 (4.6 mm × 150 mm); mobile phase A was 0.2% acetic acid in water, and mobile phase B was 100% methanol; the flow rate was 0.8 mL / min; the detection wavelength was 280 nm; the column temperature was 30°C; and the injection volume was 5 μL.

[0095] The gradient elution program is shown in Table 1:

[0096] Table 1

[0097] Time / min Mobile phase A Mobile phase B Flow rate / (mL / min) 0 65 35 0.8 9 65 35 0.8 12 5 95 0.8 18 5 95 0.8

[0098] The HPLC method for 4-vinylguaiacol and vanillin was successfully established. Figure 3 As shown in the figure, the peak positions of vanillin and 4-vinylguaiacol are 9.061min and 16.719min respectively, both of which are single strong absorption peaks. The peak positions of the test sample and the standard are consistent. The standard curves of 4-vinylguaiacol and vanillin are shown in the figure below. Figure 4 , Figure 5 As shown, the vanillin content of the sample was calculated based on the standard curve.

[0099] Example 4

[0100] This example explores the effect of reaction temperature on the yield of vanillin

[0101] A 1 g / L 4-vinylguaiacol solution was prepared, the solvent was a phosphate buffer solution containing 20% ​​glycerol (pH = 7.4); 900 μL of the 4-vinylguaiacol solution was aspirated and 100 μL of the crude enzyme solution was added; the reaction temperatures were 20°C, 30°C, 40°C, 50°C, 60°C and 70°C, respectively, the buffer was 1 M phosphate buffer solution, and the pH was 7.4; after the reaction was completed, 1 mL of methanol was added to terminate the reaction, and the sample was tested by HPLC, and the test conditions referred to Basic Experimental Example 3.

[0102] The results are as follows Figure 6 As shown, the optimum temperature for enzyme catalytic activity is 50°C, and the catalytic activity is maintained above 60% at 20-60°C, indicating that Vgo has a wide temperature adaptability.

[0103] Example 5

[0104] This example explores the effect of buffer pH on vanillin production

[0105] A 1 g / L 4-vinylguaiacol solution was prepared, the solvent was a phosphate buffer solution (pH = 7.4) containing 20% ​​glycerol; 900 μL of the 4-vinylguaiacol solution was aspirated and 100 μL of the crude enzyme solution was added; the reaction temperature was 50°C, and the pH of the buffer solution was 5, 6, 7, 8, 9, 10 and 11, respectively; after the reaction was completed, 1 mL of methanol was added to terminate the reaction, and the sample was tested by HPLC, and the detection conditions were referred to Basic Experimental Example 3.

[0106] Specific results such as Figure 7 shown.

[0107] Table 2

[0108] pH Buffer Vanillin yield / (g / L) 5 Sodium Citrate-Citric Acid 0.015 6 Sodium Citrate-Citric Acid 0.27 6 Tri-HCl 0.29 7 Tri-HCl 0.31 8 Tri-HCl 0.40 8 <![CDATA[NaH2PO4-Na2HPO4]]> 0.73 9 <![CDATA[NaH2PO4-Na2HPO4]]> 0.68 9 <![CDATA[Na2CO3-NaHCO3]]> 0.58 10 <![CDATA[Na2CO3-NaHCO3]]> 0.17 11 <![CDATA[Na2CO3-NaHCO4]]> 0.06

[0109] The results are as follows Figure 7As shown: Vgo activity is relatively stable in the range of pH = 6 to pH = 9, and the vanillin production is the highest at pH = 8. When the pH is less than 5 or greater than 10, the activity of Vgo decreases sharply.

[0110] Example 6

[0111] This example explores the conversion rate of vanillin at different times in the buffer solution.

[0112] Prepare 2 g / L 4-vinylguaiacol solution, the solvent is phosphate buffer (pH = 8) containing 20% ​​glycerol; draw 900 μL of 4-vinylguaiacol solution, add 100 μL crude enzyme solution; the reaction temperature is 50°C, the buffer is phosphate buffer with a pH of 8; after the reaction is completed, add 1 mL of methanol to terminate the reaction, take samples at 10 min, 30 min, 1 h, 2 h, 12 h, and 18 h, respectively, and test the samples with HPLC. The test conditions refer to Basic Experimental Example 3.

[0113] Specific results such as Figure 8 shown.

[0114] The results are as follows Figure 8 As shown: Vgo has the fastest conversion rate within 2 hours, with a conversion rate of 80% in 2 hours and a conversion rate of over 98% in 24 hours.

[0115] Example 7

[0116] This example explores the cumulative amount of vanillin at different times in the buffer solution

[0117] A 15 g / L 4-vinylguaiacol solution was prepared, the solvent was a phosphate buffer solution (pH=8) containing 20% ​​glycerol; 10 mL of the 4-vinylguaiacol solution was drawn and 1 mL of the crude enzyme solution was added; the reaction temperature was 50°C, and the buffer solution was a phosphate buffer solution with a pH of 8; samples were taken at 1, 2, 4, and 6 hours, and an equal volume (1 mL) of 4-vinylguaiacol solution was added after sampling; the samples were tested by HPLC, and the test conditions were referred to Basic Experimental Example 3.

[0118] Specific results such as Fig. 9 shown.

[0119] The results are as follows Fig. 9 As shown: Vgo at 6h the cumulative amount of vanillin is 11.5g.

[0120] Example 8

[0121] This example explores the effect of imidazole concentration in the eluent on the yield of vanillin

[0122] The crude enzyme solution was purified, and the protein crude enzyme solution was separated and purified by nickel column affinity chromatography. The nickel column was filled with HispurTMNi-NTAResin, and the target protein with His tag would specifically bind to the resin. Washbuffer (50mM Tris-HC1, 150mM NaC1, 20mM imidazole; pH 7.5) was used to wash away impurities, and then Elutionbuffer (50mM Tris-HC1, 150mM NaC1, 100-500mM imidazole; pH 7.5) was used to elute the protein.

[0123] After purification, the same volume of pure enzyme was loaded for SDS-PAGE verification. The results were as follows: Fig.10 As shown. In the figure, M stands for Maker; 1 stands for lane 1, which is the supernatant of E.coil (DE3)-pETDuet1-Vgo protein passed through the column with 20mM imidazole eluent; 2 stands for lane 2, which is the supernatant of E.coil (DE3)-pETDuet1-Vgo protein passed through the column with 50mM imidazole eluent; 3 stands for lane 3, which is the supernatant of E.coil (DE3)-pETDuet1-Vgo protein passed through the column with 100mM imidazole eluent; 4 stands for lane 4, which is the supernatant of E.coil (DE3)-pETDuet1-Vgo protein passed through the column with 200mM imidazole eluent; 5 stands for lane 5, which is the supernatant of E.coil (DE3)-pETDuet1-Vgo protein passed through the column with 300mM imidazole eluent; 6 stands for lane 6, which is the supernatant of E.coil (DE3)-pETDuet1-Vgo protein passed through the column with 500mM imidazole eluent.

[0124] The protein eluate obtained above was concentrated by centrifugation using Amino Ultra-15 (30 kDa) ultrafiltration tube, and the imidazole left over from the protein purification process was washed away using Desalination buffer (50 mM Tris-HCl.150 mM NaCl; pH 7.5). Glycerol was added to the obtained protein solution to a final concentration of 10%, and stored at -80°C for later use.

[0125] In summary, the present invention provides an oxygenase capable of catalyzing the synthesis of vanillin from 4-vinylguaiacol and its application, wherein the 4-vinylguaiacol oxygenase is soluble and expressed in a host bacterium. The crude enzyme solution expressed by the oxygenase stably catalyzes the conversion of 4-vinylguaiacol into vanillin, with a conversion rate of 80-98%, and can produce 11.5 g / L of vanillin in 6 hours.

[0126] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. The use of oxygenase in catalyzing the synthesis of vanillin from 4-vinylguaiacol, characterized in that: The amino acid sequence of the oxygenase includes the sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleic acid sequence of the oxygenase includes the sequence shown in SEQ ID NO.

2.

3. A method for synthesizing vanillin, characterized in that: The method comprises: using the oxygenase described in claim 1 or 2 to catalyze 4-vinylguaiacol to synthesize vanillin.

4. The method for synthesizing vanillin according to claim 3, characterized in that: The method for synthesizing vanillin specifically comprises: constructing a recombinant plasmid containing the nucleic acid sequence of the oxygenase according to claim 1 or 2, transferring the recombinant plasmid into a host cell to obtain a crude enzyme solution, adding 4-vinylguaiacol and a buffer to synthesize vanillin; Preferably, the method for synthesizing vanillin comprises the following steps: (1) connecting the nucleic acid sequence of the oxygenase according to claim 1 or 2 to a plasmid vector to construct a recombinant plasmid; (2) transferring the recombinant plasmid constructed in step (1) into a host bacterium to obtain a genetically engineered strain; (3) activating the genetically engineered strain, performing antibiotic screening, and performing seed culture to obtain seed liquid; (4) inducing expression in the seed solution; (5) collecting the genetically engineered strain by the first centrifugation, resuspending the cells and breaking them, and collecting the supernatant by the second centrifugation to obtain the genetically engineered crude enzyme solution; (6) 4-vinylguaiacol is dissolved in a buffer solution, and a crude enzyme solution of a genetically engineered strain is added to the 4-vinylguaiacol solution to react and synthesize vanillin.

5. The method for synthesizing vanillin according to claim 4, characterized in that: The plasmid vector in step (1) includes pETDuet1-Vgo; Preferably, the host bacteria in step (2) include any one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris or Saccharomyces cerevisiae.

6. The method for synthesizing vanillin according to claim 4 or 5, characterized in that: The temperature for the bacterial activation in step (3) is 25-35°C and the time is 0.5-2h; Preferably, the shaking speed of the bacterial cell activation in step (3) is 150-300 rpm; Preferably, the inoculum amount for bacterial activation in step (3) is 0.5-3%; Preferably, the antibiotics screened in step (3) include any one of ampicillin, kanamycin, streptomycin or penicillin, or a combination of at least two thereof; Preferably, the concentration of the antibiotic is 50-150 μg / mL; Preferably, the temperature of the seed culture in step (3) is 25-35°C and the time is 8-16h; Preferably, the shaking speed of the bacterial cell activation in step (3) is 150-300 rpm.

7. The method for synthesizing vanillin according to any one of claims 4 to 6, characterized in that: The step of inducing differentiation in step (4) comprises: inoculating the seed solution into a differentiation induction medium; Preferably, the inoculation amount of the seed liquid inoculation is 0.5-3%; Preferably, the differentiation induction medium is an induction composition and a basal medium; Preferably, the basal culture medium is LB culture medium; Preferably, the induction composition comprises 0.05-0.5 mM IPTG and / or 0.5-5 mM Fe2Cl; Preferably, the OD of the seed solution is 600 It is 0.1-5.

8. The method for synthesizing vanillin according to any one of claims 4 to 7, characterized in that: The speed of the first centrifugation in step (5) is 6000-10000 rpm, and the time is 2-8 min; Preferably, the second centrifugation in step (5) is performed at a speed of 6000-10000 rpm for 20-35 min; Preferably, the step (5) further includes a purification operation.

9. The method for synthesizing vanillin according to any one of claims 4 to 8, characterized in that: The purification method includes nickel column purification; Preferably, the nickel column purification step comprises: adding the protein-containing supernatant to the nickel column, adding a cleaning solution and then adding an eluent to obtain a crude enzyme solution; Preferably, the eluent comprises tris(hydroxymethylaminomethane) hydrochloride, sodium chloride and imidazole; Preferably, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 30-70 mM; Preferably, the concentration of sodium chloride is 300-800 mM; Preferably, the concentration of imidazole is 100-500 mM.

10. The method for synthesizing vanillin according to any one of claims 4 to 9, characterized in that: The buffer in step (6) comprises any one or a combination of at least two of sodium citrate-citric acid, Tris-HCl, Na2HPO4-NaH2PO4 or Na2CO3-NaHCO3; Preferably, the final concentration of the buffer in step (6) is 1.5-2.5 M; Preferably, the pH of the buffer solution in step (6) is 5.0-11.0; Preferably, the concentration of the 4-vinylguaiacol solution in step (6) is 0.5-5 g / L; Preferably, the volume ratio of the crude enzyme solution to 4-vinylguaiacol in step (6) is 1:(6-12); Preferably, the reaction time in step (6) is 10-720 min and the temperature is 20-70°C.

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