Amidase and its mutant and application

Abstract

The present invention provides an amidase and its mutants and applications, belonging to the fields of genetic engineering and enzyme catalysis technology. The amino acid sequence of the amidase is shown in SEQ ID No.1, or has more than 80% identity with the amino acid sequence shown in SEQ ID No.1 and has amidase activity; the first mutant of the amidase is obtained by mutating one or more sites in the amino acid sequence of SEQ ID No.1 at positions 77, 312, and 315; the second mutant of the amidase is obtained by mutating one or more sites in the amino acid sequence of SEQ ID No.1 at positions 76, 120, and 208. The amidase and its mutants of the present invention can hydrolyze racemic alkylamides to prepare chiral alkylanilines, and the prepared chiral alkylanilines have high optical purity and an ee value greater than 99%.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and enzyme catalysis, in particular to an amidase and a mutant thereof and application thereof. Background Art

[0002] Amidases are a class of enzymes that catalyze the hydrolysis or synthesis of amide bonds and are widely found in microorganisms, plants, and animals. Based on their primary structure and functional properties, amidases can be divided into multiple families, including the amidase family, the nitrile amidase superfamily, and the acetylamidase / formamidase family. Furthermore, based on substrate specificity, amidases can be further subdivided into carboxamidase and α-amino acid amidase types. Amidases have a very broad substrate range, encompassing a variety of compounds, including aliphatic amides, aromatic amides, heterocyclic amides, and α-amino acid amides. This broad substrate specificity makes amidases important catalytically active in a variety of biochemical reactions, such as nitrogen metabolism, drug metabolism, and the degradation of environmental pollutants.

[0003] The catalytic mechanism of amidases involves multiple steps, including substrate binding, intermediate formation, and product release. Studies have shown that enzymes in the nitrilases superfamily exist in solution as homotetrameric or homohexameric complexes, with a conserved catalytic triad of Glu, Cys, and Lys at the catalytic center. Amidases of this family form homodimeric or homooctameric complexes, characterized by a highly conserved sequence of approximately 130 amino acids, including the Ser, Ser, and Lys catalytic triad. Certain amidases exhibit different preferences for aliphatic and aromatic amides and significant differences in enantioselectivity. This characteristic holds great potential for their application in asymmetric reactions. For example, an amidase from Galactomyces geotrichum exhibits high catalytic activity towards n-butyramide, isobutyramide, and acetamide, with enantiomeric excesses (ee) reaching 99.9% for certain chiral substrates.

[0004] However, existing amidases suffer from poor stereoselectivity, failing to meet demand. Furthermore, when expressed in E. coli, amidases often exist as inclusion bodies, requiring a complex renaturation process to obtain catalytically active enzymes. This problem severely limits the application of amidases in industrial production. Summary of the Invention

[0005] To address the above-mentioned problems in the prior art, the present invention provides an amidase and its mutants and their applications. The amidase and its mutants can hydrolyze racemic alkylamides to prepare chiral alkylanilines, and the prepared chiral alkylanilines have high optical purity and an ee value greater than 99%.

[0006] The technical solutions of the present invention are as follows:

[0007] 1. The present invention first provides an amidase for hydrolyzing an alkylamide to produce an alkylaniline, wherein the amidase has an amino acid sequence as shown in SEQ ID No. 1, or has an amino acid sequence with more than 80% identity with the amino acid sequence shown in SEQ ID No. 1 and has amidase activity;

[0008] The alkylamide has a structure shown in the following formula I:

[0009]

[0010] (Formula I)

[0011] wherein R is a C1-C9 alkyl group;

[0012] The alkylaniline has the structure shown in the following formula II:

[0013]

[0014] (Formula II)

[0015] 2. The present invention also provides a first mutant of the above-mentioned amidase, which is obtained by performing site-directed mutagenesis on the amino acid sequence shown in SEQ ID No. 1, and the mutation site is one or more of the amino acids at positions 77, 312, and 315.

[0016] Furthermore, the amino acid at position 77 was mutated from G to F, the amino acid at position 312 was mutated from A to W, and the amino acid at position 315 was mutated from T to F.

[0017] 3. The present invention also provides the use of the mutant of the first amidase, which is used for selectively hydrolyzing racemic alkylamide or R-alkylamide to produce R-alkylaniline.

[0018] 4. The present invention further provides a second mutant of the above-mentioned amidase, which is obtained by site-directed mutagenesis of the amino acid sequence shown in SEQ ID No. 1, wherein the mutation site is one or more of amino acids 76, 120, and 208.

[0019] Furthermore, the amino acid at position 76 was mutated from S to A, the amino acid at position 120 was mutated from A to L, and the amino acid at position 208 was mutated from I to A.

[0020] 5. The present invention also provides the use of the second amidase mutant, i.e., for selectively hydrolyzing racemic alkylamide or S-alkylamide to produce S-alkylaniline.

[0021] 6. The present invention further provides a method for constructing a recombinant Bacillus subtilis that efficiently expresses amidase, the method comprising the following steps:

[0022] A1. Cloning a promoter, a signal peptide encoding gene, an amidase encoding gene and a terminator to construct a recombinant expression cassette. The amidase encoding gene can encode the above-mentioned amidase or two mutants of amidase;

[0023] A2. Transforming the recombinant expression cassette into Bacillus subtilis. The recombinant expression cassette exists in the Bacillus subtilis in the form of a free plasmid or is integrated into the Bacillus subtilis genome.

[0024] Furthermore, the DNA sequence of the promoter is shown in SEQ ID No. 2 of the sequence listing, the signal peptide encoding gene is shown in SEQ ID No. 3 of the sequence listing, and the DNA sequence of the terminator is shown in SEQ ID No. 4 of the sequence listing.

[0025] VII. The present invention also provides a method for preparing alkylamide by hydrolyzing alkylaniline, the method comprising the following steps:

[0026] B1. preparing a fermentation broth of the amidase or the two amidase mutants as described above, or preparing a fermentation broth of the recombinant Bacillus subtilis;

[0027] B2, adding 0.1-10 g / L of at least one fermentation broth prepared in step B1 to water, adding 0.1-100 g / L of alkylamide, and stirring the reaction at a pH of 6-10 and 25-50° C. for 0.5-48 hours;

[0028] The added form of the alkylamide includes S-alkylamide, R-alkylamide or a combination of the two in any ratio.

[0029] The beneficial technical effects of the present invention are:

[0030] 1. The present invention provides a new application of amidase in catalyzing the hydrolysis of phenylamide to prepare chiral alkylaniline, which expands the application field of amidase and has broad application prospects in the fields of pharmaceuticals, agricultural chemicals and fine chemicals.

[0031] 2. The amidase mutant prepared by the present invention has good catalytic activity, and the preparation method is simple and convenient. It can effectively hydrolyze R-alkylamide to produce R-alkylaniline, or hydrolyze S-alkylamide to produce S-alkylaniline, with high stereoselectivity. The yield of the hydrolysis product can reach more than 97%, and the purity of the obtained chiral alkylaniline is greater than 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Schematic diagram of the recombinant plasmid construction method in Example 1;

[0033] Figure 2 This is the SDS-PAGE analysis of the fermentation broth of the recombinant strain in Example 2;

[0034] Figure 3 HPLC analysis of the enantiomers of the alkylaniline in Example 3;

[0035] Figure 4 HPLC analysis of the alkylamide enantiomers in Example 3;

[0036] Figure 5 This is the optimum pH analysis for the amidase catalytic reaction in Example 4;

[0037] Figure 6 This is the optimum temperature analysis for the amidase catalytic reaction in Example 4;

[0038] Figure 7 This is an analysis of the optimal substrate addition amount in the amidase-catalyzed reaction in Example 4. DETAILED DESCRIPTION

[0039] The present invention is described in detail below with reference to the accompanying drawings and embodiments. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, rather than all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0040] In the present invention, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from commercial channels.

[0041] The term "alkylamide" refers to a compound having the structure shown in Formula I below:

[0042]

[0043] (Formula I)

[0044] The R group can be an alkyl group having 1 to 9 carbon atoms.

[0045] Alkylamides have a chiral carbon atom and are divided into S-alkylamides and R-alkylamides according to their stereostructures.

[0046] The term "alkylaniline" refers to a compound having the structure shown in Formula II below:

[0047]

[0048] (Formula ⅠI)

[0049] Alkylaniline is divided into S-alkylaniline and R-alkylaniline according to the different stereo structures, wherein R-alkylaniline has the structure shown in the following formula III:

[0050]

[0051] (Formula III)

[0052] S-alkylaniline has the structure shown in the following formula IV:

[0053]

[0054] (Formula IV)

[0055] The "amidase (EC 3.5.1.x)" described in the present disclosure refers to an enzyme that can catalyze acyl hydrolysis and acyl transfer, and the amino acid sequence of the amidase is selected from Rhodococcus sp., the amino acid sequence is shown in SEQ ID No. 1, or the amino acid sequence of the amidase is an amino acid sequence that has more than 80% identity with the amidase and has amidase activity.

[0056] Example 1: Construction of an Amidase Expression Cassette and Recombinant Genetically Engineered Bacteria

[0057] Primer pair P1 / P2 was designed based on the amidase-encoding gene DNA sequence (shown in SEQ ID No. 5, and its amino acid sequence in SEQ ID No. 1). The amidase-encoding gene was cloned from the genomic DNA of Rhodococcus opacus PD630 using a high-fidelity DNA polymerase. DNA sequences encoding the promoter, signal peptide, and terminator were obtained from the genomic DNA of B. subtilis 168 using primer pairs P3 / P4 and P5 / P6, respectively. The promoter DNA sequence is shown in SEQ ID No. 2, the signal peptide DNA sequence is shown in SEQ ID No. 3, and the terminator DNA sequence is shown in SEQ ID No. 4.

[0058] The amplified DNA sequences were connected by fusion PCR to construct an expression cassette. The expression and construction diagram is shown in Figure 1 As shown. Primer pair P7 / P8 was then used to amplify the E. coli-Bacillus subtilis shuttle vector pP43NMK plasmid backbone. The PCR product removed the shuttle vector's own promoter and target gene. The amplified expression cassette and plasmid backbone were seamlessly ligated, and the ligation product was transformed into E. coli DH5α competent cells. Positive colonies were selected for sequencing verification. The verified correct vector was named pAMI. The recombinant plasmid pAMI was transformed into B. subtilis 168 competent cells by electroporation to obtain the recombinant strain BSWT.

[0059] The primer sequences are shown in Table 1 below, where lowercase letters represent homology arms used for seamless cloning.

[0060] Table 1

[0061]

[0062] Example 2: Amidase fermentation

[0063] (1) Prepare various required culture media:

[0064] LB medium: 5 g / L yeast powder, 10 g / L tryptone, and 10 g / L sodium chloride.

[0065] Fermentation medium: yeast powder 12 g / L, tryptone 15 g / L, disodium hydrogen phosphate 8.9 g / L, dipotassium hydrogen phosphate 3.4 g / L, glycerol 10 g / L, ammonium chloride 2.67 g / L, sodium sulfate 0.71 g / L, magnesium sulfate heptahydrate 0.3 g / L.

[0066] (2) Preparation of crude enzyme solution:

[0067] Pick a single colony and place it in a test tube containing 5 mL of LB medium. Cultivate overnight at 37°C and 200 rpm. Then, transfer a 3% inoculum of the colony to LB medium and incubate at 37°C and 200 rpm for 4 hours to prepare the seed solution. Inoculate the fermentation medium at a 3% inoculum and shake the flask at 37°C and 200 rpm for 24 hours. Centrifuge the fermentation broth at 6000 rpm for 10 minutes, and the supernatant is the crude enzyme solution.

[0068] The supernatant was subjected to SDS-PAGE detection using FastPAGE protein precast gel (purchased from Beijing Qingke Biotechnology Co., Ltd.), and the supernatant of wild-type Bacillus subtilis 168 fermentation broth was used as a control. Figure 2 As shown, there is a band with the same theoretical size as the target protein in the supernatant of the strain BSWT, and there is almost no protein band at the corresponding position of the control strain, indicating that the amidase is successfully expressed.

[0069] Example 3: Construction and activity analysis of amidase mutants

[0070] (1) Construction of amidase mutants

[0071] Single-point mutations were introduced into the amino acid sequence of SEQ ID No. 1 using the primers shown in Table 2 below. The mutation sites and homology arms were designed at the 5' ends of the primers. Plasmids were then amplified using plasmid pMAI as a template. The PCR products were gel-extracted and digested with the DpnI restriction enzyme before seamless cloning and ligation. The ligated products were transformed into competent Escherichia coli DH5α cells, and transformants were verified by sequencing. The verified plasmids were then transformed into competent Bacillus subtilis 168 cells by electroporation, resulting in recombinant strains BSG77F, BSA312W, BST315F, BSS76A, BSA120L, and BSI208A.

[0072] The corresponding single-point mutation plasmids were used as templates for plasmid amplification to obtain the corresponding double-point mutation plasmids and triple-point mutation plasmids, which were then transformed into Bacillus subtilis to obtain recombinant strains BSG77F / A312W, BSA312W / T315F, BSG77F / A312W / T315F, BSS76A / A120L, BSA120L / I208A, and BSS76A / A120L / I208A.

[0073] Table 2

[0074]

[0075] (2) Detecting its performance in hydrolyzing alkylamide to generate alkylaniline:

[0076] The crude enzyme solutions of the wild type and the mutants were prepared according to the method of Example 2, and the protein concentration in the crude enzyme solution was determined using the BCA method. The catalytic activity of the amidase was analyzed as follows:

[0077] To a 50 mL Erlenmeyer flask, add 10 mL of 0.05 M glycine sodium hydroxide buffer (pH 8.5), followed by 1 g / L of racemic alkylamide and 0.2 g / L of crude enzyme. Incubate at 37°C, 200 rpm for 30 minutes. Inactivate the protein by boiling for 10 minutes. Extract the reaction solution with ethyl acetate, remove the solvent, and re-dissolve the precipitate in methanol for liquid chromatography analysis.

[0078] Liquid chromatography analysis conditions were as follows: mobile phase ratio: 0.1% phosphoric acid in water: acetonitrile = 7:3. Column: Lux Cellulose-3 chiral column (4.6 × 250 mm), detector: UV detector, flow rate: 0.4 mL / min, 230 nm, column temperature: 25°C, analysis time: 30 min, injection volume: 10 µL.

[0079] The results of alkylamide and alkylaniline standard liquid chromatography are as follows Figure 3 and Figure 4 As shown. Figure 3 and Figure 4 The catalytic activity results of the single-point mutant strain, double-point mutant strain, and triple-point mutant strain prepared in this example are shown in Table 3 below.

[0080] Table 3

[0081]

[0082] Example 4: Optimization of conditions for preparing alkylanilines by amidase hydrolysis of racemic alkylamides

[0083] The enzymatic properties of amidases from the fermentation crude enzyme solutions of strains BSWT, BSG77F / A312W / T315F, and BSS76A / A120L / I208A were analyzed according to the activity analysis method of Example 3. Figure 5 and Figure 6 As shown, the three amidases were active at pH 6-10 and 25-50°C, with the optimum reaction pH being 9.0 and the optimum reaction temperature being 40°C.

[0084] Under the conditions of the optimum reaction temperature of 40℃ and pH 9.0, 10 g / L amidase was added to catalyze the reaction for 48 h, and the effect of substrate addition on enzyme catalysis was compared. Figure 7 As shown in the figure, within the low concentration range, the three amidases can hydrolyze the substrate to a yield of more than 97% of the theoretical yield. As the substrate concentration increases, the yield begins to decrease. When the substrate concentration reaches 100 g / L, the yield is still more than 50% of the theoretical yield, and the ee value remains unchanged.

[0085] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for hydrolyzing an alkylamide to generate R-alkylaniline, characterized in that: The method comprises the following steps: A1. Fermenting to prepare an enzyme solution containing an amidase mutant, wherein the amidase mutant is obtained by subjecting the amino acid sequence of SEQ ID No. 1 to multiple mutations, with the mutation sites being amino acids 77, 312, and 315; A2. Add 0.1-10 g / L of the amidase mutant described in step A1 to water, add 0.1-100 g / L of alkylamide, and stir the reaction at a pH of 6-10 and a temperature of 25-50° C. for 0.5-48 hours; the alkylamide is added as an external digestion mixture; The amidase mutant is prepared by mutating the amino acid at position 77 of the amidase from G to F, the amino acid at position 312 from A to W, and the amino acid at position 315 from T to F.

2. A method for hydrolyzing an alkylamide to produce an S-alkylaniline, characterized in that: The method comprises the following steps: B1. Fermenting to prepare an enzyme solution containing an amidase mutant, wherein the amidase mutant is obtained by subjecting the amino acid sequence of SEQ ID No. 1 to multiple mutations, with the mutation sites being amino acids 76, 120, and 208; B2, adding 0.1-10 g / L of the amidase mutant described in step B1 to water, adding 0.1-100 g / L of alkylamide, and stirring the reaction at pH 6-10 and 25-50° C. for 0.5-48 hours; the alkylamide is added in the form of an external digestion mixture; The amidase mutant is characterized in that the amino acid at position 76 is mutated from S to A, the amino acid at position 120 is mutated from A to L, and the amino acid at position 208 is mutated from I to A.

3. Use of a recombinant Bacillus subtilis expressing amidase in the hydrolysis of alkylamide to produce alkylaniline, characterized in that: The method for constructing the recombinant Bacillus subtilis comprises the following steps: C1, cloning a promoter, a signal peptide encoding gene, an amidase encoding gene and a terminator to construct a recombinant expression cassette, wherein the amidase encoding gene encodes the amidase mutant according to any one of claims 1 or 2; C2. Transforming the recombinant expression cassette into Bacillus subtilis. The recombinant expression cassette exists in the Bacillus subtilis in the form of a free plasmid or is integrated into the Bacillus subtilis genome.

4. The use according to claim 3, characterized in that The DNA sequence of the promoter is shown in SEQ ID No. 2, the signal peptide encoding gene is shown in SEQ ID No. 3, and the DNA sequence of the terminator is shown in SEQ ID No. 4.

Images