Nitrilase mutant containing non-natural amino acid, coding gene, recombinant vector, recombinant strain and application of nitrilase mutant

Abstract

The invention relates to the technical field of bioengineering, in particular to a nitrilase mutant containing non-natural amino acid, and is characterized in that the nitrilase mutant comprises one of the following mutation modes: (1) proline at the 340th site of an amino acid sequence as shown in SEQ ID NO.1 is mutated into L-(7-hydroxycoumarin-4-yl) ethylglycine; (2) proline at the 367 site of the amino acid sequence as shown in SEQ ID NO.6 is mutated into L-(7-hydroxycoumarin-4-yl) ethyl glycine; and (3) the proline at the 314th site of the amino acid sequence as shown in SEQ ID NO.8 is mutated into L-(7-hydroxycoumarin-4-yl) ethyl glycine. The nitrilase mutant NIT-P340G-Hco with high activity and stability is successfully screened out by establishing a high-throughput screening model, constructing a large-capacity mutant library and introducing non-natural amino acid on the basis of initial enzyme for enzyme modification, the whole-cell enzyme activity of the nitrilase mutant NIT-P340G-Hco is 15.84 times that of original nitrilase, and the nitrilase mutant NIT-P340G-Hco has high activity and stability. Therefore, the enzyme activity is enhanced while the enzyme stability is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a nitrilase mutant containing non-natural amino acids, a coding gene, a recombinant vector, a recombinant strain and applications thereof. Background Art

[0002] Nitrilases (EC 3.5.5.1) are members of the nitrilase / amidase superfamily and can directly hydrolyze nitrile compounds into carboxylic acids and ammonia. Therefore, nitrilase plays a vital role in the production of carboxylic acids using nitrile substances as substrates, and nitrile substances have been widely reported to be intermediates for medicines, agricultural chemicals and other fine chemicals. Pyridine pharmaceutical intermediates have become a research hotspot for the new generation of biopharmaceuticals due to their broad spectrum and environmental friendliness. Tetrachloropyridine carboxylic acid is an important intermediate for pyridine pesticides, but the mainstream way to produce tetrachloropyridine carboxylic acid today is the chemical synthesis route, represented by Dow Chemical Company in the United States. In the process of chemical production of tetrachloropyridine carboxylic acid, a large amount of strong acids and bases are often involved, and the process conditions are complex and difficult to control, the product yield is low, the by-products are difficult to handle, and the damage to the environment is large. With the development of technologies such as protein engineering and genetic engineering, efficient enzyme catalysis methods are more conducive to the development of a sustainable green economy. As a green and natural catalyst, the catalytic reaction of enzymes often has many advantages such as few by-products, green environmental protection, mild reaction conditions, high conversion rate, and high stereoselectivity. Nitrilase can convert nitriles into carboxylic acids or amides in a one-step process, with the only by-product being ammonia gas, and the overall reaction atom utilization rate is extremely high.

[0003] However, wild-type enzymes often have low activity and are unstable under high temperature conditions. Enzyme inactivation will lead to low overall yield, which is also the main reason restricting its use as an industrial large-scale production. The patent with application number 202311747860.7 provides a mutant strain that is active on the substrate, but the mutant strain is easily inactivated under high temperature conditions. Continuous reaction under high temperature conditions can accelerate the movement and collision frequency between atoms, making it easier to reach the transition state of molecular reactions, and at the same time increase the percentage of activated molecules and increase the reaction rate. In industrial production, reducing costs and increasing yields are factors that companies must consider. Therefore, increasing the thermal stability and activity of nitrilase, increasing output under high temperature conditions, and improving substrate conversion rates are crucial for the enzyme in the field of biocatalytic applications. Summary of the invention

[0004] The present invention is conducted to solve the above-mentioned problems. In view of the fact that nitrilase is easily inactivated at high temperature, a modification strategy of nitrilase is designed. By introducing non-natural amino acids, nitrilase is modified. A nitrilase mutant containing non-natural amino acids, a coding gene, a recombinant vector, a recombinant strain and applications thereof are provided.

[0005] In a first aspect of the present invention, a nitrilase mutant containing a non-natural amino acid is provided, characterized in that the nitrilase mutant comprises one of the following mutations:

[0006] (1) mutating the proline at position 340 of the amino acid sequence shown in SEQ ID NO.1 to L-(7-hydroxycoumarin-4-yl)ethylglycine;

[0007] (2) mutating the proline at position 367 of the amino acid sequence shown in SEQ ID NO.2 to L-(7-hydroxycoumarin-4-yl)ethylglycine;

[0008] (3) The proline at position 314 of the amino acid sequence shown in SEQ ID NO. 3 is mutated to L-(7-hydroxycoumarin-4-yl)ethylglycine.

[0009] In the examples, the nucleotide sequence of the nitrilase mutant obtained by mutation method (1) is shown in SEQ ID NO.4.

[0010] In the example, the nucleotide sequence of the nitrilase mutant obtained by mutation method (2) is shown in SEQ ID NO.5.

[0011] In the example, the nucleotide sequence of the nitrilase mutant obtained by mutation method (3) is shown in SEQ ID NO.6.

[0012] Traditional enzyme engineering mostly changes the functional properties of enzymes by replacing a certain site with 20 kinds of amino acids. The present invention discloses a method for modifying nitrilase by introducing a non-natural amino acid strategy, which greatly improves its enzyme activity and thermal stability, and optimizes its fermentation process.

[0013] Compared with the original strain, the enzyme activity of the constructed nitrilase mutant NIT-P340G-Hco increased by 15.84 times. In addition, the optimal reaction temperature of the nitrilase mutant NIT-P340G-Hco increased by 5°C compared with the original template, and it was able to completely convert 100mM substrate within 8h.

[0014] Furthermore, the nitrilase mutant NIT-P340G-Hco also has improved enzyme activity when catalyzing other pesticide intermediates, thereby expanding the substrate spectrum. The nitrilase mutant NIT-P340G-Hco catalyzes the reaction at a higher temperature, increasing the reaction rate and meeting the needs of industrial production.

[0015] The present invention first mutates the proline at position 340 of the amino acid sequence shown in SEQ ID NO.1 into glycine to prepare a nitrilase mutant NIT-P340G, the amino acid sequence of which is shown in SEQ ID NO.7. Then, by using a site-specific method and exogenously evolved orthogonal aminoacyl-tRNA synthetase, the amino acid sequence at position 340 of the amino acid sequence shown in SEQ ID NO.1 is mutated into L-(7-hydroxycoumarin-4-yl)ethylglycine, thereby accurately introducing a non-natural amino acid into an enzyme molecule to prepare a nitrilase mutant NIT-P340G-Hco, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0016] The second aspect of the present invention provides a gene encoding the nitrilase mutant according to the first aspect.

[0017] The third aspect of the present invention provides a recombinant vector comprising the coding gene as described in the second aspect.

[0018] The expression host of the recombinant vector is Escherichia coli BL21 (DE3).

[0019] The fourth aspect of the present invention provides a recombinant strain comprising the encoding gene as described in the second aspect.

[0020] The fifth aspect of the present invention provides an application of the nitrilase mutant as described in the first aspect, the encoding gene as described in the second aspect, the recombinant vector as described in the third aspect, or the recombinant strain as described in the fourth aspect, characterized in that the nitrilase mutant is used to synthesize 3,4,5,6-tetrachloropyridinecarboxylic acid.

[0021] In some embodiments, the nitrilase mutant is used as a catalyst and 3,4,5,6-tetrachloropyridinecarbonitrile is used as a substrate, and the reaction is carried out at 45 to 55° C. to prepare 3,4,5,6-tetrachloropicolinic acid.

[0022] In some embodiments, the pH of the reaction system is 7 to 8.5, and the reaction system includes an additive, wherein the additive includes at least Mg 2+ , Fe 2+ , Mn 2+ 、Zn 2+ One of them.

[0023] In some embodiments, the nitrilase mutant is used as a catalyst in the form of an enzyme or a recombinant bacterium.

[0024] When the catalyst is in the form of bacterial cells, the amount of the catalyst used is 0.1 to 20 gDCW / L based on the total dry weight of the recombinant bacterial cells.

[0025] In some embodiments, the substrate concentration is 10-200 mM, and the amount of the nitrilase mutant used is 0.1-20 g DCW / L based on the total dry weight of wet cells expressing the nitrilase mutant.

[0026] The sixth aspect of the present invention provides an application of the nitrilase mutant as described in the first aspect, or the encoding gene as described in the second aspect, or the recombinant vector as described in the third aspect, or the recombinant strain as described in the fourth aspect, characterized in that the nitrilase mutant is used to catalyze 1-cyanocyclohexylacetonitrile or mandelic acid.

[0027] In the embodiments, the nitrilase mutant can also be used to catalyze different pesticide intermediates, effectively expanding the substrate spectrum of the nitrilase.

[0028] By implementing the above technical solution, the present invention has the following beneficial effects:

[0029] The present invention establishes a high-throughput screening model and constructs a large-capacity mutant library, and successfully screens out a nitrilase mutant NIT-P340G-Hco with high activity and stability, and its whole-cell enzyme activity is 15.84 times that of the original nitrilase.

[0030] The present invention introduces non-natural amino acids to modify the enzyme based on the starting enzyme, thereby improving the enzyme stability and also enhancing the enzyme activity. The nitrilase mutant NIT-P340G-Hco prepared by the present invention can achieve a conversion rate of 100% when catalyzing 100mM substrate.

[0031] The present invention also carries out high-density fermentation of the nitrilase mutant NIT-P340G-Hco in a 5L fermenter by feeding, optimizes the fermentation parameters, finally obtains high-activity bacteria, and realizes high-density fermentation in a 5L fermenter.

[0032] The nitrilase mutant NIT-P340G-Hco provided by the invention can be used for biocatalytic synthesis of 3,4,5,6-tetrachloropyridinecarboxylic acid, has high biocatalytic activity, mild reaction conditions, high yield, simple separation and purification, and has great industrial application prospects.

[0033] The enzyme modification strategy of introducing non-natural amino acids provided by the present invention can not only be used to expand the substrate spectrum of nitrilase, but also has a beneficial effect on the efficient catalysis of pharmaceutical intermediates such as 1-cyanocyclohexylacetonitrile or mandelic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a chemical synthesis method and enzyme catalytic synthesis method for synthesizing pyridine carboxylic acid green pesticides using 3,4,5,6-tetrachloropyridine carboxylic acid as raw material;

[0035] Figure 2 This is the RMSF change diagram of nitrilase under different temperature conditions simulated by molecular dynamics;

[0036] Figure 3 It is a gel image of pure enzyme protein of nitrilase mutants NIT-P340G and NIT-P340G-Hco in Example 4;

[0037] Figure 4 The effect of different temperatures on the activity of the nitrilase mutant NIT-P340G-Hco in Example 5;

[0038] Figure 5 The effect of different pH values ​​on the activity of the nitrilase mutant NIT-P340G-Hco in Example 5;

[0039] Figure 6 is the effect of different additives on the conversion of the nitrilase mutant NIT-P340G-Hco in Example 5;

[0040] Figure 7 This is the enzyme activity diagram of the nitrilase mutant NIT-P340G-Hco produced by high-density fermentation in a 5L fermenter fed-batch in Example 6;

[0041] Figure 8 This is a time course diagram of the preparation of 3,4,5,6-tetrachloropicolinic acid from 3,4,5,6-tetrachloropyridinecarbonitrile using the nitrilase mutant NIT-P340G-Hco in Example 7. DETAILED DESCRIPTION

[0042] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is described in detail below in conjunction with embodiments and drawings.

[0043] The molecular biology experiments in the embodiments, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., are mainly carried out with reference to "Molecular Cloning Experiment Guide" (3rd edition, edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0044] LB medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 2 g / L agar powder (solid culture medium), solvent is water, pH is natural.

[0045] Example 1: Construction and screening of nitrilase mutant library

[0046] In the embodiment, the preparation of the nitrilase mutant library is achieved by 4 rounds of high-throughput screening. The recombinant E. coli BL21 (DE3) / pET28a (+) -NIT containing the nitrilase gene NIT from Xanthomonadales bacterium, the nucleotide sequence of which is shown in SEQ ID NO.1, is constructed as a control strain WT, the recombinant plasmid pET28a (+) -NIT containing the nitrilase gene NIT is extracted from the recombinant E. coli, and the mutation primers are designed as shown in Table 1.

[0047] In the example, E. coli BL21 (DE3) / pET28a (+) -NIT was used as a template, and 7 residues at sites where the RMSF values ​​of the enzyme changed greatly under different temperature conditions and where there was interaction at the interface between the A chain and the B chain were selected. These selected residues were generally located in the loop region of the enzyme and were far away from the center of the active pocket of the enzyme (Arg-301, Leu-316, Arg-317, Asp-322, Phe-327, Pro-340, Asp-349). At the same time, in order to make it easier to incorporate non-natural amino acids later, amino acid sites close to the C-terminus of the enzyme are more preferred. The above sites were first subjected to alanine scanning, and the beneficial mutation sites were subjected to saturation mutation PCR, and Escherichia coli BL21 (DE3) was transformed, and high-throughput screening was performed in a 96-well plate. Through primary screening by an ELISA instrument and rescreening of liquid phase results, beneficial nitrilase mutants pET28a (+) -NIT-L316A (i.e., the isoleucine at position 316 of the amino acid shown in SEQ ID NO.1 was mutated to alanine), pET28a (+) -NIT-P340G (i.e., the proline at position 340 of the amino acid shown in SEQ ID NO.1 was mutated to glycine) and pET28a (+) -NIT-P340A (i.e., the proline at position 340 of the amino acid shown in SEQ ID NO.1 was mutated to alanine) were obtained. Finally, the above beneficial mutants were iteratively mutated, and the effects of the double mutants pET28a(+)-NIT-L316A-P340A and pET28a(+)-NIT-L316A-P340G were verified.

[0048] Table 1 Nitrilase mutation primer design

[0049]

[0050] The specific steps of PCR were as follows: PCR reaction system (50 μL): 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 25 μL 2× Phanta buffer, 1 μL dNTP mixture (10 μM each), 1 μL plasmid template, 0.5 μL DNA polymerase and 18.5 μL ultrapure water.

[0051] The PCR program set according to the Phanta Super-Fidelity DNA polymerase manual was as follows: 95°C pre-denaturation for 5 min, then 32 cycles (95°C denaturation for 15 s, 58°C annealing for 30 s, 72°C extension for 6 min 30 s), 72°C final extension for 10 min, and 16°C incubation. The obtained recombinant plasmid was transferred into E. coli BL21 (DE3) competent cells, and the clones were cultured at 37°C for 12 h. Then the clones were picked out and transferred to 8 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 180 rpm for 10 h.

[0052] The obtained mutants were screened for dominant mutants. The high-throughput screening method was as follows: a single colony on the transformation plate was picked and placed in a 96-well cell culture plate (1 mL LB liquid culture medium containing 50 mg / mL kanamycin was pre-added to each well), and cultured at 37°C, 180 rpm constant temperature shaker for 12 hours. Then take it out, add 10 μL IPTG (final concentration of 0.1 mM / L), and place it at 28°C, 180 rpm constant temperature shaker for induction for 12 hours. Centrifuge at 3000 rpm for 10 minutes. After centrifugation, the bacteria were resuspended with 1 mL 0.9% saline and transferred to a 1.5 mL Ep tube, centrifuged again, and the supernatant was removed. The initial screening reaction was carried out in a 1.5 mL Ep tube.

[0053] The reaction system is: incubate for 2 hours at a temperature of 55°C and a speed of 1000 rpm. Then add 25 μl of DMSO-PBS solution containing 100 mM substrate 3,4,5,6-tetrachloropyridine nitrile to a 96-well plate, and fully oscillate to resuspend the bacteria. Then react for 2 hours at a temperature of 37°C and a speed of 1000 rpm. Then stop by adding 15 μl HCL (6M). The reaction mixture was centrifuged at 4000 rpm for 15 minutes, and then 20 μl of the supernatant was converted to a black fluorescent microtiter plate with 150 μl of fluorescent colorimetric solution (0.1 g o-phthalaldehyde, 50 μl of mercaptoethanol and 20 ml of anhydrous ethanol mixed and diluted 10 times). The reaction was carried out at 37°C for 30 minutes, and the fluorescence intensity was detected by an enzyme reader, with an excitation wavelength of 412 nm and an emission wavelength of 467 nm. The absorbance value was higher than that of the control group and was considered to be a beneficial mutant for sequencing.

[0054] Beneficial mutants were rescreened. The rescreening method was as follows: single colonies with positive mutation potential on the transformation plate were picked and inoculated into 50 mL of LB liquid medium (containing 50 μg / mL of kanamycin resistance), and cultured at 37 ° C and 180 rpm on a shaker until OD 600When the pH reaches 0.6-0.8, add 20 μL of 0.1 mM / mL IPTG. Induce for 12 hours on a shaker at 28°C and 180 rpm. Collect the bacteria by centrifugation. React in a 50 mL Erlenmeyer flask. Reaction system: 0.1 g wet bacteria, 10 mL of pH 8.0 PB buffer, 100 mM substrate 3,4,5,6-tetrachloropyridine nitrile, react for 5 hours at 37°C and 800 rpm. Centrifuge 1 mL of the reaction solution, take the supernatant, filter with a 0.22 μm microfiltration membrane, and perform high performance liquid chromatography (HPLC) detection.

[0055] Further, due to the limitations of conventional amino acid mutation libraries, and the desired effect has not been achieved through iterative mutation. The side chain groups of non-natural amino acids have some special physicochemical properties and biological functions. The side chain groups of L-(7-hydroxycoumarin-4-yl)ethylglycine Hco are larger, which can increase hydrogen bonds and hydrophobic interactions, thereby reducing the RMSF value fluctuations of the enzyme in the kinetic simulation at different temperatures, and theoretically can improve the stability of the enzyme. In the embodiment, an attempt was made to introduce L-(7-hydroxycoumarin-4-yl)ethylglycine Hco to prepare the nitrilase mutant strain NIT-P340G-Hco.

[0056] First, the amber codons (UAG) of the used Escherichia coli strains are all mutated to ochre codons (UAA), and the release factor RF1 gene that recognizes UAG and UAA codons is knocked out, which removes the inhibitory effect on amber codons. When introducing an exogenous system, UAG is specified as a special codon so that it will not form a truncated protein during translation. Using the nitrilase mutant pET28a (+) -NIT-P340G prepared in Example 1 as a template, the 340-site codon is replaced with an amber codon for subsequent incorporation of unnatural amino acids Hco. By a site-specific method, Hco is accurately introduced into the enzyme molecule using an orthogonal aminoacyl tRNA synthetase (aaRS / tRNA pair) of exogenous evolution. Orthogonal aaRS / tRNA pairs refer to that the aaRS introduced can effectively bind to the homologous tRNA of the target UAA and respond to a specific genetic codon without cross-reacting with endogenous aaRS / tRNA pairs. Mutation was performed on the basis of the original aaRS, and the original amino acid sequence of aaRS was shown in SEQ ID NO.8, and the aaRS gene incorporated with Hco was obtained, and the amino acid sequence of the aaRS gene incorporated with Hco was shown in SEQ ID NO.9, and then the aaRS gene incorporated with Hco was inserted into the pEVOL plasmid to construct a pEVOL-aaRS-Hco plasmid. The constructed pEVOL-aaRS-Hco plasmid and pET28a(+)-NIT-P340G plasmid were co-transformed and introduced into E. coli BL21 (DE3) competent cells.

[0057] The operation of electroporation transformation is as follows: take out the prepared E. coli BL21 (DE3) electroporation competent cells from the -80°C ultra-low temperature refrigerator and place them on ice for 5 minutes. The plasmid pEVOL-aaRS-Hco (100-200 ng) and the mutated plasmid pET28a(+)-NIT-P340G (100-200 ng) were mixed evenly and transferred to the competent cells, and gently blown with a pipette to mix; then, the mixed cells were added to a 2 mm electroporation cup after an ice bath, and electroporated in Ec2, 2.5KV mode; after the electroporation, 1 mL of ice LB liquid culture medium was quickly added to the electroporation cup, and it was transferred to a 2 mL sterile centrifuge tube, and resuscitated and cultured at 30°C, 150 rpm for 3 hours; an appropriate amount of culture was spread on LB solid plate culture medium (containing a final concentration of 50 μg / mL kanamycin and 25 μg / mL chloramphenicol), and placed in a 30°C incubator for static culture overnight to obtain the nitrilase mutant strain NIT-P340G-Hco.

[0058] Since Hco is a non-natural amino acid with self-fluorescence (excitation wavelength 360nm, emission wavelength 455nm), the mutant incorporating Hco can make the protein self-fluorescent, based on which it can be verified whether the nitrilase mutant strain NIT-P340G-Hco is successfully constructed.

[0059] Example 2: Inducible expression of nitrilase mutants

[0060] The starting strain WT in Example 1 and the strains NIT-L316A, NIT-P340G, NIT-P340A, NIT-L316A-P340A, NIT-L316A-P340G and NIT-P340G-Hco of the nitrilase mutants screened in Example 1 were inoculated into LB medium (containing 34 μg / mL chloramphenicol and 50 μg / mL kanamycin) and cultured in a shaking incubator at 34°C, 180 rpm for 10 to 12 hours. Then, the inoculum was transferred to GMML medium (containing 34 μg / mL chloramphenicol and 50 μg / mL kanamycin) at a 5% inoculum and cultured at 34°C until OD 600 When the OD value reached 0.6, the inducer L-(+)-arabinose was added to a final concentration of 0.2% (w / v); the culture was continued at 34°C until the OD value reached 0.6. 600When the pH is 0.8, the target protein expression is induced by adding the inducer tetracycline (final concentration 30 ng / mL) and the corresponding non-natural amino acid (final concentration 1 mM). After 36-40 hours of induction expression at 34°C, the corresponding wet bacterial cells are obtained by centrifugation at 4°C and 8000 rpm for 10 minutes. The cells obtained above produce the corresponding nitrilase, which can be used for the preparation of pure protein enzyme solution, and can also be used for crude enzyme solution to catalyze the substrate 3,4,5,6-tetrachloropyridine nitrile to synthesize 3,4,5,6-tetrachloropyridine carboxylic acid.

[0061] Example 3: Determination of Enzymatic Activity of Original Nitrilase and Nitrilase Mutants

[0062] In this example, the enzyme activities of the original nitrilase NIT expressed by the wet micrococcus obtained in Example 2 and the nitrilase mutants NIT-L316A, NIT-P340G, NIT-P340A, NIT-L316A-P340A, NIT-L316A-P340G and NIT-P340G-Hco were detected.

[0063] Enzyme activity detection standard conditions: 10mL reaction system: 100mM 3,4,5,6-tetrachloropyridine carbonitrile substrate, pH8.0200mMPB buffer, 100μL of the above-obtained nitrilase pure enzyme solution, react for a certain time at 37°C and 800rpm. Take a sample, filter the supernatant obtained by centrifugation through a 0.22μm filter membrane, and then perform HPLC to determine the yield. At 37°C, the amount of enzyme required to catalyze the substrate to generate 1μmol of product per minute is defined as one activity unit, recorded as U.

[0064] Liquid phase detection conditions: Thermo C18 chromatographic column (250×4.6 mm; mobile phase: acetonitrile: water: 85% phosphoric acid = 60:40:0.3; flow rate: 1.0 mL / min; column temperature: 40°C; injection volume: 10 μL; UV detection wavelength 230 nm.

[0065] The protein concentration was determined using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotechnology Development Co., Ltd., Nanjing). The original strain NIT and multiple mutants obtained in Example 1, NIT-L316A (i.e., the isoleucine at position 316 of the amino acid shown in SEQ ID NO.1 mutated to alanine), NIT-P340G (i.e., the proline at position 340 of the amino acid shown in SEQ ID NO.1 mutated to glycine), NIT-P340A (i.e., the proline at position 340 of the amino acid shown in SEQ ID NO.1 mutated to alanine), NIT-L316A-P340G (i.e., the isoleucine at position 316 of the amino acid sequence shown in SEQ ID NO.1 mutated to alanine and the proline at position 340 mutated to glycine), NIT-L316A-P340A (i.e., the isoleucine at position 316 of the amino acid sequence shown in SEQ ID NO.1 mutated to alanine and the proline at position 340 mutated to alanine), and NIT-P340G-Hco (the proline at position 340 of the amino acid sequence mutated to the non-natural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine). The enzyme activity data are shown in Table 3.

[0066] Table 3 Catalytic activity of nitrilase and its mutants

[0067] Mutants Enzyme activity (U / g) NIT 5.60 NIT-L316A 18.65 NIT-P340G 40.52 NIT-P340A 20.84 NIT-L316A-P340A 31.56 NIT-L316A-P340G 36.84 NIT-P340G-Hco 88.68

[0068] The results in Table 3 show that the enzyme activity of the optimal nitrilase mutant NIT-P340G-Hco is 15.84 times that of the original NIT.

[0069] Example 4: Purification of the original nitrilase and its nitrilase mutants NIT-P340G and NIT-P340G-Hco

[0070] The nitrilase mutants NIT-P340G and NIT-P340G-Hco obtained in Example 1 were used to obtain mutant wet cells according to the method described in Example 2. The cells were collected by centrifugation at 8000 rpm and 4°C for 10 min, and washed twice with 0.9% (w / v) saline. The total amount of wet cells was 100 g / L, and resuspended in pH 8.0, 20 mM PBS buffer, and ultrasonically disrupted on an ice-water mixture for 5 min. The ultrasonic disruption conditions were: power of 250 W, disruption for 1.0 s, and pause for 2.0 s to obtain a mutant crude enzyme solution. The supernatant was collected by centrifugation at 8000 rpm and 4°C for 30 min, and after microfiltration through a 0.45 μm membrane, the mutant protein was purified using a Ni affinity column.

[0071] The mutant protein was purified using a nickel affinity column (1.6×10 cm, Bio-Rad, USA), and the specific operation was as follows: ① Pre-balance with buffer A (pH 8.0, 20 mM PBS buffer containing 0.3 M NaCl and 20 mM imidazole). ② Wash away unbound impurities with buffer A at a flow rate of 1.0 mL / min until the conductivity is stable. Then ③ Elute the target protein with buffer B (pH 8.0, 20 mM PBS buffer containing 0.3 M NaCl and 500 mM imidazole). The collected eluate was dialyzed against 20 mM PBS buffer (pH 7.0) overnight. All purification steps were carried out at 4°C. Figure 3 As shown, Figure 3 These are the protein gel images of the two best mutant pure enzymes of nitrilase. M: standard protein molecule; Lane 1: original NIT pure enzyme; Lane 2: NIT-P340G pure enzyme; Lane 3: NIT-P340G-Hco pure enzyme. Protein size was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expression of the target enzyme in the mutant strain was not much different from that in the original strain. This indicates that the increase in the enzyme activity of the mutant is not due to the increase in the expression of the enzyme itself, but because of the increase in the specific activity of the enzyme.

[0072] Example 5: Optimization of the system for synthesizing 3,4,5,6-tetrachloropicolinic acid catalyzed by nitrilase

[0073] The cells of the nitrilase E. coli BL21(DE3) / pET28a(+)-NIT mutant NIT-P340G-Hco prepared in Example 2 were used as catalysts.

[0074] Since different reaction temperatures have different effects on the rate of catalytic production of 3,4,5,6-tetrachloropyridine carboxylic acid, the atomic collision rate will be faster under high temperature conditions, but the enzyme will also be at risk of inactivation. Therefore, five temperature gradients (40°C, 45°C, 50°C, 55°C, and 60°C) were considered.

[0075] The initial reaction system was: 100 mM 3,4,5,6-tetrachloropyridine carbonitrile substrate, pH 8.0, 200 mM PB buffer, 10 g / L wet bacteria, reacted at 37°C, 800 rpm for 2 h, and samples were taken for processing and HPLC analysis.

[0076] like Figure 4 visible, Figure 4The effect of different temperature conditions on the activity of the nitrilase mutant NIT-P340G-Hco is that when the reaction temperature is between 40℃ and 50℃, the relative enzyme activity shows an upward trend, and the conversion rate reaches the highest at 50℃, and 53.7mM of product can be generated in 2h reaction. When the temperature continues to rise, the relative enzyme activity begins to decrease, and the conversion rate decreases at the same time. When the reaction temperature rises to 60℃, the relative enzyme activity is less than 60%. After testing, the generated substrate is only 19.87mM.

[0077] Furthermore, in the above resting cell reaction system, a buffer with a pH range of 5-10 was selected to investigate the effect of different pH on bacterial catalysis. The buffers used were as follows: citric acid buffer: pH 5.0, 5.5, 6.0; PBS buffer: pH 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5; PB buffer: pH 7.0, 7.5, 8.0, 8.5; borax boric acid buffer: pH 7.5, 8.0, 8.5; glycine-sodium hydroxide buffer: pH 9.0, 9.5, 10.0. After 2 hours of reaction, samples were taken for processing and HPLC analysis to compare the yield under different pH conditions.

[0078] like Figure 5 It can be seen that the catalytic efficiency of the nitrilase mutant cells for the substrate 3,4,5,6-tetrachloropyridine carbonitrile is better in the pH range of 7.0-8.5, reaching the highest at pH 8.0. When the pH is greater than 9.0 or less than 7.0, the yield decreases significantly. Therefore, the optimal pH for the catalysis of the nitrilase mutant cells is the PB buffer of pH 8.0.

[0079] Furthermore, in the above resting cell reaction system, adding different additives to the reaction system will affect the enzyme activity. The additives used are as follows: Ca 2+ Mg 2+ , K + , Fe 2+ , Mn 2+ 、Ni 2+ 、Co 2+ , Ba 2+ Cr 2+ , Cu 2+ 、Zn 2+ Sn 2+ NH4 + and EDTA to investigate the effects of different additives on bacterial catalysis. After 2 h of reaction, samples were taken and analyzed by HPLC. Figure 6 As shown, Mg was added to the reaction system 2+It can significantly increase the activity of nitrilase, and the relative enzyme activity can reach 124.24%. Magnesium ions may combine with amino acids near the active pocket to form chelates, thereby increasing enzyme activity. 2+ This will completely inactivate the enzyme.

[0080] Example 6: Fed-batch high-density fermentation of the nitrilase mutant NIT-P340G-Hco in a 5 L fermenter

[0081] In a 5L fermentation system, the fermentation basal medium was configured as follows: peptone 15g / L, yeast powder 12g / L, glycerol 15g / L, sodium chloride 10g / L, ammonium sulfate 5g / L, dipotassium hydrogen phosphate 2.28g / L, potassium dihydrogen phosphate 1.36g / L, magnesium sulfate heptahydrate 0.375g / L, and the liquid volume was 2L (including defoaming agent 1ml / L).

[0082] The feed medium was configured as follows: peptone 75 g / L, yeast powder 50 g / L, glycerol 500 g / L, citric acid monohydrate 3 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 5 g / L, and the liquid volume was 500 ml.

[0083] The recombinant genetically engineered bacteria containing the nitrilase mutant NIT-P340G-Hco gene were inoculated into LB liquid culture medium (containing 34μg / mL chloramphenicol and 50μg / mL kanamycin), cultured at 37°C for 12h, and inoculated into fresh LB liquid culture medium containing a final concentration of 50μg / mL kanamycin at a volume concentration of 2.0%, and cultured at 37°C and 180rpm for 6-8h as the secondary seed liquid. The secondary seed liquid was inoculated into a sterilized fermentation tank, and the dissolved oxygen was linked to the rotation speed. After the dissolved oxygen dropped to the lowest point and stabilized for 20min, the inducer L-(+)-arabinose, tetracycline and the corresponding non-natural amino acids were added for protein expression. The optimal dissolved oxygen for the growth of Escherichia coli is between 20% and 30%, so the dissolved oxygen is controlled at 25% after induction, and the feeding method is dissolved oxygen linked feeding. After about 16h after induction, samples were taken every two hours to detect OD 600 , cultured at 28℃ for about 36-40h 600 After it starts to drop, take the culture and centrifuge it at 4℃ and 8000rpm, collect the precipitate to obtain wet bacteria. Figure 7 ,like Figure 7 As shown, M: standard protein molecule; lane 1: shake flask fermentation control nitrilase supernatant; lane 2: shake flask control nitrilase precipitate; lane 3: 5L fermentation tank bacterial nitrilase supernatant; lane 4: 5L fermentation tank bacterial nitrilase precipitate.

[0084] Example 7: The nitrilase mutant NIT-P340G-Hco catalyzes 3,4,5,6-tetrachloropyridinecarbonitrile to produce 3,4,5,6-tetrachloropicolinic acid.

[0085] According to the description of Example 6, the wet bacterial cells of the nitrilase mutant NIT-P340G-Hco were obtained by fermentation in a 5L fermenter. In a 10mL reaction system, the bacterial cells were first resuspended with a pH 8.0, 200mM PB buffer, 0.1g of the wet bacterial cells were added to the transformation system, the feed amount of 3,4,5,6-tetrachloropyridinecarbonitrile was 100mM, and the transformation system was formed with a pH 8.0, 200mM PBS buffer as the reaction medium, and the reaction was carried out at 37°C and 800rpm. Figure 8 As shown, the final substrate conversion rate was 100% in 8 h.

[0086] Example 8: Expanding the substrate spectrum of the nitrilase mutant NIT-P340G-Hco

[0087] Nitrilase can react with substrates mainly due to the formation of halogen bonds between the key residues in the active pocket and the chlorine-containing substrates. Therefore, nitrilase also has certain activity towards similar substrates containing halogen atoms.

[0088] In the example, tetrachloropyridinecarbonitrile was first used as the lead compound, similar compounds in the PubChem library were searched, and six analogues with potential biological activity (3,6-dichloropyridinecarbonitrile, 2-cyanopyridine, 2,6-dichloropyridine-3,5-dicarbonitrile, 3-cyanopyridine, 5-bromo-3-fluoropyridinecarbonitrile and 2-chloro-6-cyanopyridine) were screened out using the FEP (free energy perturbation) calculation method.

[0089] The experimental method is as follows: resuspend 0.1g of induced wet bacteria in 10mL of buffer containing 50mM, pH 8.0 sodium phosphate to prepare a 10g / L bacterial suspension. Add 20mM of the test chemical as a substrate to a beaker, place it in a constant temperature water bath, and react at 37°C, 800r for 2h. Centrifuge the reaction solution and collect the supernatant. After appropriate dilution, pass the membrane and analyze the product content by high-performance liquid chromatography. The liquid chromatography detection method is the same as that of 3,4,5,6-tetrachloropyridine carboxylic acid. In this experiment, 1 enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the generation of 1μM of the corresponding product per minute, and the specific enzyme activity (U / g) is defined as the ratio of unit enzyme activity to unit mass.

[0090] Table 4 Catalytic activity of the original type and nitrilase mutant NIT-P340G-Hco on different substrates

[0091]

[0092] Example 9: Application of nitrilase mutants containing unnatural amino acids in the production of gabapentin (1-(aminomethyl)cyclohexaneacetic acid)

[0093] In order to verify the universality of non-natural amino acids in the strategy of nitrilase transformation, the above transformation strategy was applied to nitrilase with different catalytic substrates, and the change in enzyme activity between the mutant and the original template was determined by liquid phase detection. In this example, the nitrilase from the agile acidophilus was selected, and its amino acid sequence is shown in SEQ ID NO.2. The non-natural amino acid Hco was introduced into the nitrilase from the agile acidophilus by the method described in Example 1, and the proline at position 367 of the amino acid sequence SEQ IDNO.2 was mutated to L-(7-hydroxycoumarin-4-yl)ethylglycine, and the nucleotide sequence after mutation was shown in SEQ ID NO.5. Whether the construction was successful was detected by fluorescence. Shake flask amplification was then performed, and wet cells were obtained by centrifugation. In a 10mL reaction system, the cells were first resuspended with pH 8.0, 200mM PB buffer, 0.1g of wet cells were added to the transformation system, and then the substrate 1-cyanocyclohexylacetonitrile was added, and the reaction was carried out at 37°C and 800rpm. The enzyme catalytic activity table is shown in Table 5:

[0094] Table 5 Catalytic activity of nitrilase mutants for 1-cyanocyclohexylacetonitrile

[0095] Enzyme source Substrate Original enzyme activity (U / g) Mutant enzyme activity (U / g) Acidovorax agile 1-Cyanocyclohexylacetonitrile 350.42 460.52

[0096] Example 10: Application of nitrilase mutants containing unnatural amino acids in the production of mandelic acid

[0097] In this example, a nitrilase from Bradyrhizobium was selected, and its amino acid sequence is shown in SEQ ID NO.3. The non-natural amino acid Hco was introduced into the nitrilase from Bradyrhizobium using the method described in Example 1, and the proline at position 314 of the amino acid sequence SEQID NO.3 was mutated to L-(7-hydroxycoumarin-4-yl)ethylglycine. The nucleotide sequence after mutation is shown in SEQ ID NO.6. Fluorescence detection was used to detect whether the construction was successful. Shake flask amplification was then performed, and wet cells were obtained by centrifugation. In a 10mL reaction system, the cells were first resuspended with pH 8.0, 200mM PB buffer, 0.1g of wet cells were added to the transformation system, and then the substrate mandelonitrile was added, and the reaction was carried out at 37°C and 800rpm. The enzyme catalytic activity table is shown in Table 6:

[0098] Table 6 Catalytic activity of nitrilase mutants for mandelonitrile

[0099] Enzyme source Substrate Original enzyme activity (U / g) Mutant enzyme activity (U / g) Bradyrhizobium Mandelonitrile 85.62 254.15

[0100] The above-mentioned embodiments are preferred examples of the present invention and are not intended to limit the protection scope of the present invention.

Claims

1. A nitrilase mutant containing an unnatural amino acid, characterized in that: The nitrilase mutant comprises one of the following mutation modes: The proline at position 340 of the amino acid sequence shown in SEQ ID NO.1 is mutated to L-(7-hydroxycoumarin-4-yl)ethylglycine; The proline at position 367 of the amino acid sequence shown in SEQ ID NO.6 is mutated to L-(7-hydroxycoumarin-4-yl)ethylglycine; The proline at position 314 of the amino acid sequence shown in SEQ ID NO.8 is mutated to L-(7-hydroxycoumarin-4-yl)ethylglycine.

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

3. A recombinant vector comprising the coding gene according to claim 2.

4. A recombinant strain comprising the coding gene according to claim 3.

5. Use of the nitrilase mutant according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 3, or the recombinant strain according to claim 4, characterized in that: The nitrilase mutant is used to synthesize 3,4,5,6-tetrachloropicolinic acid.

6. The use according to claim 5, characterized in that: The nitrilase mutant is used as a catalyst and 3,4,5,6-tetrachloropyridinecarbonitrile is used as a substrate. The reaction is carried out at 45-55° C. to prepare 3,4,5,6-tetrachloropyridinecarboxylic acid.

7. The use according to claim 6, characterized in that: The pH of the reaction system is 7-8.

5.

8. The use according to claim 7, characterized in that: The reaction system includes additives, and the additives include at least Mg 2+ , Fe 2+ , Mn 2+ 、Zn 2+ One of them.

9. The use according to claim 8, characterized in that: The nitrilase mutant is used as a catalyst in the form of enzyme or recombinant bacteria.

10. Use of the nitrilase mutant according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 3, or the recombinant strain according to claim 4, characterized in that: The nitrilase mutant was used to catalyze 1-cyanocyclohexylacetonitrile or mandelic acid.

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