A nitrile hydratase mutant and use thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a nitrile hydratase mutant and application thereof. The present application is based on a wild-type nitrile hydratase from Rhodococcus ruber, and the mutation sites significantly affecting the activity of the nitrile hydratase are determined through prediction, screening and verification of key active sites. The present application provides a nitrile hydratase mutant with improved activity and application thereof in production of amide compounds. The nitrile hydratase mutant provided by the present application has a unit cell dry weight enzyme activity of up to 3649.95 U / g DCW, which is increased by 142.98% compared with the wild type. The specific enzyme activity of the purified nitrile hydratase can reach 3589.44 U / mg, which improves the efficiency of conversion of nitrile substances into amide products. The mutant provided by the present application can be used to rapidly produce 2,6-difluorobenzamide, nicotinamide, cinnamoyl amide, acrylamide and other amide substances, and has higher space-time conversion rate and shorter conversion time.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a nitrile hydratase mutant and its application. Background Technology

[0002] Amide compounds, due to their unique stability, have become a bridge connecting chemistry, agronomy, biology, and materials science. Their applications are expanding from traditional medicine and materials science to cutting-edge biotechnology. For example, 2,6-difluorobenzamide (DFBA), a chitin synthesis inhibitor, is widely used in the synthesis of environmentally friendly benzoylurea insecticides due to its high insecticidal activity, safety to humans and animals, and low likelihood of developing resistance. Furthermore, it can also serve as a chemical intermediate in pharmaceuticals, materials science, and other fields, showing great promise for future applications.

[0003] There are two main methods for synthesizing amide compounds: chemical synthesis and microbial transformation. Traditional chemical synthesis has disadvantages such as low yield, many by-products, high temperature and pressure, sulfuric acid catalytic hydration, and high equipment cost, which do not conform to the principles of green economy and can no longer meet the needs of production. The microbial transformation method, which has been developed, has been widely used because of its mild reaction conditions, high conversion rate, and less susceptibility to environmental interference.

[0004] Nitrile hydratases (NHases) are a class of metalloenzymes that catalyze the hydration reaction of nitrile compounds with ammonia at room temperature and physiological pH, producing the corresponding amide compounds. Among them, Rhodococcus rubrum (… Rhodococcus ruber Rhodococcus rubrum is an important natural host and producing strain of nitrile hydratase, but its long culture cycle and issues related to activity and stability limit its development. Therefore, heterologous expression of nitrile hydratase in Escherichia coli, which lacks nitrile metabolic enzyme systems, shortens its culture cycle and improves its spatiotemporal conversion rate, which is of great research significance for its industrial application. Nitrile hydratase is usually composed of a β subunit, an α subunit, and a helper activator protein. Its catalytic activity is highly dependent on the key metal center and unique post-translational modifications. Nitrile hydratase's active center is formed by the α / β subunit. Its catalytic ability originates from the cysteine-sulfoxy modification formed by post-translational oxidation on the α subunit. These modifications work synergistically with metal ions to polarize and activate nitrile substrates. The activator protein is the key assistant for the correct assembly and maturation of this unique active center. Based on the different metals chelated in the active center of nitrile hydratase, nitrile hydratase can be divided into iron-type nitrile hydratase (Fe-NHase) and cobalt-type nitrile hydratase (Co-NHase).

[0005] The hydration reaction of nitrile hydratase is an exothermic reaction, and the substrate product has a certain toxic effect on bacterial cells. Natural nitrile hydratases exhibit low catalytic activity, poor stability, and poor substrate product tolerance, which restricts the efficient synthesis of high-value amide compounds and the industrialization of amide product production. Therefore, obtaining nitrile hydratases with both good stability and high enzyme activity is of great significance. Enzyme modification is an effective means to improve the industrial properties of enzymes (catalytic activity, substrate product tolerance, and stability). Reported strategies for modifying the stability of nitrile hydratases mainly include strategies based on bioinformatics analysis such as salt bridge addition, free energy reduction, homologous fragment recombination, and gene fusion. Modifications based on nitrile hydratase activity mainly involve sequence alignment, channel modification, active pocket remodeling, and distal modification. However, increasing activity often leads to decreased stability. Therefore, it is crucial to improve the enzyme's tolerance to toxic substrate products and increase the utilization rate of cells and enzymes while ensuring stability meets production requirements. Therefore, there is an urgent need in this field to develop a nitrile hydratase mutant with high catalytic activity based on stability and tolerance, and to establish supporting mutant expansion, high-throughput screening, and efficient transformation and detection analysis technologies. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art, in the case of Rhodococcus ( Rhodococcus ruber Based on wild-type nitrile hydratase derived from CGMCC No. 3090, this study identifies mutation sites that significantly affect nitrile hydratase activity through prediction, screening, and verification of key active sites, and provides information on nitrile hydratase mutants with enhanced activity and their applications in the production of amide compounds.

[0007] To achieve the above objectives, the technical approach adopted by the present invention is as follows:

[0008] One of the technical solutions provided by this invention is a nitrile hydratase mutant, which is obtained by mutation based on wild-type nitrile hydratase. The wild-type nitrile hydratase comprises a β subunit, an α subunit, and an activating protein A, wherein the β subunit is shown in SEQ ID NO.1, the α subunit is shown in SEQ ID NO.2, and the activating protein A is shown in SEQ ID NO.3. The mutant comprises at least one mutation of βL34F, βN152C, or βN167G based on the wild-type β subunit shown in SEQ ID NO.1, and / or at least one mutation of αV37I, αN43D, αN43K, αN43L, αN43T, αS89E, αV101C, or αW155T based on the wild-type α subunit shown in SEQ ID NO.2. Furthermore, the activating protein A of the nitrile hydratase mutant, as shown in SEQ ID NO.3, has β subunits and α subunits that are any one of the following (1)-(11): (1) The leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 is mutated to phenylalanine; the α subunit is shown in SEQ ID NO.2; (2) Mutate leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 to phenylalanine; mutate valine at position 37 of the amino acid sequence of the α subunit shown in SEQ ID NO.2 to isoleucine; (3) Mutate leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 to phenylalanine; mutate valine at position 37 of the amino acid sequence of the α subunit shown in SEQ ID NO.2 to isoleucine and serine at position 89 to glutamic acid; (4) Mutate the leucine at position 34 of the β subunit shown in SEQ ID NO.1 to phenylalanine and the asparagine at position 167 to glycine; mutate the valine at position 37 of the α subunit shown in SEQ ID NO.2 to isoleucine and the serine at position 89 to glutamic acid. (5) Mutate the leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 to phenylalanine and the asparagine at position 167 to glycine; mutate the valine at position 37 of the amino acid sequence of the α subunit shown in SEQ ID NO.2 to isoleucine, the serine at position 89 to glutamic acid, and the valine at position 101 to cysteine. (6) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to aspartic acid, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (7) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and with the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to lysine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (8) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine, asparagine at position 167 to glycine, and asparagine at position 152 to cysteine; and with the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (9) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to leucine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (10) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to threonine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (11) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, mutating leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, mutating valine at position 37 to isoleucine, serine at position 89 to glutamic acid, valine at position 101 to cysteine, and tryptophan at position 155 to threonine.

[0009] Specifically, the mutant is NHase. M1 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.5, the amino acid sequence of the α subunit as shown in SEQ ID NO.2, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M2 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.5, the amino acid sequence of the α subunit as shown in SEQ ID NO.6, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M3 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.5, the amino acid sequence of the α subunit as shown in SEQ ID NO.7, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase.M4 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.8, the amino acid sequence of the α subunit as shown in SEQ ID NO.7, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M5 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.8, the amino acid sequence of the α subunit as shown in SEQ ID NO.9, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M6-1 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.8, the amino acid sequence of the α subunit as shown in SEQ ID NO.10, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M6-2 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.8, the amino acid sequence of the α subunit as shown in SEQ ID NO.11, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M6-3 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.12, the amino acid sequence of the α subunit as shown in SEQ ID NO.11, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M6-4 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.12, the amino acid sequence of the α subunit as shown in SEQ ID NO.13, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M6-5 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.12, the amino acid sequence of the α subunit as shown in SEQ ID NO.14, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. Specifically, the mutant is NHase. M6-6 The mutant has the amino acid sequence of the β subunit as shown in SEQ ID NO.12, the amino acid sequence of the α subunit as shown in SEQ ID NO.15, and the amino acid sequence of the activator protein A as shown in SEQ ID NO.3. This invention also relates to the coding gene of the above-mentioned nitrile hydratase mutant.

[0010] The second technical solution provided by the present invention is a recombinant vector or recombinant strain containing the nitrile hydratase mutant encoding gene described in the first technical solution; Furthermore, the expression vector used in the recombinant vector includes, but is not limited to, pET29b; Furthermore, the host cells used for the recombinant strain include, but are not limited to, Escherichia coli; preferably, Escherichia coli BL21(DE3). Preferably, the β subunit, α subunit, and activator protein A gene segments of the nitrile hydratase mutant described in one of the technical solutions are ligated and constructed into the pET-29b vector, and expressed in BL21(DE3) to obtain the recombinant strain.

[0011] The third technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the second technical solution, particularly its application in the production of the nitrile hydratase mutant described in the first technical solution.

[0012] The fourth technical solution provided by this invention is the application of the nitrile hydratase mutant or its encoding gene described in the first technical solution, or the recombinant vector or recombinant strain described in the second technical solution, particularly in the production of amide compounds; or in the catalytic generation of amide compounds from nitrile compounds. Furthermore, the nitrile compounds include, but are not limited to, at least one of: 2,6-difluorobenzonitrile, nicotinic nitrile, cinnamonitrile, and acrylonitrile; Furthermore, the amide compounds include, but are not limited to: 2,6-difluorobenzamide, nicotinamide, cinnamamide, acrylamide, and other amide substances.

[0013] Beneficial effects: 1. The nitrile hydratase mutant provided by this invention has a maximum enzyme activity of 3649.95 U / g DCW per cell dry weight, which is 142.98% higher than that of the wild type. The nitrile hydratase purified by the cobalt column purification method provided by this invention has a specific enzyme activity of up to 3589.44 U / mg, which greatly improves the efficiency of converting nitrile substances into amide products. In particular, it has a significant effect on improving the enzyme activity of toxic substrates, and the purified enzyme solution has good stability at 4°C.

[0014] 2. Taking the formation of 2,6-difluorobenzonitrile from 2,6-difluorobenzamide as an example, under the same substrate concentration (2 mol / L) and resting cell concentration (OD), 600 Under the same transformation conditions (=15.5) and conditions, the optimal mutant strain achieved a space-time yield of 5 mol / L / h, which was nearly 5 times higher than that of the wild-type strain. Liquid chromatography analysis showed that the conversion rate of 2,6-difluorobenzonitrile was 100%, the selectivity for 2,6-difluorobenzoamide was 100%, and no byproducts were generated.

[0015] 3. The resting cell transformation ability and substrate product tolerance of the nitrile hydratase mutant provided by the present invention are nearly three times higher than those of the wild-type strain. Compared with the prior art, transformation using the mutant provided by the present invention can achieve the effect of rapid production of 2,6-difluorobenzamide and other amide substances.

[0016] 4. The methodological advantage of this invention lies in the combination of multiple strategies, such as multi-cooperative auxiliary site prediction and screening, consensus sequence design, and strong coupling site co-evolution, to assist in the modification of nitrile hydratase. Furthermore, by leveraging the absorbance changes in the visible or ultraviolet regions of 2,6-difluorobenzonitrile after emulsification, a high-throughput screening method is established to rapidly screen nitrile hydratase mutants. Attached Figure Description

[0017] Figure 1 This is a map of the recombinant plasmid pET29b-NHase-WT for nitrile hydratase.

[0018] Figure 2 Wild-type NHase WT With the six-point mutant NHase M6-3 SDS-PAGE images before and after purification Where M: Marker; W: Whole bacteria; S: Supernatant; P: Precipitate; FT: Flow-through; Wash: Washing; E: Elution; Subscript 1 and the left figure represent NHase. WT Subscript 2 and the right figure show the six-point mutant NHase. M6-3 .

[0019] Figure 3 Wild-type NHase WT With the six-point mutant NHase M6-3 Native-PAGE image after purification Where M: Marker; Pro M6-3 : Six-point mutant NHase M6-3 Pure enzyme; Pro WT : Wild-type NHase WT Pure enzyme; M 6-3 -S: Six-point mutant NHase M6-3 Supernatant; M 6-3 -W: Six-point mutant NHase M6-3 Whole bacterial solution.

[0020] Figure 4 Wild-type NHase WT With the six-point mutant NHase M6-3 A comparison chart of the optimal temperatures for pure enzymes. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. The following embodiments are descriptive and not limiting, and should not be construed as limiting the scope of protection of this invention.

[0022] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0023] 1. The primer synthesis and sequencing in the embodiments of the present invention were jointly completed by Suzhou Genewiz Biotechnology Co., Ltd. and Youkang Biotechnology Co., Ltd.

[0024] 2. Some of the culture media used in this invention are as follows: LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar powder 20 g / L.

[0025] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L.

[0026] 3. The high-throughput screening method established in this invention is as follows: This invention targets nitrile substrates such as 2,6-difluorobenzonitrile, which are insoluble in water and have a melting point of 30-32℃, and amide products such as 2,6-difluorobenzoamide, which are also insoluble in water and have a melting point above 100℃. Based on the characteristic phenomena of these substrates after ultrasonic emulsification with Tween-80, a rapid screening method based on the change in absorbance before and after conversion is established. This method can be used to screen nitrile substrates by OD (Obstacle Course) changes. 600 or OD 254 The reduction in substrate consumption or the time it takes for the reaction solution to become clear is used to determine the substrate consumption or the reaction endpoint. The specific implementation is as follows: 2,6-Difluorobenzonitrile is added to Tween-80 and ultrasonically emulsified until it becomes milky white. This emulsion is then added to a 96-well plate. Different bacterial cultures or pure enzyme solutions are then added for substrate conversion. As the substrate is consumed, the emulsification gradually weakens until the conversion is complete. Simultaneously, because the product is insoluble in water and has a high melting point, it precipitates out as a solid and accumulates in the lower layer, resulting in layering with the upper layer becoming clear. Multiple experiments and liquid chromatography analysis have verified this as the reaction endpoint, successfully confirming the feasibility of this method.

[0027] 4. The method for determining the activity of nitrile hydratase used in this invention is as follows: 100 μL OD 600Add approximately 1.0 μL of bacterial culture to 400 μL of a 125 mmol / L 2,6-difluorobenzonitrile emulsion or substrate solution containing 1% Tween-80 prepared with 1× TBS solution, or add 10 μL of 0.05 mg / mL nitrile hydratase pure enzyme solution to 490 μL of a 125 mmol / L 2,6-difluorobenzonitrile emulsion or substrate solution containing 1% Tween-80 prepared with 1× TBS solution. React at 25°C and 200 rpm in a shaker for 10 min. Terminate the reaction by adding 500 μL of acetonitrile. After ultrasonic extraction for 30 min, centrifuge at 13000 rpm for 10 min and collect the supernatant. Dilute the supernatant 10-fold with the mobile phase, sonicate for 30 min, and then filter 200 μL of the solution through a 0.22 μm microporous membrane. Use C... 18 DFBN was detected by HPLC using a reversed-phase column, and its retention time and peak area at 254 nm were observed to calculate the enzyme activity per unit or the enzyme activity per unit of pure enzyme.

[0028] The HPLC analysis conditions are shown in Table 1 below: Table 1 HPLC analysis conditions

[0029] The enzyme activity of nitrile hydratase is defined as follows: (1) Nitrile hydratase activity per unit cell dry weight: the cell dry weight required to catalyze the production of 1 μmol of amide product from nitrile substrate in 1 min at 25℃;

[0030] Wherein, C is the molar concentration of the product (μmol / mL); V is the reaction volume (mL); X is the dilution factor before HPLC injection; T is the reaction time; and v is the added OD. 600 =1 - bacterial culture volume; n - bacterial culture dilution factor; 3.1586 - bacterial cell dry weight and OD 600 The formula for converting standard curves.

[0031] (2) Unit enzyme activity of nitrile hydratase: The amount of enzyme (U) required to catalyze the formation of 1 μmol of amide product from nitrile substrate in 1 min at 25℃; (3) Nitrile hydratase pure enzyme activity: The enzyme activity (U / mg) of 1 mg nitrile hydratase pure enzyme.

[0032]

[0033] Wherein, C is the molar concentration of the product (μmol / mL); V is the reaction volume (mL); X is the dilution factor before HPLC injection; T is the reaction time; v is the volume of the added pure enzyme solution; and c is the concentration of the added pure enzyme solution (mg / mL).

[0034] 5. Some of the primers involved in the embodiments of this invention are shown in Table 2 below: Table 2 Primer Table

[0035] The present invention will be further explained and illustrated below through specific embodiments.

[0036] Example 1: Establishment of a heterologous expression system for nitrile hydratase (1) Target gene: from Rhodococcus rubescens ( Rhodococcus ruber The genome of CGMCC No. 3090 was used sequentially with primers NHase β-F and NHase β-R, NHase α-F and NHase α-R, NHase AF and NHase AR to amplify the gene sequences of nitrile hydratase β subunit, α subunit and activator protein A. Some codons were optimized based on the codon bias of E. coli. After being verified by sequencing, they were used as templates for subsequent operations.

[0037] (2) Expression vector: pET29b carrying the lac Z gene and 6× His tag was selected as the final expression vector.

[0038] (3) Construction of nitrile hydratase expression vector: Since the nitrile hydratase gene sequence from Rhodococcus faecalis contains multiple terminators and the expression level of the α subunit is low in Escherichia coli, two additional RBS regions were added. The β subunit-α subunit-activator protein A gene segments were expressed in tandem using primers Nhase β-F and Nhase AR overlap PCR (the obtained sequence is shown in SEQ ID NO. 4). Then, it was expressed in the same way. Nde I and Hind The pET-29b vector obtained by double enzyme digestion was ligated using a seamless cloning method and sequentially transformed into *E. coli* DH5α and BL21(DE3) competent cells. Positive clones were selected, plasmids were extracted, and analyzed. Sequencing and subsequent transformation confirmed that nitrile hydratase was correctly folded and expressed in *E. coli*, successfully obtaining the wild-type recombinant plasmid pET29b-NHase-WT (plasmid map shown). Figure 1 (as shown), and the BL21 recombinant strain BL21-NHase containing this plasmid. WT .

[0039] Example 2: Determination of key modification sites for nitrile hydratase 1. Server-based virtual saturation mutation prediction Based on the results of virtual saturation mutations from the Fireprot 2.0 and TurNup websites, Fold X and Rosetta < -1.0 kcal / mol with Kcat values ​​higher than wild-type nitrile hydratase (NHase) were selected. WT Forty stable mutations were subjected to site-directed mutagenesis, namely: αS89Q, αD148P, αA33W, αS89L, αR153I, αS89F, αS89W, αT10S, αV37I, αE68M, αT198F, αS89M, αE168P, αA119P, αT198M, αE68P, αS89I, αS89V, and αS89Y. The mutants identified were αM124P, βR94L, βT7M, βL34F, βC218Y, βC218F, βT38I, βG213P, βA138P, βT38F, βN167G, βS228P, βN152P, βT38L, βS51M, βC218L, βS77Y, βA187L, βS51A, βS46Y, and βL34H. After further screening and verification, the obtained positive single-point mutants or their saturated mutants were freely combined to obtain positive two-point and three-point mutants.

[0040] 2. Design of Consensus Sequence Analysis and Co-evolutionary Coupled Network The interaction between the nitrile hydratase and the DFBN-binding pocket after docking was analyzed using the Protein-Ligand Interaction Profiler website. Evolutionary consensus sequences and conservation analyses were then performed on the key active sites of the pocket, identifying five key sites: αV101, αT104, αS107, αY109, and αV113. Taking the αV101 site as an example, saturation mutagenesis was performed on this site using degenerate primers αV101JB-RF and αV101JB-RR.

[0041] The evolutionary coupling network of the two subunits was predicted using the EVcouplings website. Co-evolutionary sites were selected based on the contribution of amino acids to the binding energy. Five co-evolutionary pairs were selected: α subunit: αV37-αN43, αS89-αR153, αH99-αW155; β subunit: βN167-βV150, βN167-βN152. Co-evolutionary pairs or single-point mutations involved in co-evolutionary pairs can be directly introduced.

[0042] Semi-rational degenerative primers were designed for the above sites, and positive four- to six-point mutants were obtained through screening and recombination.

[0043] Example 3: Construction of site-directed mutants of nitrile hydratase 1. Design of specific or degenerate primers Based on the structural analysis, forward and reverse primers were designed according to the location and requirements of the predicted or key sites determined in Example 2. The mutation primers corresponding to some mutation sites are shown in Table 2.

[0044] 2. Amplification and purification of mutant expression plasmids Using the wild-type recombinant plasmid pET29b-NHase-WT of nitrile hydratase constructed in Example 1 as a template, the primers designed in step 1 were used for site-directed mutagenesis or saturation mutagenesis, as follows: (1) PCR reaction system (50 μL): 25 μL 2 × Phanta Max Master Mix (Vazyme), 2 μL each of upstream and downstream primers (10 μM), 1 μL (10 ng) DNA template (plasmid pET29b-NHase-WT), 20 μL dd H2O.

[0045] (2) PCR reaction procedure: Table 3 PCR reaction procedure

[0046] (3) DpnI digestion to remove template: Add 1 μL of restriction endonuclease Dpn I (Vazyme) and 5 μL of 10× Cut Buffer to the verified correct PCR reaction product, and react in a metal bath at 37℃ for 1 h.

[0047] (4) Purification and recovery of target amplification products: The digested reaction solution was purified using the Cycle-Pure Kit (ORMGA) to obtain high-purity PCR amplification products.

[0048] 3. Construction and transformation of recombinant plasmids The purified PCR amplification product (10 μL) was ligated in a 50°C metal bath for 30 min using 2 × Clon Express Mix (Vazyme) ligase, and then sequentially transformed into E. coli. E.Coli DH5α and BL21 competent cells were cultured in an incubator at 37°C for 12–16 h.

[0049] 4. Colony PCR and sequencing verification Transformants cultured overnight at 37°C were used to select single colonies for colony PCR and double enzyme digestion or sequencing verification. The correctly verified recombinant bacteria were preserved for subsequent screening. The recombinant plasmid containing the mutant was named pET29b-NHase-X, where X represents the mutation site. For example, pET29b-NHase-βL34F represents a recombinant plasmid containing a nitrile hydratase with an L34F mutation in the β subunit, based on the wild-type nitrile hydratase.

[0050] Example 4: Screening of site-directed mutants of nitrile hydratase 1. Initial screening of nitrile hydratase mutants Preliminary screening of cells grown in 24-well plates: Add 4 mL of sterile LB liquid medium to each well of a sterilized 24-well deep-well plate, and add 2 μL of 50 μg / mL kanamycin to each well. Add 10 μL of nitrile hydratase mutant bacterial solution (transformant bacterial solution obtained in step 4 of Example 3) stored at -80 ℃ to each well, with three replicates for each mutant. Seal the wells with sterile plate membrane and incubate at 37 ℃ and 220 r / min on a shaker for 12-16 h. In a clean bench, add Co to the 24-well deep-well plate at a final concentration of 0.1 g / L. 2+ In addition to 0.2 mmol / L IPTG solution, expression was induced for 12 h at 30℃ and 200 r / min. 20 μL of the induced expression was added to a sterile 96-well plate, and simultaneously 80 μL of sterile 2,6-difluorobenzonitrile emulsified with Tween-80 was added to each well. Triple replicates were maintained, and cell transformation was performed in a 30℃ plate shaker. The OD values ​​of the transformation solution in the 96-well plate were recorded at different transformation times. 600 The conversion process continues until the conversion solution changes from a homogeneous emulsion to a clear upper layer, indicating the end of the conversion. Select solutions with short conversion completion times or high OD values. 600 Mutants with rapidly decreasing values ​​were subjected to large-scale rescreening.

[0051] 2. Rescreening of nitrile hydratase mutants The mutant recombinant strain and the wild-type recombinant strain BL21-NHase that passed the initial screening WT Three-zone activation was performed, followed by overnight incubation at 37°C. A single colony was picked and inoculated into 50 mL LB broth containing 50 μg / mL kanamycin, and incubated at 37°C in a shaker until OD (digestive activity) was reached. 600 The inoculum concentration was 0.6–1.0; then, at a 2% inoculum rate, it was transferred to 200 mL of LB medium containing kanamycin and cultured in a shaker at 37°C until the OD value reached 0.6–1.0. 600When the protein expression level is approximately 0.6–0.8, isopropyl thiogalactoside (IPTG) at a final concentration of 0.2 mmol / L is added as a protein expression inducer and CoCl2 solution at a final concentration of 0.1 g / L is added as an activator. The cells are then induced and cultured in a shaker at 30°C for 12–14 h. After harvesting the cells, they are washed twice with 1× TBS solution to obtain resting cells, which are then stored at -20°C for later use.

[0052] Add 2 mol / L 2,6-difluorobenzonitrile (DFBN) emulsified by Tween-80 sonication to a 100 mL Erlenmeyer flask, add resting bacterial cells with a DCW of 5 g / L, and add 1× TBS buffer to a total volume of 10 mL. Incubate in a shaker at 30℃ and 200 r / min for transformation, sampling every 1 h. Take 100 μL of the transformation buffer, add 600 μL of ethyl acetate and 300 μL of methanol for extraction, sonicate to mix, centrifuge, and collect 2 μL of the supernatant. Spot the sample onto an analytical thin-layer chromatography silica gel plate 1 cm below the bottom. Develop the plate in a chromatography tank containing developing solvent. Once the solvent extends to 1 cm from the top edge of the silica gel plate, remove the plate, allow it to dry naturally, and observe the spots under a 254 nm UV lamp to assess the transformation effect. After further drying, reconstitute the extract with the mobile phase, sonicate for 30 min, and then analyze the transformation using liquid chromatography. Record the transformation completion time. Simultaneously, the whole-cell catalytic activity of resting cells was measured in EP tubes, and the enzyme activity per unit dry weight was calculated. Mutants with strong substrate tolerance and high enzyme activity per unit were selected for further modification.

[0053] 3. Screening results of nitrile hydratase mutants Table 4 Comparison of enzyme activity per unit cell dry weight between wild-type and single-point mutants

[0054] As shown in Table 4 above, the positive single-point mutants identified in step 2 include: βL34F, αV37I, αD148P, αS89L, and αT10S. The optimal mutant βL34F showed a 46.23% increase in enzyme activity compared to the wild-type WT. The other positive mutants also showed varying degrees of increase in enzyme activity per unit cell dry weight. Furthermore, their resting cells exhibited good stability at 30-40℃, which can meet the transformation requirements.

[0055] The optimal single-point mutant βL34F was named NHase. M1 The mutant strain, and the recombinant strain containing the pET29b-NHase-βL34F recombinant plasmid, was named BL21-NHase. M1Based on this, it was combined with other positive single-point mutants αD148P, αV37I, αT10S, and αS89L for combined mutagenesis, resulting in 10 double-point mutants. After screening and verification, the optimal double-point mutant βL34F-αV37I was obtained and named NHase. M2 The mutant strain, and the recombinant strain containing the pET29b-NHase-βL34F-αV37I recombinant plasmid, was named BL21-NHase. M2 Based on this, saturation mutations were performed on the remaining sites αD148, αT10, and αS89, and the optimal positive three-point mutant βL34F-αV37I-αS89E was obtained and named NHase. M3 The mutant strain, and the recombinant strain containing the pET29b-NHase-βL34F-αV37I-αS89E recombinant plasmid, was named BL21-NHase. M3 .

[0056] Then, by saturating the key modification sites obtained from the consensus sequence design and introducing co-evolutionary coupling pairs or single-point mutations involved in the co-evolutionary pairs, the optimal positive four-point mutant βL34F-αV37I-αS89E-βN167G was obtained, named NHase. M4 The mutant strain, and the recombinant strain containing the pET29b-NHase-βL34F-αV37I-αS89E-βN167G recombinant plasmid, was named BL21-NHase. M4 The optimal positive five-point mutant βL34F-αV37I-αS89E-βN167G-αV101C was named NHase. M5 The mutant, and the recombinant strain containing the pET29b-NHase-βL34F-αV37I-αS89E-βN167G-αV101C recombinant plasmid, was named BL21-NHase. M5 ; and six positive six-point mutants of NHase M6-1 NHase M6 -2 NHase M6-3 NHase M6-4 NHase M6-5 NHase M6-6 And strain BL21-NHase, which contains a six-point mutant recombinant plasmid. M6-1 - BL21-NHase M6-6 For details, please see Table 5 below.

[0057] Table 5. Comparison of enzyme activity per unit cell dry weight of wild-type and recombinant bacteria containing mutants.

[0058] The results above show that, under the multi-strategy auxiliary site screening combination, the enzyme activity per unit dry weight of bacteria shows an increasing trend with the accumulation of the number of mutations. NHase containing the optimal nitrile hydratase six-point mutant is included. M6-3 The enzyme activity per unit cell dry weight reached 3649.95 U / g DCW. This is due to BL21-NHase. M6-4 BL21-NHase M6-5 BL21-NHase M6-6 The enzyme activity and space yield were not significantly different from those of the five-point mutant, so they were not included in subsequent experiments.

[0059] Example 5: Verification of the production performance of nitrile hydratase by fed-batch fermentation To BL21-NHase WT BL21-NHase M1 BL21-NHase M2 BL21-NHase M3 BL21-NHase M4 BL21-NHase M5 BL21-NHase M6-1 BL21-NHase M6-2 BL21-NHase M6-3 Escherichia coli resting cells (OD) 600 Add 2 mol / L of 2,6-difluorobenzonitrile (DFBN) ultrasonically emulsified with Tween-80 to a solution of 15.55 g / L, and then add 1× TBS buffer to a total volume of 10 mL. Place in a shaker at 30°C and 200 rpm for batch-feed conversion of DFBN (2 mol / L DFBN added per batch, adding the next batch only after the previous batch has been completely converted). Conversion rate was detected by HPLC. If conversion was complete, the substrate was added again; if incomplete conversion was observed, the conversion time and conversion rate at that feed concentration were recorded. The conversion ability of each bacterium after 16 h was statistically analyzed. Relevant data are recorded in Table 6. Table 6 Fed-batch fermentation of wild type and mutant

[0060] As shown in the table above, NHase WT The 4 mol / L DFBN could not be completely converted; the conversion rate was 59.42% after 6 h, and the final conversion rate reached 64.13% after 16 h; the mutant BL21-NHase... M1 BL21-NHase M2 BL21-NHase M3 6 mol / L LDFBN can be converted in 16 h; mutant NHaseM4 NHase M5 It can convert 8 mol / L DFBN in 16 h; mutant NHase M6-1 NHase M6-2 It can convert 10 mol / L DFBN in 16 hours; the optimal mutant strain is BL21-NHase. M6-3 12 mol / L (1668 g / L) DFBN can be converted in 16 h. After liquid phase detection and product recovery and drying, the final conversion rate of 2 mol / L DFBN can reach 98.21%. Without removing the toxic effects of the product, it can simultaneously tolerate 2 mol / L DFBN and 10 mol / L DFBA (the generated product remains in the reaction system and is not removed, but still has catalytic activity, thus it is considered to have tolerance). This is better than NHase. WT Its conversion ability and tolerance are improved by nearly 3 times.

[0061] Example 6: Purification and enzyme activity assay of wild-type and optimal six-point mutant Select the optimal expression mutant NHase M6-3 The plasmids pET29b-NHase-βL34F-βN152C-βN167G-αV37I-αS89E-αV101C and pET29b-NHase-WT were modified by adding (GS) linker+6×his tags to the N-terminus and C-terminus of their β subunits, respectively. The recombinant plasmids were then transformed into BL21 competent cells, and positive transformants were screened. Expression was induced according to the method in Example 4. Wet cells were collected at low temperature, washed twice with 1× TBS buffer, and then frozen overnight at -20°C before protein purification and enzymatic characterization.

[0062] Resuspend the bacterial pellet in 9 mL of 1× TBS buffer (pH 7.2) to ensure thorough dispersion. Add 1 mL of Hammer Super Lysis Buffer and vortex to mix thoroughly. Place the sample on ice for 10 min to lyse. Centrifuge the lysed protein buffer at 8000 rpm for 30 min. Filter the supernatant through a 0.22 μm microporous membrane and purify using a cobalt column. The main procedures are as follows: Equilibrate the packed Co NTA Beads gravity column with 5 column volumes of Lysis Buffer to ensure the packing material is in the same buffer system as the target protein. Repeat 2-3 times.

[0063] Add the sample to the balanced gravity column and retain it for at least 2 minutes, or incubate it on ice for 1 hour to ensure full contact between the sample and the medium. Collect the effluent and repeat the loading process to increase the binding efficiency.

[0064] Use 10-15 column volumes of Wash Buffer to wash away non-specific contaminating proteins and collect the wash buffer.

[0065] Elute with 5-10 column volumes of Elution Buffer, collect fractions, and analyze them separately until no protein is detected by the BCA protein concentration assay kit.

[0066] After elution, rinse with Lysis Buffer for 3 column volumes, then rinse with pure water for 5 column volumes, then rinse with 20% ethanol for 2 column volumes, and then store the medium at 2-8°C.

[0067] wild-type NHase WT and the six-point mutant NHase M6-3 The purified samples (including elution fraction, washing fraction, elution fraction, and column washing fraction), as well as the original crude enzyme solution, whole bacteria, precipitate, and supernatant, were analyzed for purification efficiency using SDS-PAGE and Native-PAGE.

[0068] SDS-PAGE results are as follows Figure 2 As shown, the left image is of wild-type NHase. WT SDS-PAGE images before and after purification; the right image shows the six-point mutant NHase. M6-3 SDS-PAGE images before and after purification. SDS-PAGE analysis showed that the theoretical molecular weights of the β and α subunits of the nitrile hydratase were 27.4 kDa and 22.8 kDa, respectively. The purification of the nitrile hydratase expressed by both strains was preliminarily completed, and related contaminating proteins were largely removed.

[0069] Since SDS-PAGE can only analyze denatured subunit proteins, native-PAGE is needed to analyze proteins with intact conformations. The results are as follows: Figure 3 As shown. The theoretical molecular weight of the nitrile hydratase tetramer is 98.2 kDa, which is... Figure 3 The results are largely consistent, indicating that the nitrile hydratase maintains a tetrameric configuration and that the protein purity is higher than 95%.

[0070] After the purified protein was concentrated by ultrafiltration and desalting, the protein concentration was adjusted to 0.05 mg / mL using a BCA protein concentration assay kit. The nitrile hydratase ratio activity was then measured by reacting the protein in a shaker at 25°C and 200 r / min for 10 min using the enzyme activity assay method described above.

[0071] Enzyme activity data are shown in Table 7 below, for the six-point mutant NHase of nitrile hydratase. M6-3 The highest enzyme activity of the pure enzyme can reach 3589.44 U / mg.

[0072] Table 7. Specific enzyme activities of wild-type and optimal mutant pure enzymes on different substrates.

[0073] Wild-type NHase WT With the optimal six-point mutant NHase M6-3 The pure enzyme solution (0.05 mg / mL) was reacted at different temperatures (20-65℃) for 10 min using the aforementioned method for determining enzyme activity. Three replicates were performed for each data set to detect enzyme activity within that temperature range. Since wild-type NHase... WT With the optimal six-point mutant NHase M6-3 Since the enzyme activity data varied considerably, the enzyme activity measured at its optimal temperature was defined as 100%, and the enzyme activity measured at other temperatures was defined as relative enzyme activity. The relative enzyme activity (%) of the corresponding enzyme at different temperatures was calculated.

[0074] pass Figure 4 It can be seen that the activity of nitrile hydratase first increases and then decreases with increasing temperature, exhibiting the highest activity of over 80% in the range of 27~40℃. When the temperature rises to 50℃, the optimal six-point mutant NHase... M6-3 It can still maintain more than 50% of its peak activity, while wild-type NHase WT The enzyme activity is less than 30%.

[0075] With wild-type NHase WT In comparison, the optimal six-point mutant NHase M6-3 The optimal temperature is 37℃, which is higher than that of wild-type NHase. WT The optimal temperature is 27℃. Increasing the optimal temperature is beneficial to substrate dissolution and catalytic reaction rate, and further confirms that the optimal six-point mutant NHase is the best. M6-3 Compared to the wild type, its temperature tolerance has been improved to some extent.

[0076] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A nitrile hydratase mutant, characterized in that, The nitrile hydratase mutant was obtained by mutation based on wild-type nitrile hydratase. The wild-type nitrile hydratase is composed of a β subunit, an α subunit, and an activating protein A. The amino acid sequence of the β subunit is shown in SEQ ID NO.1, the amino acid sequence of the α subunit is shown in SEQ ID NO.2, and the amino acid sequence of the activating protein A is shown in SEQ ID NO.

3. The amino acid sequence of the activating protein A of the nitrile hydratase mutant is shown in SEQ ID NO.3, and the β subunit and α subunit are any one of the following (1)-(11): (1) The leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 is mutated to phenylalanine; the amino acid sequence of the α subunit is shown in SEQ ID NO.2; (2) Mutate the leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 to phenylalanine; mutate the valine at position 37 of the amino acid sequence of the α subunit shown in SEQ ID NO.2 to isoleucine; (3) Mutate leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 to phenylalanine; mutate valine at position 37 of the amino acid sequence of the α subunit shown in SEQ ID NO.2 to isoleucine and serine at position 89 to glutamic acid; (4) Mutate the leucine at position 34 of the β subunit shown in SEQ ID NO.1 to phenylalanine and the asparagine at position 167 to glycine; mutate the valine at position 37 of the α subunit shown in SEQ ID NO.2 to isoleucine and the serine at position 89 to glutamic acid. (5) Mutate the leucine at position 34 of the amino acid sequence of the β subunit shown in SEQ ID NO.1 to phenylalanine and the asparagine at position 167 to glycine; mutate the valine at position 37 of the amino acid sequence of the α subunit shown in SEQ ID NO.2 to isoleucine, the serine at position 89 to glutamic acid, and the valine at position 101 to cysteine. (6) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to aspartic acid, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (7) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and with the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to lysine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (8) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine, asparagine at position 167 to glycine, and asparagine at position 152 to cysteine; and with the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (9) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to leucine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (10) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, with leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, with valine at position 37 to isoleucine, asparagine at position 43 to threonine, serine at position 89 to glutamic acid, and valine at position 101 to cysteine. (11) Mutate the amino acid sequence of the β subunit shown in SEQ ID NO.1, mutating leucine at position 34 to phenylalanine and asparagine at position 167 to glycine; and mutate the amino acid sequence of the α subunit shown in SEQ ID NO.2, mutating valine at position 37 to isoleucine, serine at position 89 to glutamic acid, valine at position 101 to cysteine, and tryptophan at position 155 to threonine.

2. The encoding gene of the nitrile hydratase mutant according to claim 1.

3. A recombinant vector or recombinant strain containing the coding gene of the nitrile hydratase mutant of claim 2.

4. The recombinant vector as described in claim 3, characterized in that, The recombinant vector uses pET29b as its expression vector.

5. The recombinant strain according to claim 3, characterized in that, The host cells used for the recombinant strain include Escherichia coli.

6. The use of the recombinant vector or recombinant strain according to claim 3 in the production of the nitrile hydratase mutant according to claim 1.

7. The use of the nitrile hydratase mutant of claim 1, or the encoding gene of claim 2, or the recombinant vector or recombinant strain of claim 3 in catalyzing the formation of amide compounds from nitrile compounds; The nitrile compound is 2,6-difluorobenzonitrile, and the amide compound generated by catalysis is 2,6-difluorobenzoamide; The nitrile compound is nicotinic nitrile, and the amide compound generated by catalysis is nicotinamide.

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