Construction and application of alanine racemase mutant and whole cell catalytic system thereof

Abstract

The application discloses an alanine racemase mutant and construction and application of a whole cell catalytic system thereof, and belongs to the technical field of biology. The known alanine racemase gene sequence is subjected to site-directed mutation to obtain mutants V32I, A58L, L78W, H100S, D164A, E79G, L122Q, G254S, P287E, M305Q and G211P. Enzyme activity of the mutants is detected through induced expression, and it is found that the enzyme activity of the mutant ALR-D164A is 1.16 times higher than that of the original enzyme. Pyridoxal phosphate kinase is introduced to be co-expressed with the alanine racemase, and D-alanine is synthesized through whole cell catalysis with L-alanine as a substrate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the construction and application of an alanine racemic enzyme mutant and its whole-cell catalytic system. Background Technology

[0002] Alanine (Ala) is one of the 20 amino acids that make up human proteins and is optically active. L-alanine is an important natural amino acid and one of the essential amino acids for the human body. It mainly provides the amino group in transamination reactions and is closely related to the metabolic pathway of sugars. D-alanine, as an important "non-natural" chiral amino acid, is a key precursor for the synthesis of various fine chemicals and drugs. In the pharmaceutical field, D-alanine is a core structural module for the synthesis of broad-spectrum antibiotics (such as vancomycin and D-cycloserine) and bioactive peptide drugs with anti-tumor and anti-inflammatory properties. In the food industry, D-alanine is an important raw material for the synthesis of novel high-intensity sweeteners (such as alitane), which are low in calories and have an excellent taste. In addition, in the cosmetics field, D-alanine can significantly promote the production of adhesion proteins and collagen in the skin's basement membrane, and has antioxidant, wrinkle-reducing, and UV-damage-repairing effects.

[0003] Currently, the main methods for preparing D-alanine include fermentation, asymmetric synthesis, chemical resolution, and enzymatic methods. Fermentation can produce D-alanine from D-cycloserine via *Lactobacillus brevis*, but the product concentration is low and the production cycle is long. Asymmetric synthesis utilizes chiral raw materials to directly synthesize optically active products; its reaction mechanism is complex, requiring pure chiral reagents or noble metal complexes as catalysts, resulting in extremely high production costs. Chemical resolution uses optically active resolving agents to achieve the separation; this method is simple, but the optical purity of the product is not high. Compared with chemical synthesis, enzymatic methods have advantages such as mild reaction conditions, high selectivity, and great application potential. Summary of the Invention

[0004] Based on the above, the purpose of this invention is to provide a racemic alanine enzyme mutant and its whole-cell catalytic system for construction and application, which can realize the synthesis of D-alanine using L-alanine as a substrate through whole-cell catalysis.

[0005] The technical solution adopted by this invention to achieve its technical objectives is as follows:

[0006] This invention provides an alanine racemic enzyme mutant, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] The present invention also provides the encoding gene of the above-mentioned alanine racemic enzyme mutant, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0008] The present invention also provides a recombinant expression vector comprising the above-mentioned coding gene.

[0009] The present invention also provides a genetically engineered strain comprising the above-described recombinant expression vector.

[0010] Furthermore, the genetically engineered strain is E. coli BL21(DE3) / pET-28a(+) / ALR-D164A, with E. coli BL21(DE3) as the host strain; the recombinant expression vector uses pET-28a(+) as the expression vector.

[0011] This invention also provides a method for constructing a whole-cell catalytic system, based on the above-mentioned genetically engineered strain, comprising:

[0012] The genetically engineered strain E. coli BL21(DE3) / pET-28a(+) / ALR-D164A was used to prepare competent cells; the PDXK gene fragment was ligated to the vector pCDFDuet to obtain the recombinant plasmid pCDFDuet-PDXK, and then the recombinant plasmid pCDFDuet-PDXK was introduced into the competent cells of the above-mentioned genetically engineered strain to obtain the recombinant strain E. coli BL21(DE3) / pET-28a(+) / ALR-D164A / pCDFDuet-PDXK; the nucleotide sequence of the PDXK gene fragment is shown in SEQ ID NO:3.

[0013] The present invention also provides a whole-cell catalytic system, which is obtained using the above-described construction method.

[0014] The present invention also provides the application of the above-mentioned whole-cell catalytic system in the production of D-alanine.

[0015] Furthermore, the application includes culturing cells used for catalysis to OD. 600 The concentration is 10-20, and L-alanine is added to catalyze the reaction to produce D-alanine.

[0016] Furthermore, in 37 o The catalytic reaction was carried out at C and 180 rpm.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention involves site-directed mutagenesis of the known alanine racemase gene sequence to obtain mutants V32I, A58L, L78W, H100S, D164A, E79G, L122Q, G254S, P287E, M305Q, and G211P. Induced expression and enzyme activity analysis revealed that the mutant ALR-D164A enzyme activity was 1.16 times higher than the original enzyme. Pyridoxal phosphate kinase was introduced and co-expressed with alanine racemase. Using L-alanine as a substrate, D-alanine was synthesized via whole-cell catalysis. When OD... 600 When the concentration was 15, after 21 hours of whole-cell catalytic reaction, the content of D-alanine reached 62%. Attached Figure Description

[0019] Figure 1 This is a basic schematic diagram of the whole-cell catalytic production process.

[0020] Figure 2 This is a colony PCR image of alanine racemic enzyme mutant. Note: M represents Marker; lanes 1-11 represent V32I, A58L, L78W, H100S, D164A, E79G, L122Q, G254S, P287E, M305Q, and G211P, respectively.

[0021] Figure 3 This is an SDS-PAGE image showing the induced expression of the alanine racemic enzyme mutant D164A. Note: M represents the marker; lane 1 represents the supernatant protein; lane 2 represents the precipitated protein.

[0022] Figure 4 This is a comparison of the relative activities of the original enzyme and the mutant enzyme.

[0023] Figure 5 This is an HPLC chromatogram of whole-cell catalysis for the production of D-alanine using genetically engineered strains. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be fully described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0025] Example 1

[0026] I. Construction of alanine racemic enzyme mutant genetically engineered strains

[0027] 1. Obtaining the genome of Escherichia coli ATCC 25922

[0028] E. coli ATCC 25922 was activated by in vitro culture. The inoculum volume was 5 mL, the inoculum size was 1% (v / v), and the culture temperature was 37°C. o C, culture time is 10-12 h, after obtaining bacterial culture, the genome of E. coli ATCC 25922 is extracted using a genomic extraction kit for Gram-negative bacteria as a PCR template.

[0029] 2. Obtaining the alanine racemic enzyme mutant gene and preparing a linearized cloning vector.

[0030] Using genomic DNA from E. coli ATCC 25922 as a template, mutant primers were designed (see Table 1), and gene fragments were obtained by PCR amplification.

[0031] Table 1 Primers and sequences for each mutant

[0032]

[0033] The PCR amplification system is shown in Table 2:

[0034] Table 2 50µL PCR amplification system

[0035]

[0036] The amplification procedure is shown in Table 3:

[0037] Table 3 PCR amplification reaction procedure

[0038]

[0039] Set the cycle number for the denaturation, annealing, and extension processes to 30. Add 1 μL of SwiftCutDpn I to the PCR product and incubate at 37°C. o The digestion reaction was carried out at C for 15 min to remove the methylated raw template. After digestion, the digestion products were purified using an agarose gel DNA recovery kit.

[0040] The pET-28a(+) plasmid was double-digested with EcoRI and SalI restriction enzymes. The digestion products were recovered by gel extraction and used as linearized cloning vectors.

[0041] 3. Construct the target strain using one-step cloning technology.

[0042] The V32I, A58L, L78W, H100S, D164A, E79G, L122Q, G254S, P287E, M305Q, and G211P gene fragments recovered in step 2, along with the linearized pET-28a(+) plasmid vector, were transformed into E. coli BL21(DE3) via one-step cloning. The specific method is as follows: 100 μL of E. coli BL21(DE3) competent cells were thawed on an ice box for 5-10 min; 10 μL of cooled one-step cloning reaction mixture (reaction mixture shown in Table 4, placed at 37°C) was added to the competent cells. o The reaction was carried out in a constant temperature incubator at 42°C for 30 min. After the reaction, it was placed on ice for 5 min. The mixture was then gently tapped with a finger to mix, and placed on ice for 30 min. Finally, it was incubated at 42°C. o Heat shock at 3°C ​​for 90 seconds, then immediately in an ice bath for 3 minutes. Add 900 μL of LB medium to the above reaction system and incubate at 37°C. o Cells were cultured at 180 rpm for 1 h at C for cell resuscitation. 100 μL of the culture was then spread onto an antibiotic plate and incubated at 37°C. o Incubate in a C incubator for 12 hours, pick single colonies, and perform colony PCR verification using the primers from step 2. The results are as follows: Figure 2 As shown, colonies that amplify the target gene fragment are the strains that have successfully expressed the plasmid. These should be preserved for later use.

[0043] Table 4 One-step cloning reaction system

[0044]

[0045] II. Induction and Expression of Alanine Racemicase Mutants

[0046] 1. Bacterial expansion culture: The bacterial culture preserved above was inoculated into 5 mL of LB liquid medium containing kanamycin at a ratio of 1:100 (bacterial culture: LB liquid medium), and incubated at 37°C. o C, 180 rpm, cultured for 12 h. Then, 1% (v / v) was inoculated into 50 mL of fermentation medium containing kanamycin (12 g / L peptone, 24 g / L yeast extract, 1.53 g / L K₂PO₄·3H₂O₂, 0.49 g / L MgSO₄, 10 g / L glycerol, 2 mL / L trace elements), and cultured until the OD of the bacterial culture reached [value missing]. 600 The value reaches 0.6-0.8.

[0047] 2. Induction of expression: Induction was performed by adding IPTG at a final concentration of 0.1 mM, 20 o C. Continue culturing at 180 rpm for 20-24 hours. The fermentation broth is then centrifuged at low temperature and high speed (4...). oCollect bacterial sludge (C, 8000 rpm, 10 min). Resuspend the bacterial sludge in PBS buffer at pH 8.0.

[0048] 3. Cell disruption and centrifugation: The resuspended bacterial solution was disrupted using an ultrasonic cell disruptor (JY92-IIN) for 15 minutes until the solution became clear. Then, the cells were centrifuged at 4... o Centrifuge at 12000 rpm for 10 min, and collect the supernatant and precipitate separately.

[0049] 4. SDS-PAGE protein gel assay: Take 30 μL of the disrupted enzyme solution, centrifuge at 12000 rpm for 1 min, separate the supernatant from the precipitate, resuspend the precipitate in 30 μL ddH2O, add 10 μL 4× protein loading buffer, boil the supernatant and precipitate for 5 min, and centrifuge at 12000 rpm for 10 min. Electrophoresis at 160 V 200 mA for 30 min. After electrophoresis, stain with Coomassie Brilliant Blue for 30 min. Remove the stained gel from the staining solution and place it in destaining solution, destaining multiple times until the background is clear. The results for mutant D164A are as follows. Figure 3 As shown, protein bands of the target size were present in both the supernatant and the precipitate, with higher protein expression levels in the supernatant. Results for other mutants were largely consistent with those for mutant D164A and can be used for further enzyme activity assays.

[0050] III. Determination of enzyme activity expressed by alanine racemic enzyme mutants

[0051] Racemization stage: The total reaction volume was 200 μL, consisting of 100 mM PBS buffer (pH 8.0), 20 μL of enzyme supernatant obtained in step two, 10 μM PLP, and 50 mM L-alanine. The mixture was incubated at 37°C. o The reaction was carried out at a constant temperature of C for 10 min, followed by the addition of 25 μL of 2 M HCl to terminate the enzymatic reaction. After incubation on ice for 2 min, the reaction was carried out at 4 °C. o C. Centrifuge at 12000 rpm for 10 min. Transfer 180 μL of the clear supernatant and mix with 20 μL of 2 M NaOH for acid-base neutralization. The blank control group uses an equal volume of buffer reagent instead of enzyme protein in the above process.

[0052] Oxidation and color development stage: A 200 μL color development system contained 100 mM Tris-HCl (pH 8.0), 4-aminoantipyrine (0.1 mg / mL), TOOS (0.1 mg / mL), horseradish peroxidase (2 U), and D-amino acid oxidase (0.1 U). 100 μL of the supernatant prepared in the aforementioned racemic stage was introduced, and the mixture was incubated at 37°C. oIncubate at C for 20 min, then monitor OD using a TecanInfinite 200 Pro multi-mode microplate reader. 550 The absorbance at that location.

[0053] The enzyme activity unit (U) is defined as follows: under specific experimental conditions, the amount of enzyme required to catalyze the production of 1 μmol of D-alanine per minute is one activity unit.

[0054] The relative activity of the original enzyme was set to 100%, and the relative activity of the mutant was calculated as: total product amount / (reaction time × enzyme volume).

[0055] Comparison of enzyme activities between the original enzyme and the mutant enzyme, as follows: Figure 4 As shown, the enzyme activity of mutant D164A is increased to 1.16 times that of the original enzyme compared to the original enzyme.

[0056] Alanine racemase (Alr, EC 5.1.1.1) catalyzes the interconversion between L-alanine and D-alanine, belonging to the 5'-phosphate pyridoxal (PLP)-dependent type and classified as an isomerase. Pyridoxal kinase (PDXK) is a key metabolic enzyme of vitamin B6, catalyzing the phosphorylation of pyridoxal (PL) to PLP. The whole-cell catalytic production process uses L-alanine as a raw material and ALR and PDXK as catalysts in a biochemical reaction. Figure 1 As shown.

[0057] The following describes the construction of engineered bacterial cells overexpressing ALR-D164A and PDXK, followed by whole-cell catalysis in the presence of L-alanine to produce D-alanine.

[0058] Example 2

[0059] I. Construction and culture of E. coli BL21(DE3) / pET-28a(+) / ALR-D164A / pCDFDuet-PDXK

[0060] (1) Prepare competent cells from E. coli BL21(DE3) / pET-28a(+) / ALR-D164A. Increase cell membrane permeability using the calcium chloride method to facilitate the introduction of the PDXK gene into the strain: Add 50 μL of E. coli BL21(DE3) / pET-28a(+) / ALR-D164A bacterial culture to a 5 mL LB test tube and incubate at 37°C. o Incubate at 37°C, 180 rpm on a shaker for 12 h. Take 1 mL of the bacterial culture from the test tube and add it to a shake flask containing 100 mL of LB medium. oC, incubate on a shaker at 180 rpm until the bacterial culture OD 600 When the concentration is 0.5-0.6, remove the shake flask and place it on ice for 10 minutes. o Centrifuge at 4100 rpm for 10 min, discard the supernatant, resuspend in 2 mL of 0.05 M CaCl2 solution containing 15% glycerol, aliquot, and incubate at -80°C. o Store in refrigerator C for later use.

[0061] (2) The PDXK (Pyridoxal Kinase) gene fragment (SEQ ID NO:4) was ligated to the linearized vector pCDFDuet (the ligation system is shown in Table 5, and the vector was placed at 37°C). o The reaction was carried out in a constant temperature incubator at 3°C ​​for 30 min, and after the reaction, it was placed on ice for 5 min before being transferred into E. coli DH5α competent cells and plated on LB agar plates containing streptomycin resistance. o C overnight culture.

[0062] Table 5 Connection System

[0063]

[0064] (3) Pick a single colony growing on the plate in (2), transfer it to LB medium containing streptomycin resistance, then extract the plasmid, and transfer the plasmid pCDFDuet-PDXK into the competent cells in step (1), and spread it on LB plates containing streptomycin resistance and kanamycin resistance, 37 o C overnight culture.

[0065] (4) Pick a single colony growing on the plate in (3), transfer it to LB medium containing streptomycin-resistant and kanamycin-resistant bacteria, and incubate for 10-12 h. Then, store it at -80°C with 30% glycerol. o The strain E. coli BL21(DE3) / pET-28a(+) / ALR-D164A / pCDFDuet-PDXK was obtained in refrigerator C.

[0066] II. Whole-cell catalysis and determination of D-alanine content

[0067] (1) The strain E. coli BL21(DE3) / pET-28a(+) / ALR-D164A / pCDFDuet-PDXK preserved in the glycerol tube in step one was inoculated into LB test tubes containing streptomycin-resistant and kanamycin-resistant strains, and incubated at 37°C. o Incubate at 180 rpm for 12 h, then transfer to a shake flask containing 50 mL of streptomycin- and kanamycin-resistant fermentation medium and incubate at 37°C. oC180 rpm shaking culture to OD 600 It is 0.6-0.8.

[0068] (2) Add IPTG to the shake flask to a final concentration of 0.1 mM for induction, and at 20°C... o After culturing the above bacterial culture at 180 rpm for 24 h, the culture was then incubated at 4°C. o Centrifuge at 8000 rpm for 10 min, discard the supernatant, and wash the collected cells twice with PBS at pH 8.0 to obtain cells for the catalytic reaction.

[0069] (3) The collected cells were resuspended and concentrated in PBS at pH 8.0 and then L-alanine at pH 8.0 was added to carry out whole-cell catalytic reaction. The cell OD in the whole-cell catalytic reaction system was... 600 The concentrations were 10, 15, and 20, respectively, with a final concentration of 100 g / L for L-alanine.

[0070] (4) The catalytic reaction system at 37 o The reaction was carried out at C, 180 rpm, and samples were taken periodically (samples were taken at 100 rpm). o Boil in water for 5 minutes to inactivate cells, then centrifuge at 12,000 rpm for 3 minutes to remove protein impurities and other insoluble impurities.

[0071] (5) Take the supernatant sample and dilute it 500 times to a concentration equal to the standard sample (0.2 g / L). Then, measure the D-alanine content in the solution using high-performance liquid chromatography (HPLC). The HPLC was performed using a Phenomenex 3126 d-penicillamine column, with an ultraviolet wavelength of 254 nm, a mobile phase of 2 mM CuSO4:IPA (95:5), and a flow rate of 0.5 mL / min. The HPLC results are as follows: Figure 5 Characteristic peaks appeared at 1.805 and 2.090 minutes, respectively, and their components were L-alanine and D-alanine.

[0072] (6) According to liquid chromatography detection, when OD 600 When the concentration was 15, after 21 hours of whole-cell catalytic reaction, the content of D-alanine reached 62%.

[0073] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A mutant of alanine racemic enzyme, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The encoding gene of the alanine racemic enzyme mutant according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 2.

4. A genetically engineered bacterial strain, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. The genetically engineered strain according to claim 4, characterized in that, The genetically engineered strain is E. coli BL21(DE3) / pET-28a(+) / ALR-D164A, with E. coli BL21(DE3) as the host strain; the recombinant expression vector uses pET-28a(+) as the expression vector.

6. A method for constructing a whole-cell catalytic system, characterized in that, Construction based on the genetically engineered strain according to claim 5 includes: The genetically engineered strain E. coli BL21(DE3) / pET-28a(+) / ALR-D164A was used to prepare competent cells; the PDXK gene fragment was ligated to the vector pCDFDuet to obtain the recombinant plasmid pCDFDuet-PDXK, and then the recombinant plasmid pCDFDuet-PDXK was introduced into the competent cells of the above-mentioned genetically engineered strain to obtain the recombinant strain E. coli BL21(DE3) / pET-28a(+) / ALR-D164A / pCDFDuet-PDXK; the nucleotide sequence of the PDXK gene fragment is shown in SEQ ID NO:

3.

7. A whole-cell catalytic system, characterized in that, Obtained using the construction method described in claim 6.

8. The application of the whole-cell catalytic system according to claim 7 in the production of D-alanine.

9. The application according to claim 8, characterized in that, The applications include culturing cells for catalysis to OD. 600 The concentration is 10-20, and L-alanine is added to catalyze the reaction to produce D-alanine.

10. The application according to claim 9, characterized in that, In 37 o The catalytic reaction was carried out at C and 180 rpm.

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