Amidase as well as mutant and application thereof

Abstract

The invention provides amidase as well as a mutant and application thereof, and belongs to the technical field of gene engineering and enzyme catalysis. The amino acid sequence of the amidase is as shown in SEQ ID No.1, or the amino acid sequence of the amidase has 80% or more consistency with the amino acid sequence as 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 77th site, the 312th site and the 315th site of an amino acid sequence of SEQ ID No.1; the second mutant of the amidase is obtained by mutating one or more sites in the 76th site, the 120th site and the 208th site of the amino acid sequence of SEQ ID No.1. The amidase and the mutant thereof can hydrolyze racemic alkylamide to prepare chiral alkylaniline, the prepared chiral alkylaniline has high optical purity, and the ee value is greater than 99%.

Description

Technical Field

[0001] The 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] Amidase is a class of enzymes that can catalyze the hydrolysis or synthesis of amide bonds and is widely found in microorganisms, plants and animals. Based on its primary structure and functional characteristics, amidase can be divided into multiple families, including the amidase characteristic family, the nitrile amidase superfamily, the acetylamidase / formamidase, etc. In addition, based on substrate specificity, amidase can be further subdivided into carboxamidase, α-amino acid amidase and other types. The substrate range of amidase is very wide, covering a variety of compounds such as aliphatic amides, aromatic amides, heterocyclic amides and α-amino acid amides. This wide substrate specificity makes amidase play an important catalytic role in a variety of biochemical reactions, such as playing a key role in nitrogen metabolism, drug metabolism and degradation of environmental pollutants.

[0003] The catalytic mechanism of amidase involves multiple steps such as substrate binding, intermediate formation and product release. Studies have shown that nitrilase superfamily enzymes exist in the form of homotetrameric or homohexameric complexes in solution and have a conserved Glu, Cys and Lys catalytic triad in the catalytic center. Amidase characteristic family enzymes form homodimers or homooctamer complexes, characterized by a highly conserved sequence of about 130 amino acids including the Ser, Ser and Lys catalytic triad. Some amidases show different preferences for aliphatic amides and aromatic amides, and show significant differences in enantiomeric selectivity. This feature makes them have great application potential in asymmetric synthesis. For example, the amidase from Galactomyces geotrichum shows high catalytic activity for n-butyramide, isobutyramide and acetamide, and the enantiomeric excess (ee) for some chiral substrates can reach 99.9%.

[0004] However, the existing amidase still has the problem of poor stereoselectivity and cannot meet the demand. In addition, when amidase is expressed in E. coli, it often exists in the form of inclusion bodies, and a complex renaturation process is required to obtain an enzyme with catalytic activity. This problem seriously limits the application of amidase in industrial production. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides an amidase and its mutant and application. The amidase and its mutant can hydrolyze racemic alkylamide to prepare chiral alkylaniline, and the prepared chiral alkylaniline has high optical purity and an ee value greater than 99%.

[0006] The technical solution of the present invention is as follows: 1. The present invention first provides an amidase for hydrolyzing alkylamide to generate alkylaniline, wherein the amino acid sequence of the amidase is as 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 alkylamide has a structure as shown in the following formula I: (Formula I) Wherein R is a C1-C9 alkyl group; The alkylaniline has a structure shown in the following formula II: (Formula II) 2. The present invention also provides a first mutant of the above-mentioned amidase, which is obtained by site-directed mutagenesis of the amino acid sequence shown in SEQID No.1, and the mutation site is one or more of the amino acids at positions 77, 312, and 315.

[0009] 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.

[0010] 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.

[0011] 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, and the mutation site is one or more of the amino acids at positions 76, 120, and 208.

[0012] 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.

[0013] 5. The present invention also provides the use of the mutant of the second amidase, i.e., for selectively hydrolyzing racemic alkylamide or S-alkylamide to produce S-alkylaniline.

[0014] 6. The present invention further provides a method for constructing a recombinant Bacillus subtilis that efficiently expresses amidase, the method comprising the following steps: A1. 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 can encode the above-mentioned amidase or two mutants of the amidase; 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 genome of Bacillus subtilis.

[0015] Furthermore, the DNA sequence of the promoter is shown in the sequence listing SEQ ID No. 2, the signal peptide encoding gene is shown in the sequence listing SEQ ID No. 3, and the DNA sequence of the terminator is shown in the sequence listing SEQ ID No. 4.

[0016] VII. The present invention also provides a method for preparing alkylamide by hydrolyzing alkylaniline, the method comprising the following steps: 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; 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; The added form of the alkyl amide includes S-alkyl amide, R-alkyl amide or a combination of the two in any ratio.

[0017] The beneficial technical effects of the present invention are: 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.

[0018] 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

[0019] Figure 1 This is a schematic diagram of the recombinant plasmid construction method in Example 1; Figure 2 This is the SDS-PAGE analysis of the fermentation broth of the recombinant strain in Example 2; Figure 3 HPLC analysis of the enantiomers of the alkylaniline in Example 3; Figure 4 is the HPLC analysis of the alkylamide enantiomers in Example 3; Figure 5 The optimum pH analysis for the amidase catalytic reaction in Example 4; Figure 6 The optimum temperature analysis for the amidase catalytic reaction in Example 4; Figure 7 This is an analysis of the most suitable amount of substrate added in the amidase-catalyzed reaction in Example 4. DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] In the present invention, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be purchased from commercial channels.

[0022] The term "alkylamide" refers to a compound having the structure shown in Formula I below: (Formula I) The R group can be an alkyl group having 1 to 9 carbon atoms.

[0024] Alkylamide has a chiral carbon atom and is divided into S-alkylamide and R-alkylamide according to the different stereostructures.

[0025] The term "alkylaniline" refers to a compound having the structure shown in Formula II below: (Formula Ⅱ) Alkylaniline is divided into S-alkylaniline and R-alkylaniline according to the different stereostructures, wherein R-alkylaniline has the structure shown in the following formula III: (Formula III) S-alkylaniline has the structure shown in the following formula IV: (Formula IV) 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.

[0029] Example 1: Construction of the amidase expression cassette and recombinant genetic engineering bacteria Primer pair P1 / P2 was designed based on the amidase encoding gene DNA sequence (as shown in SEQ ID No.5, and its amino acid sequence as shown in SEQ ID No.1), and the amidase encoding gene was cloned from the genomic DNA of Rhodococcus turbidus PD630 using a high-fidelity DNA polymerase. The DNA sequences encoding the promoter, signal peptide and terminator were obtained from the genomic DNA of B. subtilis168, and the amplification primer pairs were P3 / P4 and P5 / P6, respectively. The DNA sequence of the promoter is shown in SEQ ID No.2 of the sequence listing, the DNA sequence encoding the signal peptide 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.

[0030] The amplified DNA sequences were connected by fusion PCR to construct an expression cassette. The expression and construction schematic is shown in Figure 1 As shown. Then, primer pair P7 / P8 was used to amplify the E. coli-Bacillus subtilis shuttle vector pP43NMK plasmid backbone, and the PCR product removed the promoter and target gene carried by the shuttle vector itself. The amplified expression cassette and plasmid backbone were connected by seamless cloning, and the connection product was transformed into E. coli DH5α competent state, and positive colonies were selected for sequencing verification. The verified correct vector was named pAMI. The recombinant plasmid pAMI was transferred into the Bacillus subtilis 168 competent state by electroporation to obtain the recombinant strain BSWT.

[0031] The primer sequences are shown in Table 1 below, where lowercase letters represent homology arms used for seamless cloning.

[0032] Table 1

[0033] Example 2: Amidase fermentation (1) Prepare various required culture media: LB medium: 5 g / L yeast powder, 10 g / L tryptone, and 10 g / L sodium chloride.

[0034] 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.

[0035] (2) Preparation of crude enzyme solution: Pick a single colony in a test tube containing 5 mL LB medium, culture overnight at 37°C and 200 rpm, then transfer to LB medium at 3% inoculation and culture at 37°C and 200 rpm for 4 hours as seed solution. Inoculate the seed solution into the fermentation medium at 3% inoculation and ferment in a shake flask at 37°C and 200 rpm for 24 hours. Centrifuge the fermentation solution at 6000 rpm for 10 min, and take the supernatant as the crude enzyme solution.

[0036] 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 strain BSWT, and there is almost no protein band at the corresponding position of the control strain, indicating that the amidase is successfully expressed.

[0037] Example 3: Construction and activity analysis of amidase mutants (1) Construction of amidase mutants Use the primers shown in Table 2 below to introduce single point mutations into the amino acid sequence shown in SEQ ID No.1, design the mutation site and homology arm at the 5' end of the primer, and then amplify the plasmid using plasmid pMAI as a template. The PCR product was seamlessly cloned and connected after gel recovery and DpnI restriction enzyme digestion. The connection product was transformed into Escherichia coli DH5α competent state, and the transformant was verified by sequencing. The verified correct plasmid was transferred into Bacillus subtilis 168 competent state by electroporation transformation to obtain recombinant strains BSG77F, BSA312W, BST315F, BSS76A, BSA120L, and BSI208A.

[0038] The corresponding single-point mutation plasmid was used as a template to amplify the plasmid to obtain the corresponding double-point mutation plasmid and triple-point mutation plasmid, 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.

[0039] Table 2

[0040] (2) Detection of its ability to hydrolyze alkyl amide to generate alkyl aniline: The fermentation crude enzyme solution of the wild type and the above mutants was prepared according to the method of Example 2, and the protein concentration in the crude enzyme solution was detected by the BCA method. The catalytic activity analysis method of the amidase is as follows: Add 10 mL of 0.05M glycine sodium hydroxide buffer (pH 8.5) to a 50 mL conical flask, then add 1 g / L of racemic alkylamide and 0.2 g / L of crude enzyme solution, react at 37°C and 200 rpm for 30 minutes, and inactivate the protein by boiling for 10 minutes. After the reaction solution is extracted with ethyl acetate, the solvent is removed, and the precipitate is redissolved with methanol for liquid phase analysis.

[0041] Liquid phase analysis conditions are as follows: Mobile phase ratio: 0.1% phosphoric acid water: acetonitrile = 7:3. Chromatographic column: Lux Cellulose-3 chiral chromatographic column (4.6×250mm), detector: UV detector, flow rate 0.4 mL / min, 230 nm, column temperature 25℃, time 30 min, injection volume 10µL.

[0042] The results of the liquid phase of alkylamide and alkylaniline standards 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.

[0043] Table 3

[0044] Example 4: Optimization of conditions for preparing alkylaniline by amidase hydrolysis of racemic alkylamide 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 are 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.

[0045] Under the conditions of the optimum reaction temperature of 40℃ and pH of 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 substrates to 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.

[0046] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable 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 principle 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. An amidase for hydrolyzing an alkylamide to produce an alkylaniline, characterized in that: The amino acid sequence of the amidase is as 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 alkylamide has a structure as shown in the following formula I: (Formula I) Wherein R is a C1-C9 alkyl group; The alkylaniline has a structure shown in the following formula II: (Formula II).

2. The mutant of the amidase according to claim 1, characterized in that The mutant of the amidase 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 77th, 312th, and 315th amino acids.

3. The mutant according to claim 2, characterized in that 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.

4. Use of the mutant of amidase according to claim 2 or 3, characterized in that: The mutant is used to selectively hydrolyze racemic alkylamide or R-alkylamide to produce R-alkylaniline.

5. The mutant of the amidase according to claim 1, characterized in that The amidase mutant 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 76th, 120th, and 208th amino acids.

6. The mutant according to claim 5, characterized in that 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.

7. Use of the mutant according to claim 5 or 6, characterized in that: The mutants are used to selectively hydrolyze racemic alkylamides or S-alkylamides to produce S-alkylanilines.

8. A method for constructing a recombinant Bacillus subtilis that efficiently expresses amidase, characterized in that: The method comprises the following steps: A1. 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 can encode the amidase or amidase mutant according to any one of claims 1, 2 and 5; 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 genome of Bacillus subtilis.

9. The method according to claim 8, characterized in that The DNA sequence of the promoter is shown in SEQ ID No. 2 in the sequence list, the signal peptide encoding gene is shown in SEQ ID No. 3 in the sequence list, and the DNA sequence of the terminator is shown in SEQ ID No. 4 in the sequence list.

10. A method for preparing alkylamide by hydrolyzing alkylaniline, characterized in that: The method comprises the following steps: B1. preparing a fermentation broth of the amidase or amidase mutant according to any one of claims 1, 2, and 5, or preparing a fermentation broth of the recombinant Bacillus subtilis according to claim 8; B2, adding 0.1-10 g / L of at least one fermentation liquid 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; The added form of the alkyl amide includes S-alkyl amide, R-alkyl amide or a combination of the two in any ratio.

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