N-acetyl amino acid racemase mutant and application thereof

Abstract

The application provides a mutant of N-acetylamino acid racemase, which is obtained by site-directed mutagenesis, has higher enzymatic activity, and can improve the racemization activity of N-acetyl-L-tryptophan by 54% compared with the original N-acetylamino acid racemase; and the mutant of the N-acetylamino acid racemase has higher efficiency in preparation of D-tryptophan.

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 of various N-acetylamino acids. It can react simultaneously with stereoselective L- or D-aminoacylases to dynamically kinetically resolve N-acetylamino acids into L- or D-α-amino acids. 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 with significant applications in food, agriculture, and biopharmaceuticals. It can be used as a non-nutritive sweetener, feed additive, and plant growth enhancer. In the pharmaceutical industry, D-tryptophan is primarily used as a synthetic precursor for several drugs, such as tadalafil. N-acetyl-L-tryptophan is converted to N-acetyl-D-tryptophan by N-acetyl amino acid racemase, which in turn generates D-tryptophan by D-aminoacylase. However, a drawback of this method for producing D-tryptophan is the low catalytic activity of N-acetyl amino acid racemase for the racemization of N-acetyl-L-tryptophan. Summary of the Invention

[0003] The present invention aims to provide an N-acetylamino acid racemase mutant to address the deficiencies of existing N-acetylamino acid racemase. The mutant can improve the racemization activity of the N-acetylamino acid racemase for N-acetyl-L-tryptophan and can be further used in combination with a D-aminoacylase for the preparation of D-tryptophan. The enhancement of the catalytic activity of the 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 enzymatic activity of N-acetylamino acid racemase, which is a mutation in which the 21st threonine 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 alanine or the 21st threonine is mutated to serine.

[0005] In a second aspect, the present invention provides a mutant of N-acetylamino acid racemase, the sequence of which comprises a mutation in which the threonine at position 21 is mutated to an alanine (referred to as mutant T21A in the present invention) or a mutation in which the threonine at position 21 is mutated to a serine (referred to as mutant T21S in the present invention) from the N-terminus to the C-terminus of the amino acid sequence of the original N-acetylamino acid racemase as shown in SEQ ID NO.1.

[0006] In some embodiments of the present application, the present application also provides a nucleotide mutant affecting the enzymatic activity of N-acetyl amino acid racemase, which comprises a mutation of A at position 61 to G and C at position 63 from 5' end to 3' end direction in the nucleotide sequence as shown in SEQ ID NO. 4 encoding the original N-acetyl amino acid racemase, or a mutation of A at position 61 to T in the nucleotide sequence as shown in SEQ ID NO. 4 encoding the original N-acetyl amino acid racemase.

[0007] In some embodiments of the present application, the mutant is a No. 1 N-acetyl amino acid racemase mutant, the sequence of which comprises a mutation of threonine at position 21 from N-terminal to C-terminal in the amino acid sequence as shown in SEQ ID NO. 1 of the original N-acetyl amino acid racemase to alanine, which is also referred to as N-acetyl amino acid racemase mutant T21A in the present application.

[0008] In some specific preferred embodiments of the present application, the sequence of the No. 1 N-acetyl amino acid racemase mutant is as shown in SEQ ID NO. 2.

[0009] In some embodiments of the present application, the mutant is a No. 2 N-acetyl amino acid racemase mutant, the sequence of which comprises a mutation of threonine at position 21 from N-terminal to C-terminal in the amino acid sequence as shown in SEQ ID NO. 1 of the original N-acetyl amino acid racemase to serine, which is also referred to as N-acetyl amino acid racemase mutant T21S in the present application.

[0010] In some specific preferred embodiments of the present application, the sequence of the No. 2 N-acetyl amino acid racemase mutant is as shown in SEQ ID NO. 3.

[0011] The third aspect of the present application provides a nucleotide molecule encoding the mutant of the second aspect of the present application, the sequence of which comprises a mutation of A at position 61 to G and C at position 63 from 5' end to 3' end direction in the nucleotide sequence as shown in SEQ ID NO. 4 encoding the original N-acetyl amino acid racemase, or a mutation of A at position 61 to T in the nucleotide sequence as shown in SEQ ID NO. 4 encoding the original N-acetyl amino acid racemase.

[0012] In some embodiments of the present application, the nucleotide molecule is a nucleotide molecule encoding a No. 1 N-acetyl amino acid racemase mutant, the sequence of which comprises a mutation of A at position 61 to G and C at position 63 from 5' end to 3' end direction in the nucleotide sequence as shown in SEQ ID NO. 4 encoding the original N-acetyl amino acid racemase.

[0013] In some embodiments of the present application, the nucleotide molecule is a nucleotide molecule encoding a mutant of N-acetyl amino acid racemase 2, which comprises a mutation of A at position 61 to T in the nucleotide sequence as shown in SEQ ID NO. 4 encoding the original N-acetyl amino acid racemase in the sequence from 5' end to 3' end.

[0014] The fourth aspect of the present application provides an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleotide molecule according to the third aspect of the present application.

[0015] The fifth aspect of the present application provides the use of the mutant according to the second aspect of the present application or the mutant encoded by the nucleotide molecule according to the third aspect of the present application or the mutant prepared by the expression cassette, the recombinant vector or the recombinant microorganism according to the fourth aspect of the present application in catalyzing the preparation of D-tryptophan, which can be understood as a method for catalyzing the preparation of D-tryptophan by using the mutant according to the second aspect of the present application or the mutant encoded by the nucleotide molecule according to the third aspect of the present application or the mutant prepared by the expression cassette, the recombinant vector or the recombinant microorganism according to the fourth aspect of the present application.

[0016] According to the present application, the use includes catalyzing the preparation of D-tryptophan, improving the yield of catalyzing the preparation of D-tryptophan and improving the efficiency of preparing D-tryptophan.

[0017] The term "nucleotide mutant" used in the present application refers to the minimum unit that can be mutated in the nucleotide sequence of a gene.

[0018] The term "amino acid mutant" used in the present application refers to the minimum unit that can be mutated in the amino acid sequence of a protein.

[0019] The terms "protein" and "protein" can be used interchangeably in the present application.

[0020] To solve the problem that the racemization catalytic activity of N-acetyl amino acid racemase on N-acetyl-L-tryptophan is low in the preparation of D-tryptophan by the existing method, the present inventors have conducted a large number of studies on the mutation of N-acetyl amino acid racemase. Through a long-term and large amount of research, the present inventors have screened and found that the mutation of threonine at position 21 of N-acetyl amino acid racemase with the amino acid sequence as shown in SEQ ID NO. 1 to alanine or serine can significantly improve the racemization activity of N-acetyl amino acid racemase on N-acetyl-L-tryptophan; further used in combination with D-aminoacylase for the preparation of D-tryptophan, the enhancement of the catalytic activity of N-acetyl amino acid racemase can improve the reaction efficiency of preparing D-tryptophan and reduce the production cost of D-tryptophan.

[0021] The research result shows that the N-acetylamino acid racemase mutant protein provided by the application has higher enzyme activity, wherein the T21A mutant enzyme activity is increased by 54% than the racemization activity of the original N-acetylamino acid racemase for N-acetyl-L-tryptophan, and the production capacity per unit catalyst is increased; the N-acetylamino acid racemase mutant is applied to catalyze D-tryptophan to have higher efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0022] The application will be further described in detail below in combination with the drawings:

[0023] Figure 1 The figure shows the enzyme activity comparison results of different mutants of the 21st site of N-acetylamino acid racemase; wherein, NAAAR is the unmutated N-acetylamino acid racemase.

[0024] Figure 2 The figure shows the reaction comparison results of the T21A mutant and the unmutated N-acetylamino acid racemase for preparing D-tryptophan; wherein, NAAAR is the unmutated N-acetylamino acid racemase. DETAILED DESCRIPTION

[0025] In order to make the application easy to understand, the application will be described in detail below in combination with the drawings. However, before the detailed description of the application, it should be understood that the application is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for the purpose of describing the specific embodiments and are not restrictive.

[0026] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are now described.

[0027] EMBODIMENT

[0028] The application will be specifically described below through specific embodiments. The experimental methods described below are all laboratory routine methods unless otherwise specified. The experimental materials described below can be obtained from commercial channels unless otherwise specified.

[0029] EMBODIMENT 1

[0030] 1) Point mutation of N-acetylamino acid racemase

[0031] The pET-28a(+) recombinant plasmid containing the gene (as shown in SEQ ID NO. 4) of N-acetyl amino acid racemase (PDB accession number: 5FJR; the amino acid sequence is shown in SEQ ID NO. 1) from Amycolatopsis sp. (SEQ ID NO. 4 is located between the pET-28a sequence SEQ ID NO. 5 and the pET-28a sequence SEQ ID NO. 6) is used as a template, and point mutation PCR is performed with primer 20R (ACGGAACGGCGCAACCAG) as a forward primer, and primer 21S (AGCTTTGGCACCGCGAGC), 21A (AGCTTTGGCACCGCGAGA), and 21G (AGCTTTGGCACCGCGAGG) as reverse primers, respectively, to perform full plasmid amplification, and the Thr at the 21st site of the N-acetyl amino acid racemase is subjected to site-directed mutation, and is mutated to Ser, Ala, and Gly, respectively. TCC AGCTTTGGCACCGCGAGC), 21A (AGCTTTGGCACCGCGAGA), and 21G (AGCTTTGGCACCGCGAGG) as reverse primers, respectively, to perform full plasmid amplification, and the Thr at the 21st site of the N-acetyl amino acid racemase is subjected to site-directed mutation, and is mutated to Ser, Ala, and Gly, respectively. GCA AGCTTTGGCACCGCGAGC), 21A (AGCTTTGGCACCGCGAGA), and 21G (AGCTTTGGCACCGCGAGG) as reverse primers, respectively, to perform full plasmid amplification, and the Thr at the 21st site of the N-acetyl amino acid racemase is subjected to site-directed mutation, and is mutated to Ser, Ala, and Gly, respectively. GGT AGCTTTGGCACCGCGAGC), 21A (AGCTTTGGCACCGCGAGA), and 21G (AGCTTTGGCACCGCGAGG) as reverse primers, respectively, to perform full plasmid amplification, and the Thr at the 21st site of the N-acetyl amino acid racemase is subjected to site-directed mutation, and is mutated to Ser, Ala, and Gly, respectively.

[0032] PCR amplification system: 1 μL of template, 1 μL of forward and reverse primers, respectively, 7 μL of ddH2O, 10 μL of 2×Phanta Max MasterMix polymerase, and a total reaction system of 20 μL. PCR reaction conditions: 95 °C pre-denaturation, 3 min; 95 °C denaturation, 15 s, 56 °C annealing, 30 s, 72 °C extension, 6 min, 16 cycles; 72 °C sufficient extension, 10 min, 16 °C storage.

[0033] The PCR product is subjected to gel electrophoresis test, and then the PCR product is subjected to KLD ligation reaction, and is placed at room temperature for 2 h, and the KLD ligation system is as follows: 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).

[0034] The KLD-ligated PCR product is transformed into E. coli DH5α competent cells (Shanghai Shengong Bioengineering Co., Ltd.), and is coated on an LB plate containing 50 μg / mL of kanamycin, and is cultured at 37 °C for 16 h. Single colonies are picked from the plate for colony PCR identification and sequencing verification, and N-acetyl amino acid racemase mutant T21A, T21S, and T21G are obtained.

[0035] 2) Comparison of enzyme activities of N-acetyl amino acid racemase mutants

[0036] The mutant original plasmid containing mutant T21A, T21S, T21G plasmid was transformed into E. coli BL21 (DE3) (Shanghai SunGene Biotech Co., Ltd.) respectively, and a single colony was inoculated into 3 mL LB culture medium containing kanamycin (50 μg / mL) and cultured at 37°C and 200 rpm for 16 hours. Then, 1% inoculation was performed into 50 mL lactose induction medium (1% peptone, 0.5% yeast powder, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM Mg2SO4) containing kanamycin (50 μg / mL) to induce expression, and the culture was centrifuged after being cultured at 28°C and 200 rpm for 24 hours. The bacterial cells were washed with water twice, resuspended, and the bacterial suspension concentration OD600 was adjusted to 50 OD. A small amount of 50 OD bacterial solution was diluted to 20 OD, and the 20 OD bacterial solution was ultrasonically broken. The ultrasonic supernatant was centrifuged, and the other 50 OD bacterial solution was stored at -70°C.

[0037] The N-acetyl amino acid racemase reaction solution 1 was prepared: 25 mM N-acetyl-L-tryptophan, 50 mM Tris-HCl buffer (pH 8.0), 1 mM cobalt chloride.

[0038] HPLC detection condition 1: Poroshell 120 Chiral-T 2.7 μm 4.6 x 150 mm chiral chromatographic column; 1 mM NaH2PO4-H3PO4, 50% methanol (pH 3.0) mobile phase; column temperature 28°C; detection wavelength 280 nm; flow rate 0.4 mL / min; injection volume 10 μL.

[0039] 1 mL of enzyme reaction solution 1 was taken, 50 μL of the above ultrasonic supernatant was added, and the reaction was carried out at 37°C for 15 min. Then, 100 μL of 2M HCl was added to terminate the reaction, and the supernatant was taken after centrifugation at 12000 rpm for 2 min. After appropriate dilution, the N-acetyl-D-tryptophan content was detected by HPLC detection condition 1.

[0040] The relative enzyme activity of the unmutated N-acetyl amino acid racemase on the substrate N-acetyl-L-tryptophan was defined as 100%, and the comparison results of the racemization speed of N-acetyl-L-tryptophan by the 21st site point mutation of N-acetyl amino acid racemase were as follows: Figure 1As shown, the results show that the enzyme activity is increased by 25% when the 21st amino acid threonine is mutated to serine (T21S), increased by 54% when the 21st amino acid threonine is mutated to alanine (T21A), and greatly reduced when the 21st amino acid threonine is mutated to glycine (T21G), which is only 32% of the enzyme activity of the unmutated enzyme. The 21st site is related to the binding space of the substrate of the N-acetylamino acid racemase. When the 21st amino acid threonine is mutated to serine or alanine, the side chain becomes smaller, and the racemase can improve the catalytic activity on N-acetyl-L-tryptophan, indicating that the smaller side chain of the 21st amino acid residue leads to a larger space of the substrate binding site, which greatly improves the catalytic activity on N-acetyl-L-tryptophan. However, when the 21st amino acid is mutated to glycine, which has no side chain, the enzyme activity is the lowest.

[0041] Preparation of N-acetylamino acid racemase reaction solution 2: 25 mM N-acetyl-L-phenylalanine, 50 mM Tris-HCl buffer (pH 8.0), 1 mM cobalt chloride.

[0042] HPLC detection condition 2: Poroshell 120 Chiral-T 2.7 μm 4.6 x 150 mm chiral chromatographic column; 1 mM NaH2PO4-H3PO4, 50% methanol (pH 3.0) mobile phase; column temperature 28°C; detection wavelength 205 nm; flow rate 0.4 mL / min; injection volume 10 μL.

[0043] Take 1 mL of enzyme reaction solution 2, add 50 μL of the above ultrasonic supernatant, and react at 37°C for 15 min. Then take samples and add 100 μL of 2M HCl to terminate the reaction. Centrifuge at 12000 rpm for 2 min, take the supernatant, dilute appropriately, and detect the content of product N-acetyl-D-phenylalanine under HPLC detection condition 2.

[0044] The relative enzyme activity of the unmutated N-acetylamino acid racemase on the substrate N-acetyl-L-phenylalanine is defined as 100%. The enzyme activities of mutants T21A, T21S, and T21G on the substrate N-acetyl-L-phenylalanine are all greatly reduced. The enzyme activity of mutant T21A on the substrate N-acetyl-L-phenylalanine is 28.3% of the enzyme activity of the unmutated N-acetylamino acid racemase. The enzyme activity of mutant T21S on the substrate N-acetyl-L-phenylalanine is 15.4% of the enzyme activity of the unmutated N-acetylamino acid racemase. The enzyme activity of mutant T21G on the substrate N-acetyl-L-phenylalanine is 7.2% of the enzyme activity of the unmutated N-acetylamino acid racemase.

[0045] Example 2:

[0046] The N-acetyl amino acid racemase and D-aminoacylase double enzyme reaction solution was configured as follows: 100 mM N-acetyl-L-tryptophan, 50 mM Tris-HCl buffer (pH 8.0), 1 μM zinc sulfate, and 1 mM cobalt chloride.

[0047] The OD600 of the OD600 50 OD frozen bacteria solution in Example 1 was taken and thawed at room temperature.

[0048] The above double enzyme reaction solution was taken in 4 mL, 160 μL of unmutated N-acetyl amino acid racemase or T21A mutant thawed bacteria solution was added, and 40 U of D-aminoacylase was added, and the mixture was placed at 37°C and 200 rpm for reaction. After 1 hour and 2 hours of reaction, 100 μL of the reaction solution was taken and added to 900 μL of 0.1 M HCl to terminate the reaction. After centrifugation at 12000 rpm for 2 min, the supernatant was taken, appropriately diluted, and the D-tryptophan content was detected by HPLC.

[0049] The HPLC detection conditions were as follows: Inertsil C8-3 5 μm 4.6 x 250 mm column; 1 mM NaH2PO4-H3PO4, 50% methanol (pH 3.0) mobile phase; column temperature 28°C; detection wavelength 280 nm; flow rate 1 mL / min; injection volume 20 μL.

[0050] The reaction results are shown in Table 1. Figure 2 As can be seen, in the reaction with D-aminoacylase, T21A generates D-tryptophan faster than the unmutated NAAAR, and has higher enzyme reaction efficiency.

[0051] It should be noted that the above-described examples are only used to explain the present application and do not constitute any limitation on the present application. The present application is described by referring to typical examples, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified within the scope of the claims, and the present application can be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and examples, it does not mean that the present application is limited to the specific examples disclosed therein, but rather, the present application can be extended to all other methods and applications having the same function.

Claims

1. A mutant of N-acetyl amino acid racemase, which has a sequence of a threonine at position 21 from N-terminal to C-terminal being mutated to alanine or a threonine at position 21 from N-terminal to C-terminal being mutated to serine based on an amino acid sequence of SEQ ID NO. 1 of an original N-acetyl amino acid racemase. 2.The mutant of claim 1, wherein the mutant is a mutant of N-acetyl amino acid racemase No. 1 having a sequence of a threonine at position 21 from N-terminal to C-terminal being mutated to alanine based on an amino acid sequence of SEQ ID NO. 1 of an original N-acetyl amino acid racemase, and the sequence of the mutant of N-acetyl amino acid racemase No. 1 is shown in SEQ ID NO. 2; or the mutant is a mutant of N-acetyl amino acid racemase No. 2 having a sequence of a threonine at position 21 from N-terminal to C-terminal being mutated to serine based on an amino acid sequence of SEQ ID NO. 1 of an original N-acetyl amino acid racemase, and the sequence of the mutant of N-acetyl amino acid racemase No. 2 is shown in SEQ ID NO.

3. 3.A nucleotide molecule encoding the mutant of claim 1 or 2. 4.An expression cassette, a recombinant vector or a recombinant microorganism containing the nucleotide molecule of claim 3. 5.Use of the mutant of claim 1 or 2, the mutant encoded by the nucleotide molecule of claim 3 or the mutant prepared by the expression cassette, the recombinant vector or the recombinant microorganism of claim 4 in catalyzing preparation of D-tryptophan. The use includes catalyzing preparation of D-tryptophan, improving yield of catalyzing preparation of D-tryptophan and improving efficiency of preparing D-tryptophan. ​ 6. Use according to claim 5, characterized in that, ​

Images