A D-threonine aldolase mutant and its application

Abstract

The present invention discloses a D-threonine aldolase mutant and its application, which relates to the technical field of bioengineering. The amino acid sequence of the D-threonine aldolase mutant is selected from one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13. The method for preparing D-serine comprises the following steps: in the presence of pyridoxal phosphate and magnesium chloride, using the D-threonine aldolase mutant to catalyze the reaction of formaldehyde and glycine; the application of the D-threonine aldolase mutant in the preparation of D-serine. The highest catalytic activity of the D-threonine aldolase mutant of the present invention is 2.04 times higher than that of the wild-type strain, significantly improving the productivity of D-serine.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a D-threonine aldolase mutant and its application. Background Art

[0002] Serine, also known as β-hydroxyalanine, with the chemical formula C3H7NO3, is named after being first derived from silk. Serine is a neutral aliphatic hydroxy amino acid. D-serine is a dextrorotatory amino acid and exists in neurons, astrocytes and microglia. D-serine has a wide range of uses. As a chiral substance for synthesizing intermediates of some medicines such as lacosamide, it is also a raw material for synthesizing dipeptides, short peptides and polypeptides. D-serine is also involved in the pathogenesis of Parkinson's disease and epilepsy. In recent years, the research on D-serine in the field of neuroscience has been gradually heating up, and the market demand for D-serine has been increasing year by year.

[0003] The production methods of D-serine usually include physical resolution method, fermentation method, protein hydrolysis extraction method, chemical synthesis method, bioenzyme method, etc.

[0004] Research reports that through the action of microorganisms, N-carbamoyl-D-serine is first generated from DL-hydroxy methyl hydantoin, and then D-serine can be obtained by hydrolyzing N-carbamoyl-D-serine. The D-threonine aldolase from Arthrobacter sp DK-38 of the genus Arthrobacter has the ability to synthesize D-serine using glycine and formaldehyde, but the conversion is not complete, resulting in low optical purity and low yield of the product, and the residual formaldehyde is difficult to remove, making it difficult to meet the pharmaceutical standards.

[0005] CN1280424C discloses a method for preparing D-serine: cultivating microorganisms with L-serine deaminase (Escherichia coli sdaA or sdaB) gene in a DL-serine medium, and then extracting the remaining D-serine in the culture medium; in addition, it also discloses a method for obtaining D-serine by converting L-serine into L-tyrosine through tyrosinase, but in this method, the theoretical yield of D-serine relative to DL-serine is only 50%.

[0006] CN110373440A discloses a method for preparing DL-serine. One molecule of formaldehyde and one molecule of glycine can produce one molecule of D-serine under the action of D-threonine aldolase (Arthrobacter genus, protein sequence number: BAA31547.1), and D-serine is converted into DL-serine under the action of recombinant alanine racemase, but the optical purity of the product is not disclosed.

[0007] CN101040047B discloses that D-threonine aldolase from microorganisms of Achromobacter xylosoxidans (ATCC9220), Achromobacter xylosaccharolyticus (NBRC13495), Achromobacter denitrificans (NBRC15125), and Xanthomonas oryzae (IAM1657) can catalyze the synthesis of D-serine from formaldehyde and glycine. Meanwhile, the effects of treatment with different organic solvents on the optical purity of the product and the effects of divalent metal ions on the enzyme activity stability were screened. And a method for improving the optical purity of D-serine by using Escherichia coli with deletion of D-serine dehydrogenase and disruption of serine hydroxymethyltransferase gene as the host bacterium and introducing the L-serine deaminase gene was disclosed. However, the conversion rate of the enzyme still needs to be further improved.

[0008] The low enzyme activity and stereoselectivity have always been the bottlenecks restricting the application of D-threonine aldolase. There is an urgent need to develop new D-threonine aldolases with higher activity and higher stereoselectivity to meet the requirements of industrial applications. SUMMARY OF THE INVENTION

[0009] To solve the deficiencies of the prior art, the present invention provides a D-threonine aldolase mutant and its application. This mutant has higher activity and higher stereoselectivity.

[0010] To achieve the object of the present invention, the following scheme is proposed:

[0011] The present invention uses the D-threonine aldolase DTA (Gene ID: TVY46557.1) of Lachnellula occidentalis included in GenBank as the wild-type D-threonine aldolase. The nucleotide sequence of the gene encoding the wild-type D-threonine aldolase is shown in SEQ ID NO.1. Site-directed mutagenesis is performed on the nucleotide sequence shown in SEQ ID NO.1, and the obtained D-threonine aldolase mutants are screened to determine the key residues affecting the enzymatic properties of the wild-type D-threonine aldolase. Meanwhile, D-threonine aldolase mutants with high activity are obtained. The experimental results show that the screened D-threonine aldolase mutants exhibit good catalytic effects when applied to the catalytic reaction for preparing D-serine with glycine as the substrate.

[0012] The present invention screens six D-threonine aldolase mutants:

[0013] (1) Mutant DTA-1: Replace lysine at position 48, glutamine at position 162, and proline at position 234 in the amino acid sequence of wild-type D-threonine aldolase with histidine, asparagine, and glycine, respectively, to obtain the amino acid sequence SEQ ID NO.8 of mutant DTA-1. The nucleotide sequence corresponding to mutant DTA-1 is shown as SEQ ID NO.2.

[0014] (2) Mutant DTA-2: Replace lysine at position 48, glutamine at position 162, and proline at position 234 in the amino acid sequence of wild-type D-threonine aldolase with arginine, serine, and glycine, respectively, to obtain the amino acid sequence SEQ ID NO.9 of mutant DTA-2. The nucleotide sequence corresponding to mutant DTA-2 is shown as SEQ ID NO.3.

[0015] (3) Mutant DTA-3: Replace tyrosine at position 178, aspartic acid at position 204, and alanine at position 274 in the amino acid sequence of wild-type D-threonine aldolase with tryptophan, glutamic acid, and leucine, respectively, to obtain the amino acid sequence SEQ ID NO.10 of mutant DTA-3. The nucleotide sequence corresponding to mutant DTA-3 is shown as SEQ ID NO.4.

[0016] (4) Mutant DTA-4: Replace tyrosine at position 178, aspartic acid at position 204, and alanine at position 274 in the amino acid sequence of wild-type D-threonine aldolase with phenylalanine, glutamic acid, and valine, respectively, to obtain the amino acid sequence SEQ ID NO.11 of mutant DTA-4. The nucleotide sequence corresponding to mutant DTA-4 is shown as SEQ ID NO.5.

[0017] (5) Mutant DTA-5: Replace leucine at position 156, threonine at position 224, and phenylalanine at position 294 in the amino acid sequence of wild-type D-threonine aldolase with methionine, serine, and tryptophan, respectively, to obtain the amino acid sequence SEQ ID NO.12 of mutant DTA-5. The nucleotide sequence corresponding to mutant DTA-5 is shown as SEQ ID NO.6.

[0018] (6) Mutant DTA-6: Replace isoleucine at position 164, threonine at position 224, and glycine at position 284 in the amino acid sequence of wild-type D-threonine aldolase with valine, serine, and proline, respectively, to obtain the amino acid sequence SEQ ID NO.13 of mutant DTA-6. The nucleotide sequence corresponding to mutant DTA-6 is shown as SEQ ID NO.7.

[0019] The sequence shown in the present invention is codon-optimized in Escherichia coli by Sangon Biotech Co., Ltd., and then fully synthesized. A 6xHis fusion expression vector of the mutant gene is constructed. The expression vector uses any one of pET22b, pET28a, pET32a, and pCold TF, preferably pET28a plasmid. The host cell is Escherichia coli BL21 (DE3), which is suitable for efficient expression of exogenous genes.

[0020] The preparation method of D-serine by biocatalytic synthesis comprises the following steps:

[0021] (1) The host cells described above were subjected to ultra-low temperature freeze-thaw disruption to obtain D-threonine aldolase mutants.

[0022] (2) The obtained D-threonine aldolase mutant was added to the D-serine preparation system, the concentration of the D-threonine aldolase mutant was 20 g / L, the concentration of glycine was 150 g / L, the concentration of formaldehyde was 200 g / L, the concentration of magnesium chloride was 2 g / L, and the concentration of pyridoxal phosphate was 0.05 g / L. The transformant or its transformed product can be added at once at the beginning of the reaction, or can be added in batches or continuously during the reaction. The catalytic reaction was carried out under conditions of shaking or stirring at a temperature of 30°C-35°C; the pH was controlled at 7.2-7.5, the reaction was continued for 18 hours, and ventilation and purge were performed during the reaction. The biocatalytic conversion rate was as high as 95.2%, and the ee value of the target product was as high as 99.8%.

[0023] The beneficial effects of the present invention are: obtaining a highly active D-threonine aldolase mutant, whose maximum catalytic activity is 2.04 times higher than that of the wild-type strain, realizing efficient catalysis of D-serine production using glycine as a raw material, significantly improving the productivity of D-serine, and having good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Map of the plasmid construction for wild-type D-threonine aldolase (pET28a-DTA).

[0025] Figure 2 This is the protein electrophoresis diagram of D-threonine aldolase mutants (Y178F, D204E, A274V).

[0026] Figure 3 The figure shows the high performance liquid chromatogram of the product obtained by using D-threonine aldolase mutants (Y178F, D204E, A274V) as catalyst. DETAILED DESCRIPTION

[0027] Example 1

[0028] 1. Construction of wild-type D-threonine aldolase expression strain

[0029] Using the wild-type D-threonine aldolase gene sequence as a template, the target gene was amplified by PCR reaction, and the vector pET-28a(+) and the target gene were linearized with restriction enzymes NdeI and XhoI, and then ligated to obtain the recombinant plasmid pET-28a(+)-DTA. The plasmid construction is as Figure 1 shown.

[0030] The obtained recombinant plasmid pET-28a(+)-DTA was transferred into the intermediate cloning host E. coli DH5α by chemical transformation method. The plasmid of the verified correct positive transformant was extracted, and then the complete plasmid was transferred into the competent cells of the expression host E. coli BL21 by chemical transformation method. After recovery for 1 h, the transformation solution was centrifuged and an appropriate amount of liquid was retained to resuspend the bacteria, and then it was spread on an LB agar plate containing kanamycin sulfate (25 μg / mL) and cultured statically at 37 °C for 12 h. Finally, the clones were verified, and the successfully verified positive clones were the genetic engineering bacteria of wild-type D-threonine aldolase.

[0031] Specifically, the genetic engineering bacteria of D-threonine aldolase were activated and inoculated into LB medium, and cultured at 37 °C and 200 rpm for 12 h to obtain a seed solution; the seed solution was transferred to LB medium for fermentation at an inoculation amount of 6% v / v. When the OD of the bacteria was between 0.1 and 0.7, 0.3-1.2 mM IPTG was added to the LB medium for induction culture to obtain a fermentation broth containing D-threonine aldolase.

[0032] Among them, the formula of the LB medium is: NaCl 5-15 g / L, tryptone 5-5 g / L, yeast powder 2-7 g / L.

[0033] Among them, the fermentation conditions are: 20-37 °C, 200 rpm; the induction culture conditions are: 20-37 °C, 200 rpm, induced for 18-30 h, pH 6.0-8.0.

[0034] 2. Construction of D-threonine aldolase mutant expression strain

[0035] Site-directed mutagenesis was performed on DTA to obtain the following six mutants:

[0036] (1) DTA-1: K48H, Q162N, P234G, and the amino acid sequence is as shown in SEQ ID NO.8.

[0037] (2) DTA-2: K48R, Q162S, P234G, and the amino acid sequence is as shown in SEQ ID NO.9.

[0038] (3) DTA-3: Y178W, D204E, A274L, with the amino acid sequence shown in SEQ ID NO. 10.

[0039] (4) DTA-4: Y178F, D204E, A274V, with the amino acid sequence shown in SEQ ID NO. 11.

[0040] (5) DTA-5: L156M, T224S, F294W, with the amino acid sequence shown in SEQ ID NO. 12.

[0041] (6) DTA-6: I164V, T224S, G284P, with the amino acid sequence shown in SEQ ID NO. 13.

[0042] After the above sequences were entrusted to Sangon Biotech Co., Ltd. for codon optimization of their respective nucleotide sequences in Escherichia coli and then full gene synthesis to obtain their corresponding recombinant vectors, after being verified correct by sequencing, they were introduced into Escherichia coli BL21(DE3) to obtain the corresponding expression strains. After fermentation and expression, the best protein mutant expression was selected for electrophoresis detection. As Figure 2 shown, lane 0 is the 14 kDa - 180 kDa protein marker, lane 1 is the supernatant protein after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.2 mM IPTG at 25°C, lane 2 is the precipitated protein after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.2 mM IPTG at 25°C, lane 3 is the supernatant protein after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.4 mM IPTG at 25°C, lane 4 is the precipitated protein after high-speed centrifugation after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.4 mM IPTG at 25°C, lane 5 is the supernatant protein after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.2 mM IPTG at 30°C, lane 6 is the precipitated protein after high-speed centrifugation after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.2 mM IPTG at 30°C, lane 7 is the supernatant protein after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.4 mM IPTG at 30°C, lane 8 is the precipitated protein after high-speed centrifugation after fermentation and cell disruption of Escherichia coli BL21(DE3)-pET-28a-DTA induced by 0.4 mM IPTG at 30°C. The position and size of the protein bands are correct, the protein solubility is good, and there are few inclusion bodies.

[0043] Example 2 Determination of the enzyme activity of D-threonine aldolase mutant

[0044] Weigh 0.075 g of glycine, 0.081 g of formaldehyde solution (37% aqueous solution), 0.02 mL of 50 mM pyridoxal phosphate stock solution (PLP), and 0.0095 g of anhydrous magnesium chloride into a reactor. Add 8 mL of 200 mM Tris-HCl solution at pH 8.0, adjust the pH to 7.5 with 3 M NaOH aqueous solution, and add 0.2 g of wet whole cells of DTA obtained by the method of Example 1 (solid content 16%). React in a shaker at 30 °C and 220 rpm for 3 h. After the reaction is completed, take the reaction solution, adjust the acidity, centrifuge, take the supernatant, and detect the yield of D-serine by HPLC. Take the supernatant and adjust it to neutral, derivatize with Marfey, and detect the ee value. The results are shown in Table 1. Calculated according to the highest conversion rate, the enzyme activity is increased by 2.04 times compared with the wild strain.

[0045] The mobile phase for liquid chromatography detection is: 0.05% TFA acetonitrile solution and 0.05% TFA aqueous solution. Filter through a 0.22 μm organic filter membrane. Take 2000 mL of analytical grade acetonitrile, add 1 mL of TFA, and ultrasonicate for 30 min; take 2000 mL of ultrapure water, add 1 mL of TFA, and ultrasonicate for 30 min to remove the bubbles in it. The resulting solution is the mobile phase. The liquid chromatograph is LC-2050 Shimadzu, the detector is an ultraviolet detector, the detection chromatographic column is C18, with a specification of 250 nm × 4.6 nm, 5 μm, the flow rate of the mobile phase is 1 mL / min, the injection volume is 2 μL, the detection temperature is 30 °C, and the detection peak time is 5.848 min, as Figure 3 shown.

[0046] Table 1 Comparison of the catalytic functions of different D-threonine aldolase mutants

[0047]

[0048] Example 3 20T catalytic reaction and purification

[0049] 1. Feed and react

[0050] Add 4000 kg of water, 810 kg of D-threonine aldolase mutant, and 1 kg of pyridoxal phosphate to Reactor 1, heat to 35 °C and stir for later use. Add 9000 kg of water, 3000 kg of glycine, 45 kg of magnesium chloride hexahydrate, and 450 kg of formaldehyde to Reactor 2, heat to 35 °C, adjust the pH to 7.3 with 20% sodium hydroxide solution until all glycine is dissolved. Dropwise add formaldehyde to maintain the system pH at 7.25 - 7.35. After 2790 kg of formaldehyde is added dropwise, maintain the temperature at 35 °C and send a sample for detection after reacting for 18 hours.

[0051] 2. Heating for denaturation and decolorization

[0052] The pH of the conversion reaction solution is adjusted to 4.9 with 50% dilute sulfuric acid, heated to 55 °C and maintained for 2 hours. The reaction solution is blown off with appropriate amount of air for 8 hours. Transfer 18000 L of the reaction solution into the decolorization tank, add 200 kg of activated carbon, heat to 55 °C and decolorize for 2 - 3 hours.

[0053] 3. Ultrafiltration and nanofiltration

[0054] Approximately 22000 L of the decolorized solution after plate and frame flushing enters the ultrafiltration unit and is filtered through a 3KD ultrafiltration unit. The qualified solution enters the ultrafiltration clear liquid storage tank. The ultrafiltration concentrate needs to be backwashed twice with pure water, 1000 L of pure water each time. After the ultrafiltration unit is used up, a cleaning process is carried out according to the ultrafiltration unit cleaning operation procedures. 24000 L of the ultrafiltration clear liquid enters the nanofiltration unit and is filtered through the nanofiltration unit. The qualified solution enters the nanofiltration clear liquid storage tank. The nanofiltration concentrate needs to be backwashed twice with pure water, 1000 L of pure water each time. After the nanofiltration unit is used up, a cleaning process is carried out according to the nanofiltration unit cleaning operation procedures.

[0055] 4. Crystallization

[0056] 26000 L of the nanofiltration clear liquid enters the concentration unit for concentration, and the temperature is maintained at 50 - 55 °C. Concentration stops when 14000 L of water is removed until the system is 1000 L. The concentrated liquid is transferred to the crystallization tank for cooling crystallization, and the temperature is slowly lowered to 35 - 37 °C. After the cooling is completed, the crystallization liquid is pumped into a centrifuge for centrifugation. After centrifugation, it is rinsed with atomized pure water for 30 s - 60 s to obtain wet crude products. The mother liquor is concentrated and crystallized and centrifuged again to also obtain wet crude products. The crude product is sent for small sample drying and detection of the chiral purity of D-serine, the purity of D-serine and the glycine residue. The wet crude product is transferred to a double-cone drying device for negative pressure drying at a temperature of 70 - 80 °C, and the material can be collected when the moisture content is <0.2%.

[0057] During the whole process, the substrate conversion rate of the enzymatic reaction reached 95.2% after 18 hours of detection, which was about 6.3 hours less than that of the wild strain reaction time. Compared with the current catalytic time, the efficiency was increased by 35%. The ee value was measured to be 99.8%. The crude product reached the qualified purity after two times of concentration and crystallization. The entire 20T catalytic reaction and purification process was calculated manually and the cost was reduced by about 13% compared with the original wild enzyme catalytic process.

[0058] The above embodiments are only used to illustrate the technical ideas and characteristics of the present invention, and do not represent that they are the only or limit the present invention. Those skilled in the art should understand that without departing from the scope of the present invention, various changes or equivalent replacements made to the present invention all belong to the scope of protection of the present invention.

Claims

1. A D-threonine aldolase mutant, characterized in that, The amino acid sequence is selected from one of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.

13.

2. A gene, characterized in that, It is used to encode the D-threonine aldolase mutant described in claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence is selected from one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.

7.

4. A method for preparing D-serine, characterized in that, It includes the following steps: In the presence of pyridoxal phosphate and magnesium chloride, use the D-threonine aldolase mutant described in claim 1 to catalyze the reaction of formaldehyde and glycine.

5. The method according to claim 4, wherein In the reaction system: the concentration of glycine is 150 g / L, the concentration of formaldehyde is 200 g / L, the concentration of the D-threonine aldolase mutant is 20 g / L, the concentration of magnesium chloride is 2 g / L, the concentration of pyridoxal phosphate is 0.05 g / L, the reaction temperature is controlled at 30°C - 35°C, the pH is controlled at 7.2 - 7.5, and the reaction continues for 18 hours.

6. Use of the D-threonine aldolase mutant described in claim 1 in the preparation of D-serine.

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