N-acetyl amino acid racemase mutant and application thereof

Abstract

The invention provides a mutant protein of N-acetyl amino acid racemase, which is obtained by site-directed mutagenesis modification and has higher enzyme activity, the racemization activity of the mutant to N-acetyl-L-tryptophan is 1.44 times that of the original enzyme, and the catalytic efficiency kcat / Km is 4.6 times that of the original enzyme; when the mutant of the N-acetyl amino acid racemase is applied to preparation of D-tryptophan, the efficiency is higher.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and relates to an N-acetylamino acid racemase mutant and application thereof. Background Art

[0002] N-acetyl amino acid racemase (NAAAR) catalyzes the racemization reaction of various N-acetyl amino acids, and can react with stereoselective L- or D-aminoacylase to dynamically split and produce L- or D-amino acids using N-acetyl amino acids as raw materials. Under the racemization action of N-acetyl amino acid racemase, the theoretical reaction yield can reach 100%. D-tryptophan is an important chiral amino acid, which has important application value in food, agriculture, biomedicine, etc. It can be used as a non-nutritional sweetener, feed additive and plant growth agent. In the pharmaceutical industry, D-tryptophan is mainly used as a synthetic precursor for some drugs (such as tadalafil). N-acetyl-L-tryptophan forms N-acetyl-D-tryptophan under the action of N-acetyl amino acid racemase, and N-acetyl-D-tryptophan can generate D-tryptophan under the action of D-aminoacylase. However, the disadvantage of this method for preparing D-tryptophan is that N-acetyl amino acid racemase has low catalytic activity for the racemization of N-acetyl-L-tryptophan. Summary of the invention

[0003] The purpose of the present invention is to provide a mutant of N-acetylamino acid racemase in view of the shortcomings of the existing N-acetylamino acid racemase, wherein the mutant can improve the racemization activity of N-acetylamino acid racemase for N-acetyl-L-tryptophan, and further be used in combination with D-aminoacylase for the preparation of D-tryptophan. The enhancement of the catalytic activity of N-acetylamino acid racemase can improve the reaction efficiency of preparing D-tryptophan and reduce the production cost of D-tryptophan.

[0004] To this end, the first aspect of the present invention provides an amino acid mutant that affects the enzyme activity of N-acetylamino acid racemase, which is a mutation in which the 26th alanine in the amino acid sequence from the N-terminus to the C-terminus of the original N-acetylamino acid racemase as shown in SEQ ID NO.1 is mutated to leucine.

[0005] The second aspect of the present invention provides a mutant of N-acetylamino acid racemase, whose sequence comprises a mutation in which the 26th alanine in the amino acid sequence of the original N-acetylamino acid racemase from the N-terminus to the C-terminus is mutated to leucine (referred to as mutant A26 L in the present invention).

[0006] In some specific preferred embodiments of the present invention, the sequence of the N-acetylamino acid racemase mutant is shown as SEQ ID NO.2.

[0007] The third aspect of the present invention provides a nucleotide molecule encoding the mutant described in the second aspect of the present invention, wherein the sequence includes a mutation in which the 76th G is mutated to C and the 77th C is mutated to T in the nucleotide sequence shown in SEQ ID NO.3 encoding the original N-acetylamino acid racemase from the 5' end to the 3' end.

[0008] The fourth aspect of the present invention provides an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleotide molecule described in the third aspect of the present invention.

[0009] The fifth aspect of the present invention provides the use of the mutant as described in the second aspect of the present invention or the mutant encoded by the nucleotide molecule as described in the third aspect of the present invention or the mutant obtained by the expression cassette, recombinant vector or recombinant microorganism as described in the fourth aspect of the present invention in catalytic preparation of D-tryptophan, which can be understood as a method for catalytic preparation of D-tryptophan using the mutant as described in the second aspect of the present invention or the mutant encoded by the nucleotide molecule as described in the third aspect of the present invention or the mutant obtained by the expression cassette, recombinant vector or recombinant microorganism as described in the fourth aspect of the present invention.

[0010] According to the present invention, the application includes catalytic preparation of D-tryptophan, improving the yield of catalytic preparation of D-tryptophan and improving the efficiency of preparation of D-tryptophan.

[0011] The term "nucleotide mutant" as used in the present invention refers to the smallest unit in which a mutation can occur in the nucleotide sequence of a gene.

[0012] The term "amino acid mutant" as used in the present invention refers to the smallest unit in which a mutation may occur in the amino acid sequence of a protein.

[0013] In the present invention, the terms "protein" and "protein" can be used interchangeably.

[0014] In order to solve the problem that the racemization catalytic activity of N-acetylamino acid racemase for N-acetyl-L-tryptophan is low when preparing D-tryptophan by the existing method, the inventors have conducted a lot of research on the mutation of N-acetylamino acid racemase. The inventors screened and found that the 26th alanine of the N-acetylamino acid racemase with an amino acid sequence as shown in SEQ ID NO.1 was mutated to leucine, which can significantly improve the racemization activity of N-acetylamino acid racemase for N-acetyl-L-tryptophan; further combined with D-aminoacylase for D-tryptophan preparation, the enhancement of the catalytic activity of N-acetylamino acid racemase can improve the reaction efficiency of preparing D-tryptophan and reduce the production cost of D-tryptophan.

[0015] The research results show that the N-acetylamino acid racemase mutant protein provided by the present invention has higher enzyme activity. The A21L mutant enzyme activity for the racemization activity of N-acetyl-L-tryptophan is 1.44 times that of the original N-acetylamino acid racemase, and the catalytic efficiency kcat / Km is 4.6 times that of the original enzyme, thereby improving the production capacity of the unit catalyst; the N-acetylamino acid racemase mutant is applied to catalyze the preparation of D-tryptophan with higher efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0017] Figure 1 The results of the reaction of preparing D-tryptophan by the A26L mutant and the non-mutated N-acetylamino acid racemase are shown; wherein the original enzyme is the non-mutated N-acetylamino acid racemase. DETAILED DESCRIPTION

[0018] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0019] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0020] Example

[0021] The present invention is described in detail below through specific examples. The experimental methods described below are all routine laboratory methods unless otherwise specified. The experimental materials described below are all available from commercial channels unless otherwise specified.

[0022] Embodiment 1:

[0023] 1) Point mutation of N-acetylamino acid racemase

[0024] Using the pET-28a(+) recombinant plasmid [SEQ ID NO.3 is located between pET-28a sequence A (SEQ ID NO.4) and pET-28a sequence B (SEQ ID NO.5)] containing the gene (shown as SEQ ID NO.3) of N-acetylamino acid racemase from Amycolatopsis sp. (PDB accession number: 5FJU; amino acid sequence is shown in SEQ ID NO.1) as a template, point mutation PCR was performed using primer 25T (GGTGCCAAAGCTGGTACGG) as a forward primer and another primer in Table 1 as a reverse primer to perform full plasmid amplification, and site-directed mutation was performed on alanine at position 26 of N-acetylamino acid racemase, and mutated into another 19 amino acids respectively.

[0025] Table 1 Reverse primer list

[0026]

[0027]

[0028] PCR amplification system: template 1 μL, forward and reverse primers 1 μL each, ddH 2 O 7μL, 2×Phanta Max MasterMix polymerase 10μL, total reaction system 20μL. PCR reaction conditions: 95℃ pre-denaturation, 3min; 95℃ denaturation, 15s, 56℃ annealing, 30s, 72℃ extension, 6min, 16 cycles; 72℃ full extension, 10min, 16℃ storage.

[0029] The PCR product was checked by gel electrophoresis, and then the PCR product was subjected to KLD ligation reaction and placed at room temperature for 2 hours. The KLD ligation system included: 1 μL of PCR product, 6 μL of ddH2O, 2 μL of 5X KLD buffer (1 μL of 10×CuterSmart buffer, 1 μL of 10 mM ATP), and 1 μL of KLD Mix (0.45 μL of T4 DNA ligase, 0.4 μL of T4 polynucleotide kinase, and 0.15 μL of Dpn I).

[0030] The above KLD-linked PCR product was transformed into Escherichia coli DH5α competent cells (Shanghai Sangon Biotechnology Co., Ltd.), coated with LB plates containing 50 μg / mL kanamycin, and cultured at 37°C for 16 h. Single colonies were picked from the plates for colony PCR identification and sequencing verification to obtain 19 plasmids containing different amino acid mutants at position 26 of N-acetylamino acid racemase.

[0031] 2) Initial screening of N-acetylamino acid racemase mutants

[0032] The mutant original plasmid and the plasmid containing the 26 different amino acid mutants were transformed into Escherichia coli BL21 (DE3) (Shanghai Shenggong Biotechnology Co., Ltd.), and the single clone colony was picked and inoculated into 3 mL LB culture medium containing kanamycin (50 μg / mL) and cultured at 37°C and 200 rpm for 16 hours. Then, it was inoculated into 50 mL lactose induction medium (1% peptone, 0.5% yeast powder, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 25 mM Na 2 HPO 4 , 25 mM KH 2 PO 4 , 50 mM NH 4 Cl, 5 mM Na 2 SO 4 , 2 mM Mg 2 SO 4 ) was used to induce expression. After culturing at 28°C and 200 rpm for 24 hours, 2.5 mL of the culture was collected by centrifugation. The bacteria were washed twice with water and resuspended. The volume of the bacterial suspension was adjusted to 1 mL. The bacterial solution was then ultrasonically disrupted and the ultrasonic supernatant was collected by centrifugation.

[0033] The N-acetylamino acid racemase reaction solution was prepared: 25 mM N-acetyl-L-tryptophan, 50 mM Tris-HCl buffer (pH 8.0), and 1 mM cobalt chloride.

[0034] HPLC detection conditions 1: Poroshell 120Chiral-T 2.7μm 4.6×150mm chiral column; 1mMNaH 2 PO 4 -H 3 PO 4 , 50% methanol (pH 3.0) mobile phase; 28°C column temperature; 280nm detection wavelength; flow rate 0.4mL / min; 10μL injection volume.

[0035] Take 1 mL of enzyme reaction solution, add 50 μL of the above ultrasonic supernatant, react at 37°C for 15 min, take a sample and add 100 μL of 2M HCl to terminate the reaction, centrifuge at 12000 rpm for 2 min, take the supernatant, and after appropriate dilution, detect the N-acetyl-D-tryptophan content using HPLC detection condition 1.

[0036] Initial screening tests found that the mutation of alanine at position 26 of N-acetylamino acid racemase to leucine increased the enzyme activity by 20%, and the enzyme activities of the other 18 mutants were lower than that of the unmutated N-acetylamino acid racemase.

[0037] 3) Enzyme purification and kinetic parameter determination

[0038] The non-mutated and A26L mutated N-acetylamino acid racemase were separated and purified, and the specific enzyme activity and kinetic parameters before and after mutation were determined.

[0039] The 50mL culture after induction expression was centrifuged to collect the bacterial cells, washed with water twice and then resuspended, the bacterial suspension volume was 20mL, the bacterial liquid was ultrasonically broken, the broken liquid was placed at 55 degrees for 30 minutes, centrifuged to obtain the supernatant, and then 2mL buffer (0.5M Tris-HCl, 1M NaCl, pH 8.0) was added to the supernatant to obtain a crude enzyme solution. The crude enzyme solution was loaded onto a Q spharose FF chromatography column (1.0×10cm), washed with 10mL of washing buffer (50mM Tris-HCl, 100mM NaCl, pH 8.0), and then eluted with a 50mM Tris-HCl buffer gradient from 100mM to 500mM NaCl, and the eluate was collected in parts, and the eluate was subjected to SDS-PAGE analysis to obtain a fraction of a single band of N-acetylamino acid racemase, and ultrafiltration and desalting using a 3KDa ultrafiltration membrane to obtain a final purified enzyme solution.

[0040] Enzyme activity assay: The reaction solution consists of 25mM N-acetyl-L-tryptophan, 50mM Tris-HCl buffer (pH 8.0), 1mM cobalt chloride, and an appropriate amount of enzyme; the reaction temperature is 37 degrees and the reaction time is 15 minutes. One unit of enzyme activity is defined as the amount of enzyme required to generate 1μmol N-acetyl-D-tryptophan in 1 minute under these conditions. The specific enzyme activity is the enzyme activity per mg of protein.

[0041] Kinetic parameter determination: The reaction rates at N-acetyl-L-tryptophan concentrations of 0.8, 1.0, 1.33, 2, 2.66, 4, 8, and 12 mM were measured respectively, and the enzyme Km and Vmax values ​​were calculated using the Lineweaver-Burk double reciprocal method, and Kcat = Vmax / protein concentration and Km / Kcat were further calculated.

[0042] The results of the enzyme activity assay are shown in Table 2.

[0043] Table 2 Specific enzyme activity and kinetic parameters

[0044]

[0045] As can be seen from Table 2, the enzyme activity of the A26L mutant is 1.44 times that of the original enzyme.

[0046] Compared with the original enzyme, the A26L mutant K m decreased, indicating that the affinity of A26L for the substrate N-acetyl-L-tryptophan increased after mutation.

[0047] The catalytic efficiency kcat / Km mutant A26L is 4.6 times that of the original enzyme.

[0048] Embodiment 2:

[0049] The double enzyme reaction solution of N-acetylamino acid racemase and D-aminoacylase was prepared: 100 mM N-acetyl-L-tryptophan, 50 mM Tris-HCl buffer (pH 8.0), 1 μM zinc sulfate, and 1 mM cobalt chloride.

[0050] 4 mL of the above dual enzyme reaction solution was taken respectively, 200 μg of the unmutated N-acetylamino acid racemase or A26L mutant purified in Example 1 was added, and 30 U of D-aminoacylase was added respectively, and the mixture was reacted at 37° C. and 200 rpm. After reacting for 1 hour and 2 hours, 100 μL of the sample was added to 900 μL of 0.1 M HCl to terminate the reaction, and the supernatant was taken after centrifugation at 12000 rpm for 2 min. After appropriate dilution, the D-tryptophan content was detected by HPLC.

[0051] HPLC detection conditions: Inertsil C8-3 5μm 4.6×250mm column; 1mM NaH 2 PO 4 -H 3 PO 4 , 45% methanol (pH 3.0) mobile phase; 28°C column temperature; 280nm detection wavelength; flow rate 1mL / min; 20μL injection volume.

[0052] The reaction results are as follows Figure 1 As shown, it can be seen that in the reaction process with D-aminoacylase, when the same mass of N-acetylamino acid racemase is used, the mutant A26L generates D-tryptophan faster than the original enzyme and has a higher enzyme reaction efficiency.

[0053] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. An amino acid mutant affecting the enzyme activity of N-acetylamino acid racemase, which is a mutation in which the 26th alanine in the amino acid sequence of the original N-acetylamino acid racemase from the N-terminus to the C-terminus as shown in SEQ ID NO.1 is mutated to leucine.

2. A mutant of N-acetylamino acid racemase, the sequence of which comprises a mutation in which the 26th alanine in the amino acid sequence from the N-terminus to the C-terminus of the original N-acetylamino acid racemase as shown in SEQ ID NO.1 is mutated to leucine.

3. The mutant according to claim 2, characterized in that The sequence of the N-acetylamino acid racemase mutant is shown in SEQ ID NO.

2.

4. A nucleotide molecule encoding the mutant according to claim 2 or 3.

5. An expression cassette, recombinant vector or recombinant microorganism containing the nucleotide molecule according to claim 4.

6. Use of the mutant according to claim 2 or 3, or the mutant encoded by the nucleotide molecule according to claim 4, or the mutant obtained by the expression cassette, recombinant vector or recombinant microorganism according to claim 5 in catalytic production of D-tryptophan.

7. The use according to claim 6, characterized in that: The application includes catalytic preparation of D-tryptophan, improving the yield of catalytic preparation of D-tryptophan and improving the efficiency of preparation of D-tryptophan.

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