Nitrilase mutant with improved activity and thermal stability

Abstract

The invention discloses a nitrilase mutant with improved activity and thermal stability, and belongs to the field of enzyme engineering. The nitrilase mutant capable of improving the catalytic activity is obtained through a PROSS design tool, and the enzyme activity respectively reaches 300%, 230%, 115% and 165% of that of wild type Nit6803. Besides, after the nitrilase mutant capable of improving the thermal stability is incubated for 8 hours at the temperature of 50 DEG C, compared with 40% of relative activity of wild type Nit6803, the relative enzyme activity of the mutant is 76%, and the relative enzyme activity of the mutant is 61%. Therefore, the invention uses the enzyme to catalyze nitrile substances to produce carboxylic acid substances in industry.

Description

Technical Field

[0001] The invention relates to a nitrilase mutant with improved activity and thermal stability, belonging to the field of enzyme engineering. Background Art

[0002] Enzyme engineering is an effective means to improve the industrial properties of enzymes (activity, stability, tolerance, selectivity, etc.), mainly including strategies of directed evolution and rational design. The advantage of directed evolution is that it does not require the information of the target protein and mutations may appear in unexpected positions to obtain unexpected catalytic effects, but it requires a diverse mutation library and high-throughput screening methods as a prerequisite. The mutation methods of directed evolution mainly include error-prone PCR, DNA shuffling and site saturation mutation. Rational design is one of the classic methods of protein engineering. It changes the structure of the protein to give it different functions by performing site-directed mutations, deletions or insertions on specific amino acids of known sequences. However, it is difficult to guarantee the mutation calculation space and design accuracy due to limited computing power and the accuracy of the computational model.

[0003] Nitrilases are enzymes that can hydrolyze nitriles into carboxylic acids in one step. They can catalyze the production of many high-value amino acids and organic acids, and have been widely used in the production of a variety of chemical products and drug intermediates. However, naturally occurring nitrilases have low activity, poor thermal stability, and low stereo / regional selectivity, which are bottlenecks that limit their further application. Many reported studies have modified nitrilases based on the above-mentioned defects, which have improved the relevant performance of nitrilases to a certain extent. For example, error-prone PCR and saturation mutation were used to modify nitrilase to obtain a stable mutant with several times higher activity (Wu S, Fogiel AJ, Petrillo KL, et al. Protein engineering of nitrilase for chemoenzymatic production of glycolic acid [J]. Biotechnology and Bioengineering, 2008, 99 (3): 717-720.); rational design was used to change substrate specificity, and it was found that the 142nd tyrosine of the nitrilase of Rhodococcus ATCC33278 can determine the specificity of the catalytic substrate. After mutating it to a non-polar aliphatic amino acid, it will tend to catalyze aromatic substrates. It is worth noting that traditional engineering strategies are often based on the design and evaluation of single-point mutations, followed by multiple rounds of iterative combinations. Due to the existence of epistatic effects, its efficiency is low and it is difficult to predict the evolutionary direction of combined mutations.

[0004] The computational design of proteins is undoubtedly an emerging field. Its advantage is that it can make corresponding predictions based on structural or sequence information, thereby achieving the purpose of shortening the experimental time. In the process of enzyme molecule modification, the design performed by computer has extremely broad application prospects in enzyme engineering. Protein Repair One-Stop Shop (Protein Repair One-Stop Shop), as a tool for automatic protein design based on structure and sequence (Goldenzweig A, Goldsmith M, Hill SE, Gertman O, Laurino P, Ashani Y, DymO, Unger T, Albeck S, Prilusky J, Lieberman RL, Aharoni A, Silman I, Sussman JL, Tawfk DS, Fleishman SJ. Automated structure-and sequence-based design of proteins for high bacterial expression and stability. Mol Cell. 2016; 63: 337–46.), its effective design can enhance the performance of enzymes. However, for nitrilase, how to use PROSS to achieve its transformation and what the effect after the transformation is are still unknown. Summary of the invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a method for obtaining and applying a nitrilase mutant with improved activity and thermal stability, the purpose of which is to optimize and transform Nit6803 through the PROSS design tool to improve its activity and stability. PROSS first designs a combination of mutations through multiple sequence alignment analysis and folding free energy prediction to obtain four mutants D1, D2, D3, and D4. The whole-cell enzyme activity and thermal stability of the four mutants are screened to obtain mutants with improved activity. Further, the pure enzyme specific activity and half-life of the mutant are analyzed, and finally a mutant with improved activity and stability is obtained.

[0006] The first technical solution provided by the present invention is a nitrilase mutant, wherein the mutant is a nitrilase parent with an amino acid sequence as shown in SEQ ID NO: 1, and any one of the positions (D1) to (D4) is mutated:

[0007] (D1) asparagine at position 40, glutamic acid at position 113, glycine at position 191, alanine at position 230, and alanine at position 267;

[0008] (D2) includes the positions of (D1), as well as glutamine at position 27, lysine at position 88, and glutamine at position 244;

[0009] (D3) includes the position of (D2) and asparagine at position 223;

[0010] (D4) includes the positions of (D3), as well as methionine at position 31, threonine at position 99, and leucine at position 234.

[0011] In certain embodiments, the asparagine N corresponding to position 40 of the parent is mutated to glutamic acid E;

[0012] The glutamic acid E at position 113 of the parent is mutated to histidine H;

[0013] The glycine G at position 191 of the parent is mutated to alanine A;

[0014] Alanine A at position 230 of the parent is mutated to asparagine D;

[0015] The alanine A at position 267 of the parent is mutated to tryptophan W;

[0016] The glutamine Q at position 27 of the parent is mutated to leucine L;

[0017] The lysine K at position 88 of the parent is mutated to proline P;

[0018] The glutamine Q at position 244 of the parent is mutated to lysine K;

[0019] The asparagine N at position 223 of the parent is mutated to valine V;

[0020] The methionine M at position 31 of the parent is mutated to leucine L;

[0021] The threonine T at position 99 of the parent is mutated to arginine R;

[0022] The leucine L at position 234 of the parent was mutated to alanine A.

[0023] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0024] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0025] In certain embodiments, the recombinant vector uses pET-24a(+) as an expression vector.

[0026] The fourth technical solution provided by the present invention is a recombinant cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0027] In certain embodiments, the recombinant cell is an animal cell, a plant cell, or a microbial cell.

[0028] In certain embodiments, the microbial cell is a fungus or a bacterium.

[0029] In certain embodiments, the microbial cell uses E. coli ER2566 as an expression host.

[0030] The fifth technical solution provided by the present invention is a method for improving the activity and thermal stability of nitrilase, wherein the method comprises performing any of the following mutations on the nitrilase parent with the amino acid sequence as shown in SEQ ID NO: 1:

[0031] (a) mutating asparagine N at position 40 to glutamic acid E, glutamic acid E at position 113 to histidine H, glycine G at position 191 to alanine A, alanine A at position 230 to asparagine D, and alanine A at position 267 to tryptophan W;

[0032] (b) Asparagine N at position 40 is mutated to glutamic acid E, glutamic acid E at position 113 is mutated to histidine H, glycine G at position 191 is mutated to alanine A, alanine A at position 230 is mutated to asparagine D, alanine A at position 267 is mutated to tryptophan W, glutamine Q at position 27 is mutated to leucine L, lysine K at position 88 is mutated to proline P and glutamine Q at position 244 is mutated to lysine K.

[0033] The sixth technical solution provided by the present invention is a method for preparing nicotinic acid, wherein the mutant described in the first technical solution or the recombinant cell described in the fourth technical solution is added as a catalyst into a reaction system containing nicotinonitrile to react and prepare nicotinic acid.

[0034] The present invention provides a seventh technical solution, which is the use of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the method described in the sixth technical solution in the preparation of nicotinic acid, 2-pyridinecarboxylic acid, benzoic acid or products containing nicotinic acid, 2-pyridinecarboxylic acid or benzoic acid.

[0035] The beneficial effects of the present invention are as follows:

[0036] The present invention uses the PROSS design tool to obtain a nitrilase mutant with improved catalytic activity, and the enzyme activity reaches 300%, 230%, 115%, and 165% of the wild-type Nit6803, respectively. In addition, the nitrilase mutant with improved thermal stability, after incubation at 50° C. for 8 hours, compared with the 40% relative activity of the wild-type Nit6803, the relative enzyme activity of the mutant is 76% and 61%, respectively. Therefore, the present invention uses the enzyme to catalyze nitrile substances to produce carboxylic acid substances in industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Whole-cell thermostability screening of PROSS designed mutants.

[0038] Figure 2 : Determination of the relative enzyme activity of pure enzymes of mutants.

[0039] Figure 3 : Determination of half-life of pure enzyme of mutants. a: half-life of pure enzyme at 40℃; b: half-life of pure enzyme at 50℃. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0041] The following embodiments relate to the method:

[0042] 1. Gene amplification and fragment purification

[0043] (1) PCR reaction system (50 μL): 25 μL PrimeSTAR Max Premix (2×) (Takara), 2 μL each of upstream and downstream primers (10 μM), 0.5 μL DNA template, and 20.5 μL ddH2O.

[0044] (2) PCR reaction procedure: pre-denaturation at 98°C for 1 min; 30 cycles of 98°C for 30 s, 55°C for 15 s, and 72°C for 1 min and 30 s; and full extension at 72°C for 5 min.

[0045] (3) DpnI digestion to remove template DNA: 1 μL restriction endonuclease DpnI (Takara) and 5 μL 10× Buffer were added to the PCR reaction product and placed in a 37°C metal bath for 1 h. The digested reaction solution was purified using a Gel Extraction Kit (Kangwei Century) to obtain a high-purity PCR amplification product.

[0046] 2. Plasmid construction based on seamless cloning

[0047] (1) Fragment assembly: Prepare a DNA seamless ligation reaction system (10 μL): 5 μL 2X MultiF Seamless Assembly Mix (ABclonal), 2.5 μL each of the DNA gene fragment and the vector fragment. React in a 55°C metal bath for 30 min.

[0048] (2) Transformation: The seamless cloning reaction solution was transformed into Escherichia coli ER2566 competent cells and spread onto a screening agar plate containing the corresponding antibiotics. Transformants were cultured overnight at 37°C and single colonies were picked for subsequent verification.

[0049] 3. HPLC determination of nitrilase activity

[0050] The chromatographic column was Diamonsil C18(2) 5μm 250x 4.6mm (Dima Technology, China), and the mobile phase was a mixture of acetonitrile and water [the volume ratio was V(water):V(acetonitrile)=2:1]. The detection wavelength was 215nm, the column temperature was 40℃, the mobile phase flow rate was 1mL·min-1, and the injection volume was 10μL.

[0051] Unit enzyme activity (U) is defined as the amount of enzyme required to catalyze 3-cyanopyridine to produce 1 μmol of nicotinic acid per minute at 37°C. Specific enzyme activity (U·mg-1) is defined as the enzyme activity per milligram of nitrilase.

[0052] The raw materials used in the examples are:

[0053] 1. LB medium (L -1 ): 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, pH 7.0, 15 g of agar powder was added when preparing solid culture medium. Antibiotic concentration used during culture: kanamycin (50 μg / mL).

[0054] 2. The recombinant plasmid pET24a-Nit6803 has been disclosed in patent CN114214308A.

[0055] 3. Escherichia coli ER2566 was purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0056] Example 1 PROSS mutant design

[0057] The four mutants D1, D2, D3, and D4 in Table 1 were obtained based on nitrilase 6803 (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2) by PROSS design tool. The selected sites were mutated, and PCR amplification was performed using the pET24a-Nit6803 plasmid as a template and the corresponding primers in Table 2. After digestion and purification, the PCR product was assembled by seamless cloning, transformed into Escherichia coli competent cells ER2566, coated on LB plates containing kanamycin, and cultured at 37°C overnight. The grown colonies were sequenced to obtain mutant strains ER2566 / pET24a-Nit6803-D1, ER2566 / pET24a-Nit6803-D2, ER2566 / pET24a-Nit6803-D3, and ER2566 / pET24a-Nit6803-D4.

[0058] Table 1 Distal residue grouping

[0059]

[0060] Table 2 Primers for constructing mutations

[0061]

[0062]

[0063] Example 2: Whole-cell thermal stability screening of PROSS designed mutants

[0064] The wild-type strain ER2566 / pET24a-Nit6803 and the mutant strains ER2566 / pET24a-Nit6803-D1, ER2566 / pET24a-Nit6803-D2, ER2566 / pET24a-Nit6803-D3 and ER2566 / pET24a-Nit6803-D4 in Example 1 were inoculated into a test tube containing 3 mL of LB medium (containing kanamycin) and cultured at 37°C and 200 rpm for 12 h. 100 μL of the seed solution was transferred to a test tube containing 5 mL of LB medium (containing kanamycin) and cultured at 37°C and 200 rpm until OD 600 ≈0.6, add IPTG with a final concentration of 0.5mM, cool to 30℃, and continue culturing for 10h to fully induce the expression of nitrilase.

[0065] Take 2 ml of the induced bacterial solution, centrifuge at 12000 rpm for 3 min, discard the culture medium supernatant, and resuspend with 2 mL of 20 mM PBS buffer (pH 7.4) to obtain a cell suspension. Detect the whole cell enzyme activity, the specific steps are as follows: take 100 μL of cell suspension to a 1.5 mL EP tube, add 800 μL of PBS solution, 100 μL of 1M 3-cyanopyridine solution. After vortexing and mixing, place it in a 37 ° C metal bath for 10 min, then take it out immediately, centrifuge at 12000 rpm for 3 min, take the supernatant and filter it with a 0.22 μm filter membrane. Use high performance liquid chromatography to detect the amount of nicotinic acid produced and calculate the whole cell catalytic activity.

[0066] The cell suspension was placed at 50°C for 0 h and 8 h before reacting, and the relative enzyme activity was determined. Figure 1 After being placed at 50℃ for 0h, the relative enzyme activity of each mutant was defined as 100%. Compared with the wild-type Nit6803, the enzyme activity of mutants D1, D2, D3, and D4 were improved accordingly. The improvement of D1 was the largest, reaching about 3 times that of the wild-type. The activities of D2, D3, and D4 were 230%, 115%, and 165% of the wild-type, respectively. Figure 1 , Table 3). After being placed at 50℃ for 8h, the relative activity of the wild-type Nit6803 was 40%, while the relative enzyme activities of mutants D1 and D2 were significantly improved, reaching 76% and 61%, respectively. The relative enzyme activities of mutants D3 and D4 decreased significantly compared with the wild-type, reaching only 16% and 13% ( Figure 1 , Table 3).

[0067] Table 3 Results of whole cell heat treatment at 50℃ for 8h

[0068] Nit6803 D1 D2 D3 D4 Whole cell enzyme activity (U / mL) 6.30±0.23 17.62±0.45 14.94±0.12 7.32±0.22 10.45±0.31 50℃8h residual enzyme activity (%) 40.23±0.02 76.07±0.06 61.66±0.05 15.33±0.03 12.76±0.06

[0069] Example 3: Determination of pure enzyme properties of mutants

[0070] The wild-type strain ER2566 / pET24a-Nit6803 and the mutant strain ER2566 / pET24a-Nit6803-D1 in Example 1 were inoculated into a test tube containing 3 mL of LB medium (kanamycin) and cultured at 37°C and 200 rpm for 12 h. 1 mL of the seed solution was transferred into a test tube containing 50 mL of LB medium (containing kanamycin) and cultured at 37°C and 200 rpm until OD 600≈0.6, add IPTG with a final concentration of 0.5mM, cool to 30℃, and continue to culture for 10h to fully induce the expression of nitrilase. The induced bacterial solution was cooled on ice and centrifuged at 8000rpm for 10min in a low-temperature centrifuge (4℃), and the supernatant was discarded to collect the bacterial precipitate. Resuspend the bacterial precipitate with 20mL Binding Buffer (20mM PB, 0.5M NaCl, pH7.4), and crush it with an ultrasonic crusher for 20-30min. The bacterial suspension was always placed on ice during the crushing process. After crushing, centrifuge at 8000rpm for 20min, and the supernatant was divided into 1.5mL EP tubes and centrifuged at 12000rpm for 20min. The supernatant is the crude enzyme solution.

[0071] The expressed nitrilase / mutant was fused with 6×Hit-tag, and the nitrilase was purified using an AKTA protein purifier and a Ni-TED 6FF prepacked chromatography column (1 mL, Shanghai Biotech). The steps are as follows: (1) rinse the column with 5 to 10 column volumes of pure water at a flow rate of 2 mL / min to remove ethanol; (2) balance the column with 5 to 10 column volumes of Binding Buffer at a flow rate of 2 mL / min; (3) filter the crude enzyme through 0.45 μm and load it onto the column at a flow rate of 1 mL / min; (4) wash out impurities with 10 to 20 column volumes of Washing Buffer (20 mM PB, 0.5 M NaCl, 20 mM Imidazole, pH 7.4) at a flow rate of 2 mL / min; (5) wash out and collect the target protein with 5 to 10 column volumes of Elution Buffer (20 mM PB, 0.5 M NaCl, 500 mM Imidazole, pH 7.4) at a flow rate of 1 mL / min. (6) Rinse the column with 5 to 10 column volumes of pure water at a flow rate of 0.5 mL / min; (7) Rinse the column with 5 to 10 column volumes of 20% ethanol at a flow rate of 0.5 mL / min and store. Use 20 mM PBS (pH 7.4) to fully dialyze the recovered enzyme solution to prepare the recombinant enzyme pure enzyme dialysate.

[0072] Catalytic activity test of pure recombinant enzyme: Take 50 μL of 0.1 mg mL -1 The enzyme solution was transferred to a 1.5 mL EP tube and a 3-cyanopyridine solution with a final concentration of 100 mM was added. After vortex mixing, the mixture was placed in a 37°C metal bath for 10 min, and then 460 μL of acetonitrile was added to terminate the reaction. The supernatant was filtered with a 0.22 μm filter membrane. The nicotinic acid production was detected by high performance liquid chromatography and the whole cell catalytic activity was calculated.

[0073] The results showed that the specific enzyme activities of wild-type Nit6803 and D1 were 4.4±0.21 and 33.12±0.25 U / mg, respectively. The specific enzyme activity of D1 was 7.5 times that of the wild-type ( Figure 2 ).

[0074] Furthermore, the half-lives of wild-type Nit6803 and D1 at 40 and 50°C were determined. The results showed that the half-lives of Nit6803 and D1 at 50°C were 15 min and 51 min, respectively. At 40°C, the half-life of Nit6803 was 4.5 h, while the survival rate of D1 was 80% at 12 h. D1 had significantly improved thermal stability ( Figure 3 , Table 4).

[0075] Table 4 Characterization of mutant enzymes

[0076] Mutants Half-life at 40℃ Half-life at 50℃ Nit6803 4.5h 15min D1 Not Detected 51min

[0077] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A nitrilase mutant, characterized in that The mutant is a nitrilase parent with an amino acid sequence as shown in SEQ ID NO: 1, and a site as shown in any one of (D1) to (D4) is mutated: (D1) asparagine at position 40, glutamic acid at position 113, glycine at position 191, alanine at position 230, and alanine at position 267; (D2) includes the positions of (D1), as well as glutamine at position 27, lysine at position 88, and glutamine at position 244; (D3) includes the position of (D2) and asparagine at position 223; (D4) includes the positions of (D3), as well as methionine at position 31, threonine at position 99, and leucine at position 234.

2. The mutant according to claim 1, characterized in that The asparagine N at position 40 of the parent is mutated to glutamic acid E; The glutamic acid E at position 113 of the parent is mutated to histidine H; The glycine G at position 191 of the parent is mutated to alanine A; Alanine A at position 230 of the parent is mutated to asparagine D; The alanine A at position 267 of the parent is mutated to tryptophan W; The glutamine Q at position 27 of the parent is mutated to leucine L; The lysine K at position 88 of the parent is mutated to proline P; The glutamine Q at position 244 of the parent is mutated to lysine K; The asparagine N at position 223 of the parent is mutated to valine V; The methionine M at position 31 of the parent is mutated to leucine L; The threonine T at position 99 of the parent is mutated to arginine R; The leucine L at position 234 of the parent was mutated to alanine A.

3. A gene encoding the mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector uses pET-24a(+) as an expression vector.

6. A recombinant cell expressing the mutant according to claim 1 or 2, or containing the gene according to claim 3, or transformed with the recombinant vector according to claim 4 or 5.

7. The recombinant cell according to claim 6, characterized in that The recombinant cell is an animal cell, a plant cell or a microbial cell.

8. A method for improving the activity and thermal stability of nitrilase, characterized in that: The method is to perform any of the following mutations on a nitrilase parent with an amino acid sequence as shown in SEQ ID NO: 1: (a) mutating asparagine N at position 40 to glutamic acid E, glutamic acid E at position 113 to histidine H, glycine G at position 191 to alanine A, alanine A at position 230 to asparagine D, and alanine A at position 267 to tryptophan W; (b) Asparagine N at position 40 is mutated to glutamic acid E, glutamic acid E at position 113 is mutated to histidine H, glycine G at position 191 is mutated to alanine A, alanine A at position 230 is mutated to asparagine D, alanine A at position 267 is mutated to tryptophan W, glutamine Q at position 27 is mutated to leucine L, lysine K at position 88 is mutated to proline P and glutamine Q at position 244 is mutated to lysine K.

9. A method for preparing nicotinic acid, characterized in that: The mutant according to claim 1 or 2, or the recombinant cell according to claim 6 or 7 is added as a catalyst into a reaction system containing nicotinonitrile to react and prepare nicotinic acid.

10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant vector according to claim 4 or 5, or the recombinant cell according to claim 6 or 7, or the method according to claim 8, or the method according to claim 9 in the preparation of nicotinic acid, 2-pyridinecarboxylic acid, benzoic acid or products containing nicotinic acid, 2-pyridinecarboxylic acid or benzoic acid.

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