A nitrile hydratase mutant, genetically engineered bacteria and application in preparation of acrylamide

Abstract

The application discloses a nitrile hydratase mutant, a genetically engineered bacterium and application thereof in preparation of acrylamide. Through a semi-rational design method, the molecular of a beta subunit and an alpha subunit are reformed, and the stability of the modified nitrile hydratase is obviously improved. With acrylonitrile as a substrate, the product tolerance of the nitrile hydratase mutant of the application is all enhanced, and the enzyme activity of some mutants is also improved. The initial type NHAM and the mutants B-G41D, B-S51E, B-R55C and B-R98I with the most obvious improvement in enzyme activity and stability are simultaneously subjected to catalytic reaction, and it is found that after the product tolerance of the mutants is improved, the final concentration of the product of B-R98I can reach 523 g / L, which is obviously improved compared with 322 g / L of the initial type, and the application provides convenience for industrial application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering, and particularly relates to a nitrile hydratase mutant, a genetically engineered bacterium and application thereof in preparation of acrylamide. BACKGROUND

[0002] Nitrile hydratase (NHase) is a kind of biological catalyst that can catalyze the hydration of nitrile compounds to amides. The nitrile hydratase produced by microorganisms can efficiently catalyze the hydration of acrylonitrile to acrylamide. The polyacrylamide produced by the polymerization of acrylamide is widely used as a drag-reducing agent, flocculant and thickening agent in oil exploitation, water treatment and papermaking.

[0003] Compared with the traditional chemical catalytic method, the biological enzyme catalytic method for preparing acrylamide has the advantages of high conversion efficiency, high product purity, mild reaction conditions and environmental friendliness, and has the trend of gradually replacing the chemical method. Mitsubishi Company of Japan uses rose-colored red bacterium J1 (CN91101323.7) containing nitrile hydratase to catalyze acrylonitrile to produce acrylamide, with an annual output of more than 200,000 tons. At present, the research focus of the biological enzyme catalytic method for producing acrylamide is the discovery of high-yield nitrile hydratase strains and the improvement of the performance of nitrile hydratase itself. In the hydration reaction process, the nitrile hydratase in the free cells is inhibited by the added acrylonitrile and high-concentration acrylamide solution, and the catalytic activity rapidly decreases, so it is impossible to obtain a high product concentration in a batch of hydration reaction. Most of the reported wild-type nitrile hydratases have problems such as poor catalytic stability and insufficient catalytic activity, and most of the improvements in stability only involve thermal stability, but in practical application, it is very important to improve the product tolerance of nitrile hydratase while ensuring the enzyme activity.

[0004] By constructing a genetically engineered strain with nitrile hydratase catalytic activity and protein engineering of nitrile hydratase, the practical problems in acrylamide production can be solved to a certain extent. The patent document with the application publication number CN107881163A has disclosed a nitrile hydratase (NHAM) derived from Aurantimonas manganoxydans SI859A (Aurantimonas manganoxydans SI859A), which has high catalytic activity for acrylonitrile and good thermal stability, but the product tolerance still needs to be improved. SUMMARY

[0005] The present application provides a nitrile hydratase mutant, a genetically engineered bacterium and application thereof in preparation of acrylamide. The nitrile hydratase mutant not only has high catalytic activity for acrylonitrile and good thermal stability, but also has high product tolerance, and the enzyme activity of the nitrile hydratase mutant is not lost under high product concentration.

[0006] The specific technical solutions are as follows:

[0007] The application provides a nitrile hydratase mutant, which is any one of the following mutants based on a wild-type nitrile hydratase with a nucleotide sequence as shown in SEQ ID No. 7:

[0008] (1) a nitrile hydratase beta subunit with a 41st glycine in an amino acid sequence as shown in SEQ ID No. 2 is mutated to obtain;

[0009] (2) a nitrile hydratase beta subunit with a 51st serine in an amino acid sequence as shown in SEQ ID No. 2 is mutated to obtain;

[0010] (3) a nitrile hydratase beta subunit with a 55th arginine in an amino acid sequence as shown in SEQ ID No. 2 is mutated to obtain;

[0011] (4) a nitrile hydratase alpha subunit with a 98th arginine in an amino acid sequence as shown in SEQ ID No. 1 is mutated to obtain.

[0012] The nitrile hydratase NHAM derived from Aurantimonas manganoxydans SI859A (Aurantimonas manganoxydans SI859A) is composed of an alpha subunit, a beta subunit and a regulatory protein, and the amino acid sequences are shown in SEQ ID No. 1, No. 2 and No. 3. The nitrile hydratase mutant is obtained by mutating the wild-type nitrile hydratase with a beta subunit amino acid sequence as shown in SEQ ID No. 2 or a wild-type nitrile hydratase with an alpha subunit amino acid sequence as shown in SEQ ID No. 1.

[0013] Then, the gene (SEQ ID No. 5) encoding the mutation of the beta subunit or the gene (SEQ ID No. 4) encoding the mutation of the alpha subunit is combined with the gene (SEQ ID No. 6) of the regulatory protein to obtain a nitrile hydratase gene cluster alpha-beta-p14k, and the connection sequence between the beta subunit and the regulatory protein p14k is shown in SEQ ID No. 7; the gene cluster is constructed into an expression vector pET-28a (+), and is introduced into an E. coli genetic engineering bacterium E. coli BL21 (DE3) to obtain a mutant strain.

[0014] Further, the mutant is any one of the following:

[0015] (1) the 41st glycine in the nitrile hydratase beta subunit with an amino acid sequence as shown in SEQ ID No. 2 is respectively mutated into any one of aspartic acid and glutamic acid;

[0016] (2) the serine at position 51 of the beta subunit of the hydratase is mutated to aspartic acid, wherein the amino acid sequence is shown in SEQ ID NO. 2;

[0017] (3) the arginine at position 55 of the beta subunit of the hydratase is mutated to any one of cysteine, isoleucine, leucine, methionine, asparagine, valine, respectively, wherein the amino acid sequence is shown in SEQ ID NO. 2;

[0018] (4) the arginine at position 98 of the alpha subunit of the hydratase is mutated to any one of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, tryptophan, tyrosine, respectively, wherein the amino acid sequence is shown in SEQ ID NO. 1.

[0019] The application further provides a gene encoding the hydratase mutant.

[0020] The application further provides a recombinant expression vector comprising the gene.

[0021] Further, the original expression vector of the recombinant expression vector is pET-28a(+).

[0022] The application further provides a genetically engineered bacterium comprising a host cell and a target gene introduced into the host cell, wherein the target gene comprises the gene.

[0023] Further, the host cell is E. coli BL21 (DE3).

[0024] The application further provides an application of the hydratase mutant, or the gene, or the recombinant expression vector, or the genetically engineered bacterium in catalyzing production of acrylamide from acrylonitrile.

[0025] The application further provides a method for catalyzing production of acrylamide from acrylonitrile, comprising: using acrylonitrile as a substrate, and using a catalyst to continuously catalyze hydration reaction of the substrate in a buffer solution to obtain acrylamide, wherein the catalyst is the hydratase mutant or the immobilized enzyme thereof, or the genetically engineered bacterium.

[0026] Further, the hydration reaction can reach a product concentration of 425-523 g / L.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] The application is directed to nitrile hydratase (NHAM) derived from Aurantimonas manganoxydans SI859A, the alpha and beta subunits are respectively subjected to semi-rational design, the stability expression of nitrile hydratase NHAM in genetically engineered bacteria E.coli BL21(DE3) is significantly improved, so that the tolerance of nitrile hydratase mutant in 300g / L acrylamide solution is significantly improved, wherein the tolerance of beta subunit mutant B-R55C is improved, at the same time, the enzyme activity is increased by 2.1 times compared with the wild type, which has very great industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The gas phase detection spectrum of nitrile hydratase NHAM catalyzing acrylonitrile to generate acrylamide is shown, and the peak order is substrate, internal standard and product in turn, wherein the retention time of substrate acrylonitrile is 1.5min, the retention time of internal standard acetamide is 4.4min, and the retention time of product acrylamide is 8.8min.

[0030] Figure 2 The result graph of initial relative enzyme activity of nitrile hydratase initial type WT and mutant and residual enzyme activity after 300g / L acrylamide solution immersion treatment for 1h.

[0031] Figure 3 The residual enzyme activity curve graph of nitrile hydratase initial type WT and beta subunit 55th site mutant B-R55C in 300g / L acrylamide solution immersion incubation for different time.

[0032] Figure 4 The progress graph of nitrile hydratase initial type WT and beta subunit mutants B-G41D, B-S51E, B-R55C, B-R98I batch reaction. DETAILED DESCRIPTION

[0033] The application will be further described below in combination with specific embodiments, and the following enumeration is only a specific embodiment of the application, but the protection scope of the application is not limited to this.

[0034] The recombinant E.coli with nitrile hydratase gene involved in the application uses pET-28a(+) as the carrier and E.coli E.coli BL21(DE3) as the host. The reagent kit for preparing competent cells is purchased from TAKARA company.

[0035] Reagents for downstream catalytic process: Acrylonitrile was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; acetamide was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.; acrylamide was purchased from Aladdin Reagent Co., Ltd.; other commonly used reagents were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. The three-letter or one-letter representation of amino acids used in the text of the present application adopts the IUPAC code for amino acids (Eur. J. Biochem., 138: 9-37, 1984).

[0036] Definition of enzyme activity (U / mL): the amount of enzyme required to catalyze the production of 1 μmol of acrylamide from acrylonitrile per minute.

[0037] Standard detection system for nitrile hydratase enzyme activity: an appropriate amount of enzyme solution, 4 mM substrate, total system 500 μL, reaction medium 0.25 M phosphate buffer at pH 7.5. Reaction at 28°C for 5 min, gas phase detection of product acrylamide production.

[0038] Detection of acrylamide: gas chromatography, carrier gas nitrogen, flow rate 0.24 mL / min, column oven temperature 210°C, detection temperature 250°C, vaporization temperature 250°C. The chromatographic column is a Propack Q packed column.

[0039] Determination of product tolerance: the initial NHAM and mutants were soaked in a 300 g / L acrylamide solution and incubated at 28°C, and the enzyme activity was determined after 1 h. The enzyme activity of the enzyme without soaking treatment was defined as 100%, and the tolerance result in 300 g / L acrylamide solution was obtained.

[0040] Example 1 Construction of initial NHAM and mutant recombinant bacteria

[0041] The nitrile hydratase NHAM derived from Aurantimonas manganoxydans SI859A was subjected to semi-rational design, and mutations were designed at positions 41, 51 and 55 of the β subunit and at position 98 of the α subunit.

[0042] The codon optimization and gene synthesis service was provided by a commissioned company, and the genes of the initial NHAM and mutants of nitrile hydratase NHAM derived from Aurantimonas manganoxydans SI859A were cloned on the pET-28a(+) plasmid, and the target gene was placed between the restriction sites BamH I and Hind III. The pET-28a(+)-NHAM recombinant plasmid was obtained.

[0043] The primers shown in Table 1 were used for PCR under the conditions shown in Table 2, and the PCR product was transformed into E. coli BL21 (DE3). The plasmid with correct sequencing result was used for expression to obtain the recombinant strain.

[0044] Table 1 Primers used for PCR

[0045]

[0046]

[0047]

[0048] Table 2 PCR amplification system

[0049] Component Volume (μL) PrimeSTAR 25 Upstream primer 1.0 Downstream primer 1.0 Plasmid template 0.5 ddH2O 22.5

[0050] PCR amplification conditions:

[0051] 1) pre-denaturation: 98°C for 5 min;

[0052] 2) denaturation: 98°C for 10 s; annealing: 60°C for 15 s; extension: 72°C for 1 min 30 s; total 35 cycles;

[0053] 3) extension: 72°C for 10 min;

[0054] 4) storage at 4°C for 2.0 h.

[0055] After the PCR amplification, the amplification product was detected by 1.0% agarose gel electrophoresis, and the target band was purified and recovered by a DNA recovery purification kit.

[0056] The recovered PCR product was digested with DPNI to remove the template. The digestion system is shown in Table 3.

[0057] Table 3 Digestion system

[0058]

[0059]

[0060] Digestion conditions:

[0061] 1) 37°C: 1 h;

[0062] 2) 75°C: 15 min;

[0063] 3) storage at 4°C for 2.0 h.

[0064] The product after digestion was transformed into E. coli BL21 competent cells.

[0065] Example 2

[0066] I. Microbial culture and enzyme activity determination

[0067] (1) Cultivation of microorganism

[0068] Composition of LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, dissolved with deionized water, and then constant volume, sterilized at 121°C for 20 min, and ready for use. LB solid medium (plate culture dish): 20 g / L of agar powder was added to the LB liquid medium, sterilized at 121°C, and then introduced into the culture dish after cooling, to make a plate.

[0069] The engineered bacteria E. coli BL21 (DE3) containing the relevant genes were inoculated into 5 mL of LB liquid medium containing 50 μg / mL of kanamycin, and cultured at 37°C for 12 h with shaking. The culture was transferred into 500 mL of fresh LB liquid medium containing 50 μg / mL of kanamycin, and cultured at 37°C with shaking until the OD600 reached about 0.8, then IPTG was added to a concentration of 0.5 mM, and cobalt chloride was added to a final concentration of 0.4 mM, and the culture was induced at 18°C for 16-18 h. After the culture was completed, the culture was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected and stored in a -70°C ultra-low temperature freezer for later use.

[0070] (2) Determination of enzyme activity

[0071] The bacterial cells collected after the culture was completed were washed twice with 0.25 M PB buffer (pH 7.5). Then the bacterial cells were resuspended in 0.5 times the volume of the fermentation broth of 0.25 M PB buffer (pH 7.5), and the cell suspension was used for subsequent determination.

[0072] Definition of enzyme activity: According to the International Enzyme Conference in 1961, 1 unit of enzyme activity refers to the amount of enzyme that can convert 1 micromole of substrate in 1 minute under specific conditions, or the amount of enzyme that can convert 1 micromole of related groups in the substrate.

[0073] Cyanohydrinase enzyme activity determination system: the total system is 500 μL, the reaction medium is 0.25 M phosphate buffer at pH 7.5, the substrate is 425 μL of 50 g / L acrylonitrile, 50 μL of OD 600 2 bacterial solution is added, and the reaction is carried out at 28°C for 5 min, then 25 μL of 4 mol / L HCl is added to terminate the reaction.

[0074] The reaction solution was taken and centrifuged at 12000 rpm for 2 min, and the supernatant was taken and an equal amount of internal standard (20 g / L of acetamide solution) was added, and the amount of product acrylamide was determined by gas phase internal standard method. The liquid chromatogram is shown in Figure 1 .

[0075] II. Tolerance determination of NHAB initial type and mutants

[0076] Product tolerance of enzyme: the collected cells were resuspended with 300 g / L acrylamide solution, soaked in 28°C water bath for 1 h, and then the residual enzyme activity was determined by using enzyme activity detection system, and the enzyme activity of untreated enzyme was defined as 100%.

[0077] The results are shown in Figure 2 As shown in the figure, the residual enzyme activity of NHAM initial type was 45% after soaking in 300 g / L acrylamide solution for 1 h, the residual enzyme activity of mutants G41D and G41E at site 41 of β subunit could reach about 70%, and the enzyme activity was significantly improved compared with wild type. The tolerance of β subunit S51E mutant was also improved to 73%, and the initial enzyme activity was increased by 2.4 times compared with wild type. The tolerance of six mutants at site 55 of β subunit was significantly improved, among which the mutant R55C, the enzyme activity was increased by 2.1 times and the residual enzyme activity was 63.3%. For the mutants at site 98 of α subunit, the tolerance was significantly improved by replacing glutamic acid, valine and other seventeen kinds of amino acids, but the initial enzyme activity was slightly lost.

[0078] Product tolerance curve of enzyme: the collected cells were resuspended with 300 g / L acrylamide solution, soaked in 28°C water bath, and sampled every 30 min, then the residual enzyme activity was determined by using enzyme activity detection system, and the enzyme activity of untreated enzyme was defined as 100%.

[0079] The results are shown in Figure 3 As shown in the figure, the tolerance of B-R55C was significantly better than that of initial type WT, and the residual enzyme activity of B-R55C mutant after 2 h was twice that of wild type.

[0080] Example 3 B-G41D catalyzing acrylonitrile to generate acrylamide

[0081] The fermentation broth of genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)-NHAM-B-G41D constructed in Example 2 was centrifuged at 4000 rpm for 10 min to collect the bacterial cells, 2 g of wet bacterial cells were resuspended with 140 mL of 0.25 M phosphate buffer (pH 7.5), and the hydration reaction was carried out at 20°C, 40 g / L of acrylonitrile was added to each batch, and the next batch was added after the substrate was completely consumed. Then the content of acrylonitrile, acrylamide and acrylic acid in the reaction system was detected by gas chromatography. The substrate conversion rate was more than 95%, the acrylamide concentration reached 464 g / L, and no acrylic acid was generated in the reaction system.

[0082] Example 4 B-S51E catalyzing acrylonitrile to generate acrylamide

[0083] The fermentation broth of the genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)-NHAM-B-S51E constructed in Example 2 was centrifuged at 4000 rpm for 10 min to collect the bacterial cells, 2 g of the wet bacterial cells was weighed and resuspended in 140 mL of 0.25 M phosphate buffer (pH 7.5), and a hydration reaction was carried out at 20°C, 40 g / L of acrylonitrile was added in each batch, and the next batch was added after the substrate was completely consumed. Then the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system were detected by gas chromatography. The substrate conversion rate was greater than 95%, the acrylamide concentration reached 518 g / L, and no acrylic acid was generated in the reaction system.

[0084] Example 5 B-R55C catalyzes acrylonitrile to generate acrylamide

[0085] The fermentation broth of the genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)-NHAM-B-S51E constructed in Example 2 was centrifuged at 4000 rpm for 10 min to collect the bacterial cells, 2 g of the wet bacterial cells was weighed and resuspended in 140 mL of 0.25 M phosphate buffer (pH 7.5), and a hydration reaction was carried out at 20°C, 40 g / L of acrylonitrile was added in each batch, and the next batch was added after the substrate was completely consumed. Then the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system were detected by gas chromatography. The substrate conversion rate was greater than 95%, the acrylamide concentration reached 425 g / L, and no acrylic acid was generated in the reaction system.

[0086] Example 6 A-R98I catalyzes acrylonitrile to generate acrylamide

[0087] The fermentation broth of the genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)-NHAM-B-S51E constructed in Example 2 was centrifuged at 4000 rpm for 10 min to collect the bacterial cells, 2 g of the wet bacterial cells was weighed and resuspended in 140 mL of 0.25 M phosphate buffer (pH 7.5), and a hydration reaction was carried out at 20°C, 40 g / L of acrylonitrile was added in each batch, and the next batch was added after the substrate was completely consumed. Then the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system were detected by gas chromatography. The substrate conversion rate was greater than 95%, the acrylamide concentration reached 523 g / L, and no acrylic acid was generated in the reaction system.

[0088] Comparative Example 1 NHAM catalyzes acrylonitrile to generate acrylamide

[0089] The fermentation broth of genetically engineered bacteria E. coli BL21(DE3)-pET-28a(+)-NHAM constructed in Example 2 was centrifuged at 4000 rpm for 10 min to collect the bacterial cells, 2 g of wet bacterial cells was weighed and resuspended in 140 mL of 0.25 M phosphate buffer (pH 7.5), and a hydration reaction was carried out at 20°C, 40 g / L of acrylonitrile was added in each batch, and the next batch was added after the substrate was completely consumed. Then the content of acrylonitrile, acrylamide and acrylic acid in the reaction system was detected by gas chromatography. The substrate conversion rate was greater than 95%, the acrylamide concentration reached 322 g / L, and no acrylic acid was generated in the reaction system.

Claims

1. A nitrile hydratase mutant characterized in that, The mutant is any one of the following mutations based on the wild-type nitrile hydratase of the nucleotide sequence shown in SEQ ID NO. 7: (1) the 41st glycine of the nitrile hydratase β subunit with the amino acid sequence shown in SEQ ID NO. 2 is mutated into any one of aspartic acid and glutamic acid; (2) the 51st serine of the nitrile hydratase β subunit with the amino acid sequence shown in SEQ ID NO. 2 is mutated into glutamic acid; (3) the 55th arginine of the nitrile hydratase β subunit with the amino acid sequence shown in SEQ ID NO. 2 is mutated into any one of cysteine, isoleucine, leucine, methionine, asparagine and valine; (4) the 98th arginine of the nitrile hydratase α subunit with the amino acid sequence shown in SEQ ID NO. 1 is mutated into any one of glycine, isoleucine and proline.

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

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

4. A genetically engineered bacterium comprising a host cell and a target gene introduced into the host cell, characterized in that, The gene of claim 2.

5. Use of the nitrile hydratase mutant of claim 1, the gene of claim 2, the recombinant expression vector of claim 3 or the genetically engineered bacterium of claim 4 in catalyzing the production of acrylamide from acrylonitrile.

6. A process for the catalytic production of acrylamide from acrylonitrile, characterized in that, It comprises: using a catalyst to continuously catalyze the hydration reaction of the substrate in a buffer solution to obtain acrylamide, with acrylonitrile as the substrate; The catalyst is the nitrile hydratase mutant of claim 1 or its immobilized enzyme, or the genetically engineered bacterium of claim 4.

7. The method of claim 6, wherein, The product concentration of the acrylamide can reach 425-523 g / L.

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