A nitrilase mutant with improved activity and thermal stability

Abstract

The application discloses a nitrilase mutant with improved activity and thermal stability, and belongs to the field of enzyme engineering. The nitrilase mutant with improved catalytic activity is obtained through a PROSS design tool, and the enzyme activity reaches 300%, 230%, 115% and 165% of that of wild type Nit6803 respectively. In addition, the nitrilase mutant with improved thermal stability has a relative enzyme activity of 76% and 61% after incubation at 50 DEG C for 8 hours, compared with 40% of the relative activity of wild type Nit6803. Therefore, the application of the enzyme catalyzed nitrile substances to produce carboxylic acid substances in industry is utilized.

Description

TECHNICAL FIELD

[0001] The present application relates to a nitrilase mutant with improved activity and thermal stability, belonging to the field of enzyme engineering. BACKGROUND

[0002] Enzyme engineering is an effective means to improve the properties of enzymes (activity, stability, tolerance, selectivity, etc.) in the enzyme industry, mainly including strategies of directed evolution and rational design. The advantage of directed evolution is that it does not need to obtain the information of the target protein and the mutation can occur in unexpected positions to obtain unexpected catalytic effect, but it needs to obtain a mutation library with diversity and a high-throughput screening method as a prerequisite. The mutation methods of directed evolution mainly include error-prone PCR, DNA shuffling and site-saturated mutagenesis. Rational design is one of the classic methods of protein engineering, which changes the structure of the protein by site-directed mutagenesis, deletion or insertion of specific amino acids of known sequences, so as to make the protein have different functions, but it is limited by the limited computing power and the accuracy of the calculation model, and it is difficult to guarantee the mutation calculation space and design accuracy.

[0003] Nitrilase is a kind of enzyme that can hydrolyze nitrile to carboxylic acid in one step, and can catalyze many high-value amino acids and organic acids, which has been widely used in the production of many chemical products and drug intermediates. However, naturally occurring nitrilases have low activity, poor thermal stability, and low stereoselectivity / region selectivity, which is a bottleneck that limits their further application. Many reported studies focus on the above-mentioned defects of nitrilases and modify nitrilases to improve the performance of nitrilases to some extent. For example, error-prone PCR and saturation mutagenesis are used to modify nitrilases, and stable mutants with several times higher activity are obtained (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 is used to change the substrate specificity, and it is found that the tyrosine at position 142 of the nitrilase of the genus Rhodococcus ATCC33278 can determine the specificity of the catalyzed substrate, and 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 single-point mutation design and evaluation, followed by multiple rounds of iterative combination, and due to the existence of epistatic effects, the efficiency is low and it is difficult to predict the evolution direction of combined mutations.

[0004] Computational protein design is undoubtedly an emerging field. Its advantage lies in the ability to make predictions based on structure or sequence information, thereby shortening experimental time. In the process of modifying enzyme molecules, computer-based design has extremely broad application prospects in enzyme engineering. Protein Repair One-Stop Shop (Protein Repair One-Stop Shop), a tool for automated protein design based on structure and sequence (Goldenzweig A, Goldsmith M, Hill SE, Gertman O, Laurino P, Ashani Y, Dym O, 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.), can effectively enhance enzyme performance. However, for nitrilase, how to apply PROSS to achieve its transformation and what the effect of the transformation will be is unknown. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention provides the development and application of nitrilase mutants with enhanced activity and thermostability. The goal is to optimize and modify Nit6803 using the PROSS design tool to improve its activity and stability. PROSS first designed mutation combinations through multiple sequence alignment analysis and folding free energy prediction, resulting in four mutants: D1, D2, D3, and D4. The four mutants were screened for whole-cell enzyme activity and thermostability to obtain mutants with enhanced activity. Furthermore, the pure enzyme specific activity and half-life of these mutants were analyzed, ultimately yielding mutants with enhanced activity and stability.

[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 sites of (D1), and the 27th glutamine, the 88th lysine and the 244th glutamine;

[0009] (D3) includes the sites of (D2), and the 223rd asparagine;

[0010] (D4) includes the sites of (D3), and the 31st methionine, the 99th threonine and the 234th leucine.

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

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

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

[0014] The alanine A corresponding to the 230th of the parent is mutated to asparagine D;

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

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

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

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

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

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

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

[0022] The leucine L corresponding to the 234th of the parent is mutated to alanine A.

[0023] The second technical solution provided by the present application is a gene encoding the mutant of the first technical solution.

[0024] The third technical solution provided by the present application is a recombinant vector carrying the gene of the second technical solution.

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

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

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

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

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

[0030] The fifth technical solution provided by the present application is a method for improving the activity and thermal stability of a nitrilase, which is to mutate the nitrilase parent with an amino acid sequence as shown in SEQ ID NO: 1 as follows:

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

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

[0033] The sixth technical solution provided by the present application is a method for preparing nicotinic acid, which adds the mutant of the first technical solution or the recombinant cell of the fourth technical solution as a catalyst into a reaction system containing nicotinonitrile to prepare nicotinic acid by reaction.

[0034] The seventh technical solution provided by the present application is the use of the mutant of the first technical solution, or the gene of the second technical solution, or the recombinant vector of the third technical solution, or the recombinant cell of the fourth technical solution, or the method of the fifth technical solution, or the method of the sixth technical solution in the preparation of nicotinic acid, 2-picolinic acid, benzoic acid, or a product containing nicotinic acid, 2-picolinic acid, or benzoic acid.

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

[0036] The application obtains a nitrilase mutant with improved catalytic activity by a PROSS design tool, and the enzyme activity reaches 300%, 230%, 115% and 165% of that of wild type Nit6803, respectively. In addition, the nitrilase mutant with improved thermal stability has a relative enzyme activity of 76% and 61% after incubation at 50℃ for 8h, compared with 40% of wild type Nit6803. Therefore, the application has industrial application of the enzyme catalyzing nitrile substances to produce carboxylic acid substances. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 : PROSS design mutant whole cell thermal stability screening.

[0038] Figure 2 : Mutant pure enzyme specific enzyme activity determination.

[0039] Figure 3 : Mutant pure enzyme half-life determination.a: Pure enzyme 40℃ half-life determination;b: Pure enzyme 50℃ half-life determination. DETAILED DESCRIPTION

[0040] The preferred embodiments of the application are described below, and it should be understood that the embodiments are used to better explain the application, and are not used to limit the application.

[0041] The method involved in the following examples is as follows:

[0042] 1. Gene amplification and fragment purification

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

[0044] (2) PCR reaction program: 98℃ 1min pre-denaturation; 98℃ 30s, 55℃ 15s, 72℃ 1min 30s, cycle 30 times; 72℃ 5min sufficient extension.

[0045] (3) DpnI digestion to remove template DNA: 1 μL of restriction endonuclease DpnI (Takara) and 5 μL of 10x Buffer were added to the PCR reaction product, and the reaction was carried out in a 37℃ metal bath for 1h. The purified reaction solution after digestion was obtained by using the reagent kit Gel Extraction Kit (Kangwei Century), and high-purity PCR amplification product was obtained.

[0046] 2. Plasmid construction based on seamless cloning

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

[0048] (2) Transformation: transform the seamless cloning reaction solution into E. coli ER2566 competent cells, and coat on the screening agar plate containing the corresponding antibiotic. Incubate at 37°C overnight to obtain transformants, and pick single colonies for subsequent verification.

[0049] 3. HPLC determination of nitrile hydrolase activity

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

[0051] The unit enzyme activity (U) is defined as the amount of enzyme required to catalyze the generation of 1 μmol of nicotinic acid from 3-cyanopyridine per minute at 37°C. The specific enzyme activity (U·mg-1) is defined as the enzyme activity possessed by each milligram of nitrile hydrolase.

[0052] Raw materials used in the examples:

[0053] 1. LB medium (L -1 ): tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0, add agar powder 15 g when preparing solid medium. The concentration of the antibiotic used in the culture is kanamycin (50 μg / mL).

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

[0055] 3. E. coli ER2566 was purchased from Wuhan Moliang Biotechnology Co., Ltd.

[0056] Example 1 PROSS design mutant

[0057] Four mutants D1, D2, D3, D4 in Table 1 were obtained by PROSS design tool based on nitrilase 6803 (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2). The selected sites were mutated, and the pET24a-Nit6803 plasmid was used as a template for PCR amplification using the corresponding primers in Table 2. The PCR product was digested, purified, and assembled by seamless cloning, transformed into E. coli competent cells ER2566, spread on LB plates containing kanamycin, and incubated at 37°C overnight. The resulting 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 groupings

[0059]

[0060] Table 2 primers for constructing mutations

[0061]

[0062]

[0063] Example 2: PROSS design mutant whole cell thermostability screening

[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 test tubes containing 3 mL of LB medium (containing kanamycin) and incubated at 37°C, 200 rpm for 12 h. 100 μL of seed liquid was transferred into test tubes containing 5 mL of LB medium (containing kanamycin) and incubated at 37°C, 200 rpm until the OD 600 ≈0.6, IPTG was added to a final concentration of 0.5 mM, the temperature was lowered to 30°C, and the incubation was continued for 10 h to allow full induction of nitrilase expression.

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

[0066] The cell suspension was placed at 50℃ for 0h and 8h respectively before reaction, and the relative enzyme activity was determined, and the results are shown in Figure 1 After placing at 50℃ for 0h, the relative enzyme activity of each mutant was defined as 100%. Compared with wild type Nit6803, the enzyme activity of mutants D1, D2, D3 and D4 was improved, and D1 was the largest, about 3 times of the wild type, D2, D3 and D4 were 230%, 115% and 165% of the wild type respectively (Table 3). Figure 1 After placing at 50℃ for 8h, the relative activity of wild type Nit6803 was 40%, and the relative enzyme activity of mutants D1 and D2 was significantly improved, which was 76% and 61% respectively. Compared with wild type, the relative enzyme activity of mutants D3 and D4 decreased significantly, which was only 16% and 13% respectively (Table 3). Figure 1

[0067] Table 3: Results of whole cell 50℃ heat treatment 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 Residual enzyme activity at 50°C for 8h (%) 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 mutant pure enzyme properties

[0070] The wild type strain ER2566 / pET24a-Nit6803 and the mutant strain ER2566 / pET24a-Nit6803-D1 in Example 1 were inoculated into test tubes containing 3 mL of LB medium (kanamycin), and cultured at 37℃, 200 rpm for 12h. 1 mL of seed liquid was transferred into test tubes containing 50 mL of LB medium (containing kanamycin), and cultured at 37℃, 200 rpm until OD 600 ​≈0.6, IPTG was added to a final concentration of 0.5 mM, the temperature was reduced to 30 °C, and the culture was continued for 10 h to allow for full induction of the nitrilase. The induced bacterial solution was cooled on ice and centrifuged at 8000 rpm for 10 min in a low-temperature centrifuge (4 °C). The supernatant was discarded to collect the bacterial precipitate. The bacterial precipitate was resuspended in 20 mL of Binding Buffer (20 mM PB, 0.5 M NaCl, pH 7.4), and was broken up using an ultrasonic disrupter for 20-30 min, with the bacterial suspension always being placed on ice during the breaking process. After breaking, the solution was centrifuged at 8000 rpm for 20 min, and the supernatant was divided into 1.5 mL EP tubes and centrifuged at 12000 rpm for 20 min. The supernatant was the crude enzyme solution.

[0071] The expressed nitrilase / mutant fusion had a 6xHit-tag, and the nitrilase was purified using an AKTA protein purification instrument and a Ni-TED 6FF pre-packed chromatography column (1 mL, Shanghai Generay Biotech). The steps were as follows: (1) the column was rinsed with 5-10 column volumes of pure water at a flow rate of 2 mL / min to remove ethanol; (2) the column was equilibrated with 5-10 column volumes of Binding Buffer at a flow rate of 2 mL / min; (3) the crude enzyme was filtered through a 0.45 μm filter and loaded onto the column at a flow rate of 1 mL / min; (4) the column was washed with 10-20 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 to remove impurities; (5) the target protein was eluted and collected using 5-10 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) the column was rinsed with 5-10 column volumes of pure water at a flow rate of 0.5 mL / min; and (7) the column was rinsed with 5-10 column volumes of 20% ethanol at a flow rate of 0.5 mL / min for storage. The recovered enzyme solution was dialyzed against 20 mM PBS (pH 7.4) to prepare the recombinant enzyme pure enzyme dialysate.

[0072] The catalytic activity of the recombinant enzyme pure enzyme was detected as follows: 50 μL of enzyme solution with a concentration of 0.1 mg·mL-1was taken into a 1.5 mL EP tube, and 3-cyanopyridine solution was added to a final concentration of 100 mM. After mixing and shaking, the tube was placed in a 37 °C metal bath for 10 min, 460 μL of acetonitrile was added to terminate the reaction, and the supernatant was filtered through a 0.22 μm filter membrane. The amount of nicotinic acid produced was detected using high-performance liquid chromatography, and the whole-cell catalytic activity was calculated. -1

[0073] ​The results show that the specific enzyme activity of wild type Nit6803 and D1 is 4.4±0.21 and 33.12±0.25 U / mg, respectively, and the specific enzyme activity of D1 is 7.5 times that of the wild type. Figure 2

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

[0075] Table 4 Characterization of mutant enzymes

[0076] Mutant Half-life at 40°C Half-life at 50°C Nit6803 4.5h 15 min D1 Not determined 51 min

[0077] Although the present application has been disclosed in preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the scope of protection of the present application should be defined by the claims.​​

Claims

1. A nitrilase mutant, characterized in that The mutant is obtained by mutating the nitrilase parent with the amino acid sequence shown in SEQ ID NO: 1 at any one of the positions (D1) to (D4): (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 sites of (D1), as well as glutamine at position 27, lysine at position 88, and glutamine at position 244; (D3) includes the sites of (D2) and asparagine at position 223; (D4) includes the sites of (D3), as well as methionine at position 31, threonine at position 99, and leucine at position 234; 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; 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.

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

3. A recombinant vector carrying the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector uses pET-24a(+) as an expression vector.

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

6. The recombinant cell according to claim 5, characterized in that The recombinant cell is an animal cell or a microbial cell.

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

8. A method for preparing nicotinic acid, characterized in that: The mutant according to claim 1 or the recombinant cell according to claim 5 or 6 is added as a catalyst into a reaction system containing nicotinonitrile to react and prepare nicotinic acid.

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

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