CN114277022B - Nitrile hydratase mutant with high activity and high thermal stability - Google Patents

Abstract

The invention discloses a nitrile hydratase mutant with high activity and high thermal stability, belonging to the technical field of bioengineering. The invention mutates the 50 th glutamic acid on the beta subunit in the amino acid motif of the nitrile hydratase into leucine to obtain mutant E50L, and the specific enzyme activity of the mutant E50L catalytic nicotinonitrile provided by the invention is improved to 353.53 +/-22.34U/mg, which is improved by 5.83 times compared with a wild type. While the enzyme activity is improved, the mutant keeps the advantage of good thermal stability of Pt NHase and has a certain improvement on tolerance. The successful construction of the mutant with higher enzyme activity, good heat stability and substrate tolerance enhances the confidence of researchers in improving the enzyme activity by modifying the protein by a rational design method, and further promotes the subsequent industrial production and application of Pt NHase.

Description

Nitrile hydratase mutant with high activity and high thermal stability

Technical Field

The invention relates to a nitrile hydratase mutant with high activity and high thermal stability, belonging to the technical field of bioengineering.

Background

The amide products have high industrial value, wherein the most representative products are nicotinamide, which is vitamin B ₃ Can affect cell differentiation and aging, and has antioxidant and antiinflammatory effects. In the dermatological field, nicotinamide can treat diseases such as brown skin diseases, acne, psoriasis, pigmentation disorders, etc.; the cosmetic has effects of relieving skin aging and brightening skin by adding appropriate amount of nicotinamide. The traditional industrial production method of amide products is chemical synthesis, but the method has the defects of harsh reaction conditions, low conversion rate and high byproduct yield. With the discovery and research of nitrile hydratase, a biocatalysis method with mild conditions and strong selectivity is gradually replacing a chemical synthesis method, and is successfully applied to the industrial production of amide products such as nicotinamide, acrylamide, 5-cyanovaleramide and the like.

Nitrile hydratase (nitrile hydratase, NHase for short, EC 4.2.1.84) is a metalloenzyme that hydrates nitrile substrates and converts them to the corresponding amide products. Since 1985, the first generation strain N-774 has been used for the bioconversion of acrylonitrile to acrylamide, which is also the first successful case of biotechnology for the manufacture of commercial chemicals. Currently, mitsubishi corporation has been able to achieve annual yields of acrylamide in excess of 200000 tons.

Nitrile hydratase has been used in industrial production, but since nitrile hydration is an exothermic reaction, and most nitrile hydratase is rapidly inactivated at a temperature exceeding 50 ℃, the contradiction between the two limits the wider application of nitrile hydratase to some extent. Therefore, it is of great importance to obtain nitrile hydratase having both higher enzyme activity and good thermostability.

The thermonitrites hydratase (PtNHase) derived from Pseudonocardia thermophila Pseudonocardia thermophila JCM3095,3095 still has about 90% of enzyme activity after being treated at 60 ℃ for 5 hours, and has excellent heat stability, but the wild type catalytic enzyme activity is lower. Therefore, it is of great importance to obtain nitrile hydratase having both good thermostability and high enzymatic activity.

Disclosure of Invention

Aiming at the prior art difficulties and problems, the invention provides an amino acid motif of nitrile hydratase derived from Pseudonocardia thermophila P.thermophila JCM3095 and application thereof in substrate catalytic production.

A first object of the present invention is to provide nitrile hydratase mutants containing an alpha subunit, a beta subunit and a regulatory protein.

In one embodiment of the invention, the amino acid sequence of the alpha subunit is shown as SEQ ID NO.1.

In one embodiment of the invention, the amino acid sequence of the beta subunit is shown as SEQ ID NO.2.

In one embodiment of the invention, the amino acid sequence of the regulatory protein is shown as SEQ ID NO.3.

It is a second object of the present invention to provide a gene encoding the mutant.

It is a third object of the present invention to provide a vector containing the gene.

It is a fourth object of the present invention to provide microbial cells of the nitrile hydratase mutants.

In one embodiment, the microbial cells comprise E.coli.

In one embodiment of the invention, the cells are host E.coli BL21 and vector pET series plasmid.

In one embodiment of the invention, the plasmid is pET-24 (+).

The fifth object of the present invention is to provide a method for improving the enzyme activity of nitrile hydratase, which is to mutate the 50 th glutamic acid of the beta subunit of nitrile hydratase with the amino acid sequence shown as SEQ ID NO.2 into leucine.

A sixth object of the present invention is to provide a method for producing nitrile hydratase, comprising inoculating the above microbial cells in a medium, culturing at 35-38deg.C to OD ₆₀₀ When the temperature is 0.6-0.8, adding inducer IPTG to induce for 12-16h at 22-24 ℃.

In one embodiment of the invention, the method isInoculating the microorganism cells into LB culture medium, culturing at 37deg.C to OD ₆₀₀ When the temperature is 0.6-0.8, adding inducer IPTG to induce for 12-16h at 24 ℃.

A seventh object of the present invention is to provide a method for producing nicotinamide by catalyzing nicotinamide with a nitrile hydratase mutant or a microbial cell using nicotinamide as a substrate.

The invention also provides application of the nitrile hydratase mutant, the gene, the vector or the microbial cell in preparation of amide substances.

The invention also provides application of the nitrile hydratase mutant, the gene, the vector or the microbial cell in preparing nicotinamide-containing products.

The beneficial effects are that:

1. the specific enzyme activity of the single-point mutant E50L constructed on the No. 50 subunit of the beta subunit in the amino acid motif of the nitrile hydratase is 353.53 +/-22.34U/mg, and compared with the specific enzyme activity of the unmodified wild type nitrile hydratase of 60.61+/-1.60U/mg, the specific enzyme activity of the single-point mutant E50L is obviously improved and is 5.83 times that of the wild type nitrile hydratase. The mutation obviously improves the catalytic activity of the nitrile hydratase, is beneficial to the production of industrial fine chemicals such as nicotinamide, improves the catalytic efficiency and reduces the production cost.

2. The mutant E50L provided by the invention has good substrate tolerance and product tolerance, and after the pure enzyme is treated for 30min by 50% nicotinamide, 64% of residual enzyme activity is still remained, and the wild type residual enzyme activity is only 7%; after the whole cells were treated with 1M nicotinamide for 30min, the enzyme activity was not affected, whereas the wild-type residual 89% of enzyme activity. After the whole cells are treated by 0.50M, 1M and 2M nicotinonitrile, the residual enzyme activities of the wild type enzymes are 33%, 27% and 17%, respectively, the residual enzyme activities corresponding to the mutants are 41%, 37% and 19%, and the substrate tolerance of the mutants is improved to a certain extent compared with the wild type.

Drawings

Fig. 1: SDS-PAGE electrophoresis of pure enzyme, M: protein Marker, WT: wild type, E50K: pET24a (+) -E50K, E50L: pET24a (+) -E50L, E50Q: pET24a (+) -E50Q.

Fig. 2: specific enzyme activity of PtNHase wild-type enzyme WT and mutant E50K, E50L, E50Q.

Fig. 3: ptNHase mutants have thermostability and tolerance to wild type.

Detailed Description

1. Culture medium:

LB medium (g/L): 10 parts of sodium chloride, 10 parts of tryptone and 5 parts of yeast powder.

2YT medium (g/L): tryptone 16, yeast extract 10, sodium chloride 10.

2. Buffer solution:

strep affinity chromatography Binding buffer: 3.58g of disodium hydrogen phosphate, 8.18g of sodium chloride and 0.22g of potassium chloride are weighed, the pH=7.40 is regulated by hydrochloric acid, ultrapure water is added to fix the volume to 500mL, and 0.50mM DTT is added during the experiment;

strep affinity chromatography elution buffer (wash buffer): 0.27g of desthiobiotin was added per 500mL of the strep affinity chromatography binding buffer, the ph=7.40 was adjusted with hydrochloric acid, ultrapure water was added to a volume of 500mL, and 0.50mM DTT was added at the time of the experiment.

3. Enzyme activity determination method

Enzyme activity unit (U): the amount of enzyme required to catalyze the formation of 1. Mu. Mol nicotinamide per minute of nicotinonitrile at 25 ℃;

specific enzyme activity (U/mg): number of units of enzyme activity per mg of pure enzyme.

Example 1: plasmid construction of nitrile hydratase mutants

The mutation site E50L was designed on a primer using the wild-type plasmid pET24a (+) -PtNHase WT (described in Cheng et al, computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability, 2020) as a template, and the plasmid pET24a (+) -E50L having a mutated nucleotide sequence was amplified by whole-plasmid PCR, and the primer sequences used are shown in Table 1.

PCR reaction system: the volume of the total system was 50. Mu.L, which was 1. Mu.L for each of the upstream primer and the downstream primer and 25. Mu.L for each of the template plasmid and 25. Mu.L for each of the Primer STAR Max DNA polymerase, and the system was supplemented with sterilized ultrapure water.

PCR reaction conditions: the pre-denaturation temperature is 98 ℃ for 10min; denaturation at the same temperature for 30s, annealing conditions were set at 56℃for 30s, extension at 72℃for 90s, and 34 cycles were repeated; finally, the extension was carried out at 72℃for 10min to ensure complete extension.

The whole plasmid PCR product requires removal of the master plasmid by DpnI digestive enzymes. The digestion system was 5. Mu. L Quick Cut Buffer and 1. Mu.LDpnI digestive enzymes were added per 44. Mu.L of PCR product and reacted in a constant temperature metal bath at 37℃for 2 hours during digestion. Subsequently, the digested PCR product was transformed into E.coli DH 5. Alpha. And plated on LB medium plates containing 50mg/L kanamycin, and incubated at 37℃overnight in an inverted state.

Single colony is selected and inoculated in 5mL LB culture medium, shaking culture is carried out at 37 ℃ and 200rpm for overnight, a commercial plasmid extraction kit is used for obtaining recombinant plasmid, sequencing verification is carried out by Suzhou Jin Weizhi biotechnology limited company, and finally the recombinant plasmid pET24a (+) -E50L is obtained.

TABLE 1 primer sequences

(Note: F means upstream primer, R means downstream primer)

Example 2: expression and purification of wild enzyme WT and mutants

Step 1: e.coli BL21 (DE 3) was transformed with the wild-type plasmid pET24a (+) -PtNHase WT and the recombinant plasmid pET24a (+) -E50L of Pt NHase of example 1, respectively, and single colonies were picked up to 5mL of LB medium and cultured at 37℃and 200rpm for 7-8 hours. The seed solution was transferred to 100mL of 2YT medium at 1% (v/v) and cultured at 37℃and 200rpm to OD ₆₀₀ To 0.6-0.8, isopropyl thiogalactoside (IPTG) at a final concentration of 0.4mM and CoCl at 0.1g/L ₂ ·6H ₂ O, changing the culture temperature to 24 ℃, and carrying out induced expression for 12-16h to obtain fermentation bacteria liquid.

Step 2: the wild WT and its mutant E50L are purified by affinity chromatography, and the purification column is StrepTrap HP 1mL column of GE company. The cells were collected by centrifugation at 10000rpm for 3min, resuspended in 20mL of binding buffer and sonicated in an ice-water mixture. The crushed solution was centrifuged at 12000rpm at 4℃for 30min, and the supernatant was filtered through a 0.22 μm organic filter. After the purification column is balanced by the binding buffer solution, loading is carried out, then the binding buffer solution is used for washing off the impurity protein, the target protein is eluted by 100% of the eluting buffer solution and collected, and the pure enzyme solution of the WT and the mutant E50L thereof is obtained. Protein concentration was quantified using the Brandford protein concentration detection kit. The purification quality of the target protein is detected by SDS-PAGE, and the detection is shown in figure 1, so that the protein expressed by the wild type and the mutant thereof has single protein band after purification and high purification quality.

Example 3: detection of catalytic efficiency of wild-type and mutant of Pt NHase

10. Mu.L of purified 0.05mg/mL WT and mutant E50L thereof of example 2 were placed in a centrifuge tube, incubated at 25℃for 5min in a metal bath, 490. Mu.L of 200mM nicotinonitrile substrate was added, and after mixing, the reaction was carried out at 25℃for 10min, and finally 500. Mu.L of acetonitrile was added to terminate the reaction, the final volume of the reaction solution being 1mL. After the reaction, the reaction solution was filtered through a 0.22 μm organic filter membrane, and the product yield was measured by High Performance Liquid Chromatography (HPLC), the detector was an ultraviolet detector (detection wavelength: 210 nm), the column temperature was C18 (column temperature: 40 ℃ C.), the mobile phase was a mixed solution of acetonitrile and water (volume ratio of acetonitrile to water: 1:2), the flow rate was 0.60mL/min, the sample injection amount was 10. Mu.L, and the sample injection time was 10min.

The results of specific enzyme activity calculation of WT and mutant are shown in FIG. 2, the specific enzyme activity of wild-type enzyme WT is 60.61+ -1.60U/mg, and the specific enzyme activity of mutant E50L is 353.53 + -22.34U/mg. The specific enzyme activity of the mutant catalytic nicotinonitrile is 5.83 times that of the wild type.

Example 4: thermal stability of nitrile hydratase wild type and mutant

The metal bath temperature is set to be 60 ℃, the temperature is kept for 0, 2, 3 and 5 hours respectively, then sampling is carried out, the residual enzyme activity is measured, the enzyme activity of a sample (enzyme which is not subjected to heat treatment) treated for 0 hour is defined as 100% by taking the sample as a reference, the relative enzyme activity of the other samples is calculated in sequence, and the thermal stability of the pure enzyme at 60 ℃ is analyzed.

As shown in FIG. 3-a, the wild type enzyme activity is still about 90% after 5h treatment at 60 ℃, and the mutant E50L treatment is performed for 62% of the same time, so that the heat stability is reduced to a certain extent compared with the wild type enzyme activity, but the heat stability is still better.

Example 5: substrate tolerance and product tolerance of nitrile hydratase wild type and mutant

(1) Nicotinamide tolerance:

(a) Pure enzyme tolerance to nicotinamide:

50% nicotinamide (50 g nicotinamide per hundred milliliters of water) was formulated for subsequent reactions.

Taking 10 mu L of purified pure enzyme solution of 0.05mg/mL WT and mutant E50L thereof in example 2, adding the pure enzyme solution into a 1.5mL centrifuge tube in advance, and placing the centrifuge tube in a constant-temperature metal bath at 25 ℃ for preheating for 5min; 250 mu L of 50% nicotinamide is added into the reaction group, and an equal volume of 10mM KPB buffer solution is added into the control group at the same time for 30min; after that, 250. Mu.L of 200mM nicotinonitrile was added and reacted for 10min, and 500. Mu.L of acetonitrile was used as a terminator. The substrate reduction was measured by high performance liquid chromatography after filtration through a 0.22 μm organic filter, and the corresponding enzyme activity was calculated, comparing the product (nicotinamide) tolerance of the pure enzyme.

As shown in FIG. 3-b, after 30min of treatment with 50% nicotinamide, the wild-type residual enzyme activity was only 7%, while the mutant still had 64% residual enzyme activity, which retained more than half of the enzyme activity; the results show that the product tolerance of the E50L mutant pure enzyme is greatly improved, and the E50L mutant pure enzyme has excellent product (nicotinamide) tolerance.

(b) Tolerance of cells to nicotinamide:

a nicotinamide solution with a concentration of 0, 1M was prepared. A fermentation broth was obtained as in step 1 of example 2, and was uniformly diluted to OD ₆₀₀ The fermentation broths of=3 are respectively placed in nicotinamide systems with different concentrations, the reaction system comprises 100 mu L of fermentation broths and 900 mu L of nicotinamide with a certain concentration, and the metal bath is kept at 25 ℃ for 30min. After centrifugation at 6000rpm for 10min in the ultra-high speed centrifuge, the cell supernatant was retained, resuspended in 10mM KPB buffer (pH=7.40), and repeated 2 times, and the resulting cell was resuspended in 100. Mu.LKPB buffer (pH=7.40). Taking 10 mu L of bacterial liquid for enzyme activity determination, determining the product yield of a sample by high performance liquid chromatography, defining the whole cell enzyme activity of an untreated control group as 100%, and calculating other samplesThe product (nicotinamide) tolerance of the cells was compared with respect to enzyme activity.

The results are shown in FIG. 3-d, with 89% enzyme activity remaining after treatment of the wild type with 1M nicotinamide; the residual enzyme activity was 100% after the mutant was treated with the same concentrations as described above. The product (nicotinamide) tolerance of the mutated cells is increased relative to the wild type in a 1M nicotinamide environment.

(2) Cell tolerance to nicotinonitrile:

nicotine solutions were prepared at concentrations of 0, 0.50, 1, 2M. A fermentation broth was obtained by the method of example 2, step 1, and was uniformly diluted to OD ₆₀₀ The fermentation broths of=3 are respectively placed in nicotinamide systems with different concentrations, the system comprises 100 mu L of fermentation broths and 900 mu L of nicotinonitrile with certain concentration, and the treatment is carried out for 30min at the constant temperature of a metal bath at 25 ℃. After centrifugation at 6000rpm for 10min in the ultra-high speed centrifuge, the cell supernatant was retained, resuspended in 10mM KPB buffer (pH=7.40), and repeated 2 times, and the resulting cell was resuspended in 100. Mu.LKPB buffer (pH=7.40). Taking 10 mu L of bacterial liquid for enzyme activity measurement, measuring the product yield of a sample by high performance liquid chromatography, defining the whole cell enzyme activity of an untreated control group as 100%, calculating the relative enzyme activities of other samples, and comparing the substrate (nicotinonitrile) tolerance of cells.

As a result, as shown in FIG. 3-c, the residual enzyme activities after the wild-type treatment with 0.50M, 1M and 2M nicotinonitrile were 33%, 27% and 17%, respectively; the residual enzyme activities corresponding to the mutants were 41%, 37% and 19%. The substrate tolerance of the mutant is improved to a certain extent compared with the wild type.

Comparative example 1: comparison of the catalytic efficiency of mutant pET24a (+) -E50L with other 50-position mutants

The specific embodiment is the same as example 1, except that the glutamic acid at 50 th site is mutated into other 2 amino acids (lysine, glutamine), enzyme mutants are prepared according to example 2 and used for the catalytic reaction of nicotinonitrile, and as shown in fig. 2, the specific enzyme activities of the mutants E50K and E50Q are significantly lower than pET24a (+) -e50L and only equal to the wild type.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> a nitrile hydratase mutant having high activity and high thermostability

<130> BAA211581A

<160> 3

<170> PatentIn version 3.3

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<212> PRT

<213> Pseudonocardia thermophila

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Asp Leu Phe Arg Phe Gly Ile Glu Gln Met Asn Pro Ala Glu Tyr Leu

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Claims (9)

1. A nitrile hydratase mutant, which is characterized by consisting of an alpha subunit, a beta subunit and regulatory proteins;

the amino acid sequence of the alpha subunit is shown as SEQ ID NO. 1;

the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2;

the amino acid sequence of the regulatory protein is shown as SEQ ID NO.3.

2. A gene encoding the nitrile hydratase mutant of claim 1.

3. A recombinant vector comprising the gene of claim 2.

4. A microbial cell expressing the nitrile hydratase mutant of claim 1.

5. The microbial cell of claim 4, wherein the microbial cell comprises escherichia coli.

6. The microbial cell according to claim 4 or 5, wherein the cell is a cell using escherichia coli BL21 as a host and pET series plasmid as a vector.

7. The microbial cell of claim 6, wherein said vector is pET-24 (+).

8. A method for producing nitrile hydratase, characterized in that the microbial cells of any one of claims 4 to 7 are inoculated into a culture medium and cultured at 35 to 38 ℃ to OD ₆₀₀ When the temperature is 0.6-0.8, adding an inducer IPTG to induce 12-16h at 22-24 ℃.

9. Use of a nitrile hydratase mutant according to claim 1, or a gene according to claim 2, or a vector according to claim 3, or a microbial cell according to any of claims 4 to 7, for the preparation of a nicotinamide-containing product.