D-threonine aldolase mutant and application thereof

Abstract

The invention discloses a D-threonine aldolase mutant and application thereof, and 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 the D-serine comprises the following steps: in the presence of pyridoxal phosphate and magnesium chloride, catalyzing formaldehyde and glycine to react by using the D-threonine aldolase mutant; the invention further discloses the application of the D-threonine aldolase mutant in preparation of D-serine. The highest catalytic activity of the D-threonine aldolase mutant is improved by 2.04 times compared with the enzyme activity of a wild strain, and the productivity of D-serine is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a D-threonine aldolase mutant and application thereof. Background Art

[0002] Serine, also known as β-hydroxyalanine, has a chemical formula of C3H7NO3. It is named after silk, which was first derived from it. Serine is a neutral aliphatic hydroxyl-containing amino acid. D-serine is a right-handed amino acid that exists in neurons, astrocytes, and microglia. D-serine has a wide range of uses. As a chiral substance that is an intermediate for synthetic medicines such as lacosamide, it is also a raw material for the synthesis of 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 become increasingly popular, and the market demand for D-serine has increased year by year.

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

[0004] Research reports that, through microbial action, N-carbamoyl-D-serine is first generated from DL-hydroxymethylhydantoin, and then hydrolyzed to obtain D-serine. The D-threonine aldolase from Arthrobacter spDK-38 of the genus Arthrobacter has the ability to synthesize D-serine from glycine and formaldehyde, but the conversion is not complete, resulting in low optical purity of the product, low yield, and difficult to remove residual formaldehyde, making it difficult to meet pharmaceutical standards.

[0005] CN1280424C discloses a method for preparing D-serine: a microorganism having an L-serine deaminase (Escherichia coli sdaA or sdaB) gene is cultured on a DL-serine medium to extract the remaining D-serine in the culture medium; in addition, a method for obtaining D-serine by converting L-serine into L-tyrosine by tyrosinase is also disclosed, 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, wherein one molecule of formaldehyde and one molecule of glycine can produce one molecule of D-serine under the action of D-threonine aldolase (Arthrobacter, 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 Achromobacter xylosoxidans (ATCC9220), Achromobacter xylosylating (NBRC13495), Achromobacter denitrifying (NBRC15125), and Xanthomonas oryzae (IAM1657) can catalyze the synthesis of D-serine from formaldehyde and glycine, and screens the effects of different organic solvent treatments on the optical purity of the product, as well as the effects of divalent metal ions on the stability of enzyme activity. It also discloses a method for improving the optical purity of D-serine by using Escherichia coli with a D-serine dehydrogenase deficiency and a serine hydroxymethyltransferase gene destroyed as a host bacterium and introducing an L-serine deaminase gene. However, the conversion rate of the enzyme needs to be further improved.

[0008] Low enzyme activity and stereoselectivity have always been the bottleneck restricting the application of D-threonine aldolase. It is urgent to develop new D-threonine aldolase with higher activity and higher stereoselectivity to meet the requirements of industrial application. Summary of the invention

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

[0010] In order to achieve the purpose of the present invention, the following scheme is proposed: The present invention uses Lachnellula occidentalis D-threonine aldolase DTA (Gene ID: TVY46557.1) included in GenBank as a wild-type D-threonine aldolase, and the gene nucleotide sequence encoding the wild-type D-threonine aldolase is shown in SEQ ID NO.1. The nucleotide sequence shown in SEQ ID NO.1 is subjected to site-directed mutagenesis, and the obtained D-threonine aldolase mutant is screened to determine the key residues affecting the enzymatic properties of the wild-type D-threonine aldolase, and at the same time obtain a D-threonine aldolase mutant with high activity. The experimental results show that the D-threonine aldolase mutant obtained by screening is applied to the catalytic reaction of preparing D-serine with a glycine substrate, and exhibits a good catalytic effect.

[0011] The present invention screened out six D-threonine aldolase mutants: (1) Mutant DTA-1: In the amino acid sequence of the wild-type D-threonine aldolase, lysine at position 48 is replaced by histidine, glutamine at position 162 is replaced by asparagine, and proline at position 234 is replaced by glycine, that is, the amino acid sequence of mutant DTA-1 is SEQ ID NO.8. The corresponding nucleotide sequence of mutant DTA-1 is shown in SEQ ID NO.2.

[0012] (2) Mutant DTA-2: In the amino acid sequence of the wild-type D-threonine aldolase, the lysine at position 48 is replaced by arginine, the glutamine at position 162 is replaced by serine, and the proline at position 234 is replaced by glycine. The amino acid sequence of the mutant DTA-2 is SEQ ID NO.9. The nucleotide sequence corresponding to the mutant DTA-2 is shown in SEQ ID NO.3.

[0013] (3) Mutant DTA-3: In the amino acid sequence of the wild-type D-threonine aldolase, the tyrosine at position 178 is replaced by tryptophan, the aspartic acid at position 204 is replaced by glutamic acid, and the alanine at position 274 is replaced by leucine, which is the amino acid sequence of the mutant DTA-3 (SEQ ID NO.10). The nucleotide sequence corresponding to the mutant DTA-3 is shown in SEQ ID NO.4.

[0014] (4) Mutant DTA-4: In the amino acid sequence of the wild-type D-threonine aldolase, the tyrosine at position 178 is replaced by phenylalanine, the aspartic acid at position 204 is replaced by glutamic acid, and the alanine at position 274 is replaced by valine. The amino acid sequence of the mutant DTA-4 is SEQ ID NO.11. The nucleotide sequence corresponding to the mutant DTA-4 is shown in SEQ ID NO.5.

[0015] (5) Mutant DTA-5: In the amino acid sequence of the wild-type D-threonine aldolase, the leucine at position 156 is replaced by methionine, the threonine at position 224 is replaced by serine, and the phenylalanine at position 294 is replaced by tryptophan. The amino acid sequence of the mutant DTA-5 is SEQ ID NO.12. The nucleotide sequence corresponding to the mutant DTA-5 is shown in SEQ ID NO.6.

[0016] (6) Mutant DTA-6: In the amino acid sequence of the wild-type D-threonine aldolase, the isoleucine at position 164 is replaced by valine, the threonine at position 224 is replaced by serine, and the glycine at position 284 is replaced by proline. The amino acid sequence of the mutant DTA-6 is SEQ ID NO.13. The nucleotide sequence corresponding to the mutant DTA-6 is shown in SEQ ID NO.7.

[0017] The sequence shown in the present invention is optimized for Escherichia coli codons by Sangon Biotechnology Co., Ltd., and then the whole gene is synthesized, and 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, and the host cell is Escherichia coli BL21 (DE3), which is suitable for efficient expression of foreign genes.

[0018] The preparation method of biocatalytic synthesis of D-serine comprises the following steps: (1) The host cells described above are subjected to ultra-low temperature freeze-thaw disruption to obtain a D-threonine aldolase mutant.

[0019] (2) Add the obtained D-threonine aldolase mutant to the D-serine preparation system, the concentration of the D-threonine aldolase mutant is 20 g / L, the concentration of glycine is 150 g / L, the concentration of formaldehyde is 200 g / L, the concentration of magnesium chloride is 2 g / L, and the concentration of pyridoxal phosphate is 0.05 g / L. The transformant or its processed 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 is carried out under shaking or stirring at a temperature of 30°C-35°C; the pH is controlled at 7.2-7.5, the reaction is continued for 18 hours, ventilation and purge are carried out during the reaction, and the biocatalytic conversion rate is as high as 95.2%, and the ee value of the target product is as high as 99.8%.

[0020] The beneficial effects of the present invention are: obtaining a D-threonine aldolase mutant with high activity, the maximum catalytic activity of which is 2.04 times higher than that of the wild-type strain, realizing efficient catalysis of producing D-serine 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

[0021] Figure 1 Map of the plasmid construction for wild-type D-threonine aldolase (pET28a-DTA).

[0022] Figure 2 This is the protein electrophoresis diagram of D-threonine aldolase mutants (Y178F, D204E, A274V).

[0023] Figure 3 The figure is a high performance liquid chromatogram of the product obtained by using D-threonine aldolase mutants (Y178F, D204E, A274V) as catalyst. DETAILED DESCRIPTION

[0024] Example 1 1. Construction of wild-type D-threonine aldolase expression strain The wild-type D-threonine aldolase gene sequence was used as a template to amplify the target gene 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 was constructed as follows: Figure 1 shown.

[0025] The obtained recombinant plasmid pET-28a(+)-DTA was transferred into the intermediate cloning host E.coliDH5a by chemical transformation, and the positive transformants that were verified to be correct were subjected to plasmid extraction. The complete plasmid was then transferred into the competent cells of the expression host E.coliBL21 by chemical transformation. After 1 hour of recovery, the transformation liquid was centrifuged and appropriate liquid was retained to resuspend the bacteria, which were then spread on LB agar plates containing kanamycin sulfate (25ug / mL) and cultured at 37°C for 12 hours. Finally, the clones were verified, and the successfully verified positive clones were wild-type D-threonine aldolase genetic engineering bacteria.

[0026] Specifically, the D-threonine aldolase gene engineering bacteria are activated and inoculated into LB medium, and seed culture is carried out at 37° C. and 200 rpm for 12 hours to obtain seed liquid; the seed liquid is transferred to LB medium at an inoculation amount of 6% v / v for fermentation, and when the 0D of the bacteria is between 0.1 and 0.7, 0.3-1.2 mM IPTG is added to the LB medium for induction culture to obtain a fermentation liquid containing D-threonine aldolase.

[0027] Wherein, the LB culture medium has a formula of: NaCl 5-15g / L, trypsin 5-5g / L, and yeast powder 2-7g / L.

[0028] Wherein, the fermentation conditions are: 20-37°C, 200rpm; the induction culture conditions are: 20-37°C, 200rpm, induction 18-30h, pH 6.0-8.0.

[0029] 2. Construction of D-threonine aldolase mutant expression strain Site-directed mutagenesis of DTA resulted in the following six mutants: (1) DTA-1: K48H, Q162N, P234G, the amino acid sequence is shown in SEQ ID NO.8.

[0030] (2) DTA-2: K48R, Q162S, P234G, the amino acid sequence is shown in SEQ ID NO.9.

[0031] (3) DTA-3: Y178W, D204E, A274L, the amino acid sequence is shown in SEQ ID NO.10.

[0032] (4) DTA-4: Y178F, D204E, A274V, the amino acid sequence is shown in SEQ ID NO.11.

[0033] (5) DTA-5: L156M, T224S, F294W, the amino acid sequence is shown in SEQ ID NO.12.

[0034] (6) DTA-6: I164V, T224S, G284P, the amino acid sequence is shown in SEQ ID NO.13.

[0035] The above sequences were commissioned by Sangon Biotech Co., Ltd. to optimize the E. coli codons for their respective nucleotide sequences, and then synthesized into full genes to obtain their respective recombinant vectors. After sequencing verification, they were introduced into E. coli BL21 (DE3) to obtain the corresponding expression strains. After fermentation expression, the best protein mutation expression was selected for electrophoresis detection, such as Figure 2 As shown, lane 0 is a 14kDa-180kDa protein marker, lane 1 is the supernatant protein after E. coli BL21(DE3)-pET-28a-DTA was fermented at 25°C and 0.2 mM IPTG was induced to break the cells, lane 2 is the precipitated protein after E. coli BL21(DE3)-pET-28a-DTA was fermented at 25°C and 0.2 mM IPTG was induced to break the cells, lane 3 is the supernatant protein after E. coli BL21(DE3)-pET-28a-DTA was fermented at 25°C and 0.4 mM IPTG was induced to break the cells, lane 4 is the precipitated protein after high-speed centrifugation after E. coli BL21(DE3)-pET-28a-DTA was fermented at 25°C and 0.4 mM IPTG was induced to break the cells, lane 5 is the supernatant protein after E. coli BL21(DE3)-pET-28a-DTA was fermented at 30°C and 0.2 mM The supernatant protein after IPTG-induced fermentation and cell lysis, lane 6 is the precipitated protein after high-speed centrifugation of Escherichia coli BL21 (DE3) -pET-28a-DTA at 30°C and 0.2mM IPTG-induced fermentation and cell lysis, lane 7 is the supernatant protein after Escherichia coli BL21 (DE3) -pET-28a-DTA at 30°C and 0.4mM IPTG-induced fermentation and cell lysis, lane 8 is the precipitated protein after high-speed centrifugation of Escherichia coli BL21 (DE3) -pET-28a-DTA at 30°C and 0.4mM IPTG-induced fermentation and cell lysis, the protein band position and size are correct, the protein solubility is good, and there are few inclusion bodies.

[0036] Example 2 Determination of D-threonine aldolase mutant enzyme activity Quantitatively weigh 0.075g glycine, 0.081g formaldehyde solution (37% aqueous solution), 0.02mL of 50mM pyridoxal phosphate mother solution (PLP), and 0.0095g anhydrous magnesium chloride into the reactor, add 8ml 200mM Tris-HCl solution of pH 8.0, adjust the pH to 7.5 with 3MNaOH aqueous solution, and add 0.2g DTA whole cell wet bacteria (solid content 16%) obtained by the method of Example 1. Shake at 30°C and 220rpm for 3h. After the reaction is completed, take the reaction solution, adjust the acid, centrifuge, take the supernatant, and HPLC to detect the D-serine yield. Take the supernatant to adjust neutrality, Marfey derivatization, and detect the ee value. The results are shown in Table 1. According to the highest conversion rate, the enzyme activity is increased by 2.04 times compared with the wild strain.

[0037] The mobile phase for liquid chromatography detection is: 0.05% TFA acetonitrile solution and 0.05% TFA aqueous solution. Use a 0.22um organic filter membrane to filter through the membrane. Take 2000ml of analytical pure acetonitrile, add 1ml TFA, and ultrasonicate for 30min; take 2000ml of ultrapure water, add 1ml TFA, and ultrasonicate for 30min to remove the bubbles. The result is the mobile phase. The liquid chromatograph is LC-2050 Shimadzu, the detector is an ultraviolet detector, the detection column is C18, the specification is 250nm×4.6nm, 5μm, the mobile phase flow rate is 1mL / min, the injection volume is 2μL, the detection temperature is 30℃, and the peak detection time is 5.848min. Figure 3 shown.

[0038] Table 1 Comparison of catalytic functions of different D-threonine aldolase mutants Example 3 20T catalytic reaction and purification 1. Feeding reaction Add 4000kg water, 810kg D-threonine aldolase mutant and 1kg pyridoxal phosphate to reactor 1, heat to 35℃, stir and set aside. Add 9000kg water, 3000kg glycine, 45kg magnesium chloride hexahydrate and 450kg formaldehyde to reactor 2, heat to 35℃, adjust pH to 7.3 with 20% sodium hydroxide solution, and dissolve all glycine. Add formaldehyde dropwise to keep the system pH at 7.25-7.35. After the addition of 2790kg formaldehyde, keep the temperature at 35℃ and react for 18 hours before sending the sample for testing.

[0039] 2. Denaturation and discoloration due to temperature rise The reaction liquid was adjusted to pH 4.9 with 50% dilute sulfuric acid, and the temperature was raised to 55°C for 2 hours. The reaction liquid was stripped with an appropriate amount of air for 8 hours. 18,000L of the reaction liquid was transferred to a decolorization tank, 200kg of activated carbon was added, the temperature was raised to 55°C, and the decolorization was carried out for 2-3 hours.

[0040] 3. Ultrafiltration and nanofiltration About 22,000L of decolorized liquid after plate and frame washing enters the ultrafiltration unit, filtered by the 3KD ultrafiltration unit, and qualified liquid enters the ultrafiltration clear liquid storage tank. The ultrafiltration concentrate needs to be top washed with pure water twice, 1000L of pure water each time. After the ultrafiltration unit is used up, the cleaning process is carried out according to the ultrafiltration unit cleaning operation procedures. 24,000L of ultrafiltration clear liquid enters the nanofiltration unit, filtered by the nanofiltration unit, and qualified liquid enters the nanofiltration clear liquid storage tank. The nanofiltration concentrate needs to be top washed with pure water twice, 1000L of pure water each time. After the nanofiltration unit is used up, the cleaning process is carried out according to the nanofiltration unit cleaning operation procedures.

[0041] 4. Crystallization 26000L of nanofiltration clear liquid enters the concentrator unit for concentration, and the temperature is maintained at 50-55℃. 14000L of water is concentrated and removed until the system is 10000L, and the concentration is stopped. The concentrated liquid is transferred to the crystallization tank for cooling and crystallization, and the temperature slowly drops to 35-37℃. After cooling, the crystallization liquid is pumped into the centrifuge for centrifugation. After centrifugation, it is rinsed with atomized pure water for 30s-60s to obtain a wet crude product. The mother liquor is concentrated again, crystallized and centrifuged to obtain a wet crude product. A small sample of the crude product is dried and sent for testing of the hand purity of D-serine, the purity of D-serine and the residue of glycine. The wet crude product is transferred to the double cone drying equipment for negative pressure drying at a temperature of 70-80℃, and the material can be collected when the moisture content is less than 0.2%.

[0042] 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 shorter than the reaction time of the wild strain. Compared with the current catalytic time, the efficiency was increased by 35%, and the ee value was measured to be 99.8%. The crude product reached the qualified purity after two concentration and crystallization. According to manual calculation, the entire 20T catalytic reaction and purification process reduced the cost by about 13% compared with the original wild enzyme catalytic process.

[0043] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within 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 Used to encode the D-threonine aldolase mutant according to 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: The following steps are involved: The D-threonine aldolase mutant of claim 1 is used to catalyze the reaction of formaldehyde and glycine in the presence of pyridoxal phosphate and magnesium chloride.

5. The method according to claim 4, characterized in that In the reaction system: the concentration of glycine is 150 g / L, the concentration of formaldehyde is 200 g / L, the concentration of 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 is continued for 18 hours.

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

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