Nitrilase mutant and application thereof in biosynthesis of nicotinic acid

Abstract

The invention discloses a nitrilase mutant and application thereof in nicotinic acid biosynthesis, and belongs to the technical field of enzyme engineering. Through mutation design based on sequence fitness, the nitrilase mutant with improved nitrilase activity, thermal stability and soluble expression level is constructed, and the half-life period of the mutant D1F4 at 50 DEG C can reach 123.40 + / -5.94 min. The high-performance mutant obtained through construction is used for preparing nicotinic acid through whole-cell catalysis of 3-cyanopyridine, the final concentration of the product can be remarkably increased, and the final concentration of the product after 520 min of reaction can reach 2.21 M.

Description

TECHNICAL FIELD

[0001] The present application relates to a nitrilase mutant and its application in nicotinic acid biosynthesis, belonging to the technical field of enzyme engineering. BACKGROUND

[0002] At present, the commonly used enzyme engineering modification methods mainly include directed evolution, semi-rational design and rational design, etc., and most enzymes can be improved in enzyme industrial properties (activity, stability, tolerance, selectivity, etc.) on this basis. A large number of practical cases of directed evolution have fully shown that, with the help of high-throughput screening technology, large-scale screening of random mutations of each site of the enzyme molecule can successfully obtain the target mutant. However, due to the complex structure of the protein itself, the large molecular weight, and the extremely time-consuming screening process, the directed evolution strategy taking error-prone PCR as the core means often fails to achieve the desired efficiency. As a traditional and classic method of protein engineering, rational design mainly operates on the known protein sequence to implement site-directed mutation, deletion or insertion of specific amino acids, so as to change the spatial structure of the protein and endow it with new functional characteristics. However, due to the limitations of computing power and the imperfect accuracy of the calculation model, it is difficult to fully cover all possible mutation calculation spaces and ensure the accuracy of the design results in actual application.

[0003] Nitrilase (EC 3.5.5.1) as an important member of the nitrilase superfamily is an industrial enzyme that can convert nitrile compounds into carboxylic acids and ammonia in one step. Carboxylic acids are widely used in the fields of bulk chemicals and pharmaceutical intermediates, and nicotinic acid and mandelic acid have been produced on an industrial scale. Compared with chemical methods, enzyme synthesis has the advantages of mild reaction conditions, high stereoselectivity, and no need for expensive catalysts, and has both economic and environmental benefits. However, natural nitrilases have low enzyme activity, poor stability and tolerance, and poor thermal stability, and have low stereoselectivity and regioselectivity, which seriously restricts their further application in biocatalysis. In addition, nitrilases expressed in heterologous microbial expression systems tend to form inactive inclusion bodies, and the soluble expression level is low, making the preparation cost of nitrilases high. SUMMARY

[0004] The present application provides a nitrilase mutant, which has one or more mutations based on the parent shown in SEQ ID NO. 1 as follows:

[0005] (1) the 7th leucine is mutated to proline;

[0006] (2) the 80th glutamine is mutated to glutamic acid;

[0007] (3) the 88th lysine is mutated to proline;

[0008] (4) the 160th threonine is mutated to alanine.

[0009] In one embodiment, the nitrilase mutant is based on the parent shown in SEQ ID NO. 1, wherein the 80th glutamine is mutated to glutamic acid, and the 88th lysine is mutated to proline.

[0010] In one embodiment, the nitrilase mutant is based on the parent shown in SEQ ID NO. 1, wherein the 7th leucine is mutated to proline, the 80th glutamine is mutated to glutamic acid, the 88th lysine is mutated to proline, and the 240th glutamic acid is mutated to proline.

[0011] The present application also provides a gene encoding the nitrilase mutant.

[0012] The present application also provides an expression vector carrying the gene.

[0013] In one embodiment, the vector is pET series plasmid.

[0014] In one embodiment, the plasmid includes but is not limited to pET24a.

[0015] The present application also provides an engineered bacterium expressing the nitrilase mutant.

[0016] In one embodiment, the nitrilase mutant is expressed in E. coli BL21 (DE3) with pET24a as the vector.

[0017] The present application also provides a method for preparing the nitrilase mutant, wherein the engineered bacterium is cultured in a medium for a period of time, and the nitrilase mutant is collected.

[0018] In one embodiment, the culture is at 35-40℃ to OD 0.5-0.8, and the expression of the enzyme is induced by IPTG.

[0019] In one embodiment, the culture is at 37℃, 200 rpm to OD 600 0.6, and the temperature is lowered to 28-30℃, and the culture is continued for 10 h with IPTG at a final concentration of 0.5 mM.

[0020] The present application also provides an enzyme preparation containing the nitrilase mutant or the engineered bacterium cells.

[0021] The present application also provides a method for preparing nicotinic acid by enzymatic method with 3-cyanopyridine as the substrate.

[0022] In one embodiment, the method involves reacting the nitrile hydrolase mutant or the engineered bacterial cells in a reaction system containing 3-cyanopyridine at 36–40°C for at least 30 min.

[0023] In one embodiment, the reaction takes 30 to 60 minutes.

[0024] The present invention also provides the use of the nitrile hydrolase, or the enzyme preparation, or the method in the preparation of nicotinic acid or nicotinic acid-containing products.

[0025] Beneficial effects:

[0026] (1) This invention improves the activity, thermal stability and soluble expression level of nitrile hydrolase by mutation design based on sequence fitness. The mutant D1F4 has a half-life of 123.40±5.94 min at 50℃, which can increase the content of active nitrile hydrolase per unit cell, thereby greatly improving the whole cell activity.

[0027] (2) This invention utilizes a high-performance mutant to achieve efficient whole-cell catalysis of 3-cyanopyridine to nicotinic acid, significantly increasing the final product concentration. The final product concentration can reach 2.21M after 520 min of reaction. Attached Figure Description

[0028] Figure 1 The results are SDS-PAGE analysis of different proteins; C represents whole cells, S represents lysate supernatant, and P represents lysate.

[0029] Figure 2 : Scale-up of whole-cell catalytic reaction; a: D1 whole-cell catalysis; b: D1F4 whole-cell catalysis. Detailed Implementation

[0030] 1. Gene amplification and fragment purification

[0031] (1) PCR reaction system (50μL): 25μL PrimeSTAR Max Premix (2×) (Takara), 2μL each of upstream and downstream primers (10μM), 0.5μL DNA template, 20.5μL ddH2O.

[0032] (2) PCR reaction program: 98℃ for 1 min pre-denaturation; 98℃ for 30 s, 55℃ for 15 s, 72℃ for 1 min and 30 s, 30 cycles; 72℃ for 5 min for full extension.

[0033] (3) DpnI digestion to remove template DNA: 1 μL of restriction enzyme DpnI (Takara) and 5 μL of 10x Buffer were added to the PCR reaction product, which was placed in a 37°C metal bath for 1 h. The digested reaction solution was purified using the kit Gel Extraction Kit (Kangwei Century) to obtain a high-purity PCR amplification product.

[0034] 2. Plasmid construction based on seamless cloning

[0035] (1) Fragment assembly. A DNA seamless ligation reaction system (10 μL) was prepared: 5 μL of 2X MultiF Seamless Assembly Mix (ABclonal), 2.5 μL of DNA gene fragments, and 2.5 μL of vector fragments. The reaction was performed in a 55°C metal bath for 30 min.

[0036] (2) Transformation. The seamless cloning reaction solution was transformed into E. coli BL21 (DE3) competent cells, and was plated on a selection agar plate containing the corresponding antibiotic, which was incubated at 37°C overnight to obtain transformants, and a single colony was picked for subsequent verification.

[0037] 3. Culture medium

[0038] LB medium (L -1 ): tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0, and agar powder 15 g was added when preparing a solid culture medium.

[0039] The concentration of the antibiotic used during culture: kanamycin (50 μg / mL).

[0040] 4. Mutant screening based on whole-cell, crude enzyme, and pure enzyme activity determination

[0041] Whole-cell thermostability determination: equal OD of bacterial solution was placed in a 55°C metal bath for different times, 100 μL of bacterial solution treated at 55°C for different times was taken to a 1.5 mL EP tube, and the remaining steps were consistent with the whole-cell enzyme activity determination steps.

[0042] Pure enzyme activity determination: 50 μL of enzyme solution with a concentration of 0.1 mg·mL -1 was taken to a 1.5 mL EP tube, and 3-cyanopyridine solution with a final concentration of 100 mM was added. After vortexing and mixing, the reaction 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 using a 0.22 μm filter membrane. The amount of nicotinic acid generated was detected using high-performance liquid chromatography, and the whole-cell catalytic activity was calculated.

[0043] Half-life determination: 50 μL of enzyme solution with a concentration of 0.1 mg·mL -1Enzyme solution was treated at 55℃ for 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8h, then 3-cyanopyridine solution was added to a final concentration of 100mM, and the remaining steps were consistent with the first step of the pure enzyme activity assay.

[0044] 5. Protein induction expression and purification

[0045] Protein induction expression and ultrasonic disruption: inoculate into a test tube containing 3mL LB medium (kanamycin), incubate at 37℃, 200rpm for 12h. Transfer 1mL seed liquid into a test tube containing 50mL LB medium (containing kanamycin), incubate at 37℃, 200rpm until OD 600 =0.6, add IPTG to a final concentration of 0.5mM, cool to 30℃, continue to cultivate for 10h to induce the expression of nitrile hydratase.

[0046] Cool the induced bacterial liquid on ice, and centrifuge at 8000rpm for 10min in a low-temperature centrifuge (4℃), pour off the supernatant to collect the bacterial precipitate. Resuspend the bacterial precipitate with 20mL Binding Buffer (20mM PB, 0.5M NaCl, pH 7.4), and disrupt it with an ultrasonic disrupter for 20-30min, with the bacterial suspension always placed on ice during the disruption process. Centrifuge at 8000rpm for 20min after disruption, and centrifuge the supernatant at 12000rpm for 20min, and the supernatant is the crude enzyme solution.

[0047] Metal nucleophilic chromatography purification of protein: the expressed nitrile hydratase is fused with 6×Hit-tag, and AKTA protein purification instrument and Ni-TED 6FF pre-packed chromatography column (1mL, Shanghai Generay) are used to purify the nitrile hydratase. The steps are as follows: (1) flush the column with 5-10 times the column volume of pure water at a flow rate of 2mL / min to remove ethanol; (2) equilibrate the column with 5-10 times the column volume of Binding Buffer at a flow rate of 2mL / min; (3) filter the crude enzyme at 0.45μm, and load it onto the column at a flow rate of 1mL / min; (4) wash away the impurities with 10-20 times the column volume of Washing Buffer (20mM PB, 0.5M NaCl, 20mM Imidazole, pH 7.4) at a flow rate of 2mL / min; (5) wash out and collect the target protein with 5-10 times the column volume of Elution Buffer (20mM PB, 0.5M NaCl, 500mM Imidazole, pH 7.4) at a flow rate of 1mL / min; (6) flush the column with 5-10 times the column volume of pure water at a flow rate of 0.5mL / min; (7) flush the column with 5-10 times the column volume of 20% ethanol at a flow rate of 0.5mL / min and store it.

[0048] Dialysis: To further remove high concentration of salt in the pure enzyme, the sample was dialyzed against 20 mM PBS (pH 7.4) sufficiently. The protein purity was detected by SDS-PAGE.

[0049] 6. HPLC determination of nitrile hydratase activity

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

[0051] The unit enzyme activity (U) was defined as the amount of enzyme required to catalyze 1 μmol of nicotinic acid from 3-cyanopyridine per minute at 37 °C; the specific enzyme activity (U·mg -1 ) was defined as the enzyme activity per milligram of nitrile hydratase; and the whole-cell activity (U / mL) was defined as the catalytic activity per mL of cells (OD600 = 4 as the final concentration of the reaction system).

[0052] Example 1: Design of mutants

[0053] The sequence of the nitrile hydratase D1 shown in SEQ ID NO. 1 was scored for fitness using the SPIRED-Fitness tool (published in the paper “An end-to-end framework for the prediction of protein structure and fitness from single sequence”), and 29 residues were not the optimal amino acid species for fitness, of which 22 residues had a fitness change of ≥1 after mutation. Therefore, mutations with a significant improvement in fitness were selected as candidate single-point mutations.

[0054] The recombinant plasmid pET24a-D1 carrying the D1 sequence shown in SEQ ID NO. 2 was used as a template, and the corresponding primers were used for PCR amplification. The PCR product was digested and purified, and then assembled by seamless cloning and transformed into E. coli competent cells BL21 (DE3) to construct single-point mutant recombinant bacteria. The constructed mutations and the primers used are shown in Table 1 and Table 2, respectively.

[0055] Table 1 Grouping of mutant residues

[0056] Name Mutation Fitness change Name Mutation Fitness change FN1 G3D 4.99 FN12 T99Q 1.74 FN2 M6E 1.44 FN13 M102I 7.07 FN3 L7P 21.71 FN14 T156V 2.31 FN4 Y9P 7.10 FN15 T160A 2.41 FN5 A15V 4.93 FN16 L164I 3.23 FN6 A17V 2.99 FN17 M210I 1.69 FN7 A18V 3.36 FN18 T238G 9.04 FN8 M31I 3.88 FN19 E240P 1.01 FN9 S74E 1.74 FN20 Y251S 1.70 FN10 Q80E 1.16 FN21 A253V 1.10 FN11 K88P 2.89 FN22 G269E 1.57

[0057] Table 2 Primers for constructing mutations

[0058]

[0059]

[0060]

[0061] Example 2: Single point mutation screening

[0062] (1) Whole cell catalytic activity determination:

[0063] The recombinant bacteria containing single point mutations constructed in Example 1 were inoculated into test tubes containing 3 mL of LB medium (containing kanamycin) and cultured 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 cultured at 37°C, 200 rpm until OD 600 ≈0.6, 0.5 mM IPTG was added, and the culture was continued at 37°C for 12 h to induce the expression of nitrile hydratase.

[0064] 2 ml of the induced bacterial liquid was taken and centrifuged at 12000 rpm for 3 min, the culture medium supernatant was discarded, and 2 mL of 20 mM PBS buffer (pH 7.4) was used to resuspend the cells to obtain a cell suspension.

[0065] 100 μL of the cell suspension with OD 600 = 4 was taken into a 1.5 mL EP tube, 800 μL of PBS solution and 100 μL of 1 M 3-cyanopyridine solution were added. After vortexing and mixing, the tube was immediately taken out of the 37°C metal bath after 10 min of reaction and centrifuged at 12000 rpm for 3 min. The supernatant was filtered with a 0.22 μm filter membrane. The amount of nicotinic acid produced was detected by high performance liquid chromatography, and the whole cell catalytic activity was calculated.

[0066] The mutant strains were induced for expression, and the catalytic activity of the whole cells was determined. The results are shown in Table 3. The whole cell activity of FN3, FN10, FN11, FN15 and FN16 was higher than that of the control D1.

[0067] (2) Thermal stability determination:

[0068] The cell suspension of recombinant cells expressing different single mutations was prepared according to the method of step (1), and the residual activity was determined after incubation at 55°C for 2 h, which represented the thermal stability. The results are shown in Table 3. Among the 7 positive single point mutations with increased activity, the whole cell activity and thermal stability of FN10, FN11 and FN15 were significantly improved compared with D1.

[0069] Table 3: Whole cell screening of single point mutations

[0070]

[0071]

[0072] Example 3: Iterative combination to prepare combination mutants

[0073] Using the single mutant FN10 constructed in Example 1 as the starting template, the mutations of FN3, FN11, FN12, FN15, FN16 or FN19 were introduced step by step according to Example 1 to construct double-point mutants. The whole-cell activity of the double-point mutants was determined according to the method of Example 2, and the residual activity after incubation at 55°C for 4h was determined. The results are shown in Table 4. The activity of FN10 / 3 reached 16.30±1.15 U / mL, and the thermal stability was slightly lower than that of FN10. The thermal stability of FN10 / 11 was the highest, and the activity was not significantly different from that of FN10.

[0074] Since thermal stability is more important for long-time catalytic processes, FN10 / 11 was selected as the starting template, and the mutations of FN3, FN12, FN15, FN16 or FN19 were introduced to construct triple-point combination mutants. The whole-cell activity of the triple-point mutants was determined according to the method of Example 2, and the residual activity after incubation at 55°C for 4h was determined. The results are shown in Table 4. FN10 / 11 / 3 had an activity comparable to that of FN10 / 3, and a thermal stability comparable to that of FN10 / 11. The thermal stability of FN10 / 11 / 19 was significantly higher than that of FN10 / 11, while the activity and thermal stability of FN10 / 11 / 15 and FN10 / 11 / 16 were both decreased compared to those of FN10 / 11. Therefore, FN10 / 11 / 3 and FN19 were combined to construct the four-point combination mutant FN10 / 11 / 3 / 19, which further improved the whole-cell activity and thermal stability. SDS-PAGE analysis showed that the soluble expression of FN10 / 11 / 3 / 19 was significantly improved compared to that of D1. Figure 1 ) The mutant FN10 / 11 / 3 / 19 obtained by screening was named D1F4 for short.

[0075] Table 4: Iterative combination screening

[0076]

[0077]

[0078] Example 4: Determination of the properties of pure enzymes of mutants

[0079] The starting enzyme D1 and the mutant D1F4 constructed in Example 3 were respectively induced for overexpression, metal affinity chromatography purification, and determination of the enzymatic properties of the pure enzymes. The results showed that the specific enzyme activities of the mutant D1 and the mutant D1F4 were 105.34±2.85 U / mg and 101.50±2.72 U / mg, respectively, and both had comparable specific enzyme activities. The half-lives of D1 and the mutant D1F4 at 50°C were further determined. The results showed that the half-lives of D1 and D1F4 at 50°C were 40.70±1.58 min and 123.40±5.94 min, respectively, and D1F4 had significantly improved thermal stability (Table 5). The above results showed that D1F4 was a preferred combined mutation with high activity and high thermal stability.

[0080] Table 5 Characterization of mutant enzymes

[0081] Mutant 50°C half-life (min) D1 40.70±1.58 D1F4 123.40±5.94

[0082] Example 5: Preparation of nicotinic acid by whole-cell catalysis

[0083] The whole cells expressing the single mutant D1 constructed in Example 2 and the whole cells expressing the combined mutant D1F4 prepared in Example 3 were respectively used for scale-up catalytic verification. The volume of the reaction system was 100 mL, the cell OD600 was 10 (the wet cell concentration was about 4 g / L), 3-cyanopyridine was used as the substrate at a final concentration of 200 mM, the substrate was added in batches (200 mM each time), the reaction was carried out on a magnetic stirrer, and the reaction temperature was maintained at 37°C. The pre-experiment showed that D1 and D1F4 could completely react with 200 mM substrate in 60 min and 30 min, respectively. Therefore, the time interval for substrate addition in the reaction system of the cells expressing D1 was set to 60 min, and the time interval for substrate addition in the early stage (within 60 min after the start of the reaction) of the cells expressing D1F4 was set to 30 min, and the time interval for substrate addition was appropriately increased (45 min to 60 min) in the middle and late stages of the reaction. The results, as shown in Table 6, showed that the final concentration of the product of mutant D1 after 600 min of reaction was 1.77 M, and the final concentration of the product of D1F4 after 520 min of catalysis reached 2.21 M, significantly improving the catalytic efficiency and achieving a production rate of 4.25 mmol / min per unit time, which had a strong application prospect. Figure 2

[0084] Although the present application has been disclosed in the above preferred embodiments, 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 protection scope of the present application should be defined by the claims.​

Claims

1. A nitrile hydrolase mutant, characterized in that, Based on the parent shown in SEQ ID NO.1, it has one or more of the following mutations: (1) Mutate the 7th leucine to proline; (2) Mutate the glutamine at position 80 to glutamic acid; (3) Mutate the 88th lysine to proline; (4) Mutate the threonine at position 160 to alanine.

2. The nitrile hydrolase mutant according to claim 1, characterized in that, The nitrile hydrolase mutant is (a) or (b): (a) Based on the parental line shown in SEQ ID NO.1, glutamine at position 80 was mutated to glutamic acid, and lysine at position 88 was mutated to proline; (b) Based on the parental form shown in SEQ ID NO.1, leucine at position 7 was mutated to proline, glutamine at position 80 was mutated to glutamic acid, lysine at position 88 was mutated to proline, and glutamic acid at position 240 was mutated to proline.

3. A gene encoding the nitrile hydrolase mutant of claim 1 or 2.

4. An expression vector carrying the gene of claim 3.

5. Genetically engineered bacteria expressing the nitrile hydrolase mutant of claim 1 or 2.

6. Recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET24a as the vector, the nitrile hydrolase mutant described in claim 1 or 2 is expressed.

7. A method for preparing the nitrile hydrolase mutant according to claim 1 or 2, characterized in that, The recombinant Escherichia coli of claim 6 was cultured in a culture medium for a period of time, and the nitrile hydrolase mutant was collected.

8. An enzyme preparation containing the nitrile hydrolase mutant of claim 1 or 2, or the recombinant Escherichia coli cell of claim 6.

9. A method for preparing nicotinic acid, characterized in that, The nitrile hydrolase mutant of claim 1 or 2 or the recombinant Escherichia coli cells of claim 6 are reacted in a reaction system containing 3-cyanopyridine at 36-40°C for at least 30 min.

10. The use of the nitrile hydrolase mutant of claim 1 or 2, or the genetically engineered bacteria of claim 5, or the recombinant Escherichia coli of claim 6, or the enzyme preparation of claim 8, or the method of claim 9 in the preparation of nicotinic acid or nicotinic acid-containing products.

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