A method for efficiently expressing amidase and its application
By constructing a recombinant expression vector with a fusion tag in Escherichia coli, the problems of narrow substrate spectrum of amidase and inclusion body expression were solved, and efficient expression and production of chiral compounds with high optical purity were achieved.
Patent Information
- Application Number
- CN202510519890.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2045-04-22
AI Technical Summary
The substrate spectrum of existing amidases is not broad enough, and when expressed in Escherichia coli, they often exist in the form of inclusion bodies, requiring a renaturation process, which limits their industrial application.
The fusion tag was used to significantly increase the expression level of amidase. The amidase and fusion tag coding sequences were cloned into an Escherichia coli expression vector to construct a recombinant expression plasmid, and the amidase was expressed in Escherichia coli competent cells.
The high optical purity expression of amidase was achieved, and it catalyzed the high efficiency of preparing chiral compounds from racemic alkylanilines and alkylamides, with an ee value of more than 97%.
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Abstract
Description
Technical Field
[0001] The present invention relates to the fields of enzyme technology and genetic engineering technology, in particular to a method for efficiently expressing amidase and application thereof. Background Art
[0002] Amidases are an important class of biocatalysts that catalyze the hydrolysis of amide bonds to form the corresponding carboxylic acids and ammonia. This catalytic activity plays a crucial role in the pharmaceutical industry and the synthesis of agrochemicals, primarily due to their broad substrate spectrum and high chemical, regio, and stereoselectivity. These properties make amidases crucial for the preparation of pharmaceuticals and agrochemicals, particularly in the synthesis of chiral carboxylic acids, α-amino acids, and amides, where their high stereoselectivity makes them valuable tool enzymes.
[0003] Due to the growing demand for chiral drugs and intermediates, the biosynthesis of chiral compounds using biological or enzymatic methods is gaining increasing attention. Biosynthesis utilizes oxidoreductases, synthetases, lyases, and hydrolases to catalyze asymmetric synthesis reactions, converting chemically synthesized precursors into complex chiral compounds such as alcohols, ketones, aldehydes, amines, acids, esters, and amides. Enzymatic resolution utilizes the high stereo, site, and regioselectivity of hydrolases to catalyze the hydrolysis of a single enantiomer of a chemically synthesized racemate or derivative, resulting in a mixture of reacted and unreacted optical isomers. The two enantiomers are then separated based on their physicochemical properties to yield a single, optically active product. This method is primarily used to prepare chiral alcohols, acids, amines, esters, and amides.
[0004] Amidases studied in existing technologies generally suffer from a limited substrate spectrum. Most amidases can only catalyze the reaction of alkylamines (amides), with fewer capable of catalyzing aromatic amines (amides). 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 aforementioned issues with existing technologies, the present invention provides a method for efficiently expressing amidase and its application. The method utilizes a fusion tag to significantly increase the expression of amidase. The resulting amidase can be used to generate chiral alkylanilines with high optical purity, resulting in compounds with ee values exceeding 97%.
[0006] The technical solutions of the present invention are as follows:
[0007] A method for efficiently expressing amidase, comprising the following steps:
[0008] A1. Clone a fusion tag encoding sequence for enhancing amidase expression into an Escherichia coli expression vector to obtain a first recombinant expression vector, wherein the fusion tag is encoded by the DNA sequence shown in SEQ ID No. 1;
[0009] A2. Clone the amidase coding sequence into the first recombinant expression vector obtained in step A1 to construct a second recombinant expression vector, wherein the amidase is fused with the fusion tag and the amidase is located downstream of the fusion tag;
[0010] A3. Introducing the second recombinant expression vector constructed in step A2 into competent E. coli cells to construct a recombinant genetically engineered bacterium;
[0011] A4. Fermenting the recombinant genetically engineered bacteria obtained in step A3 to express amidase.
[0012] Preferably, the amino acid sequence of the amidase is selected from any one of SEQ ID No.2-SEQ ID No.14, or has more than 80% identity with any one of the amino acid sequences of SEQ ID No.2-SEQ ID No.14 and has amidase activity.
[0013] The present invention also provides a method for preparing chiral alkylaniline by enzymatically catalyzing racemic alkylaniline, the method comprising the following steps:
[0014] B1, preparing amidase by the above method;
[0015] B2. Under the catalysis of any one or more of the amidases, selectively reacting an acyl donor with racemic alkylaniline to form an alkylamide, with the remaining chiral alkylaniline; the acyl donor is a carboxylic acid or a carboxylate containing 2 to 10 carbon atoms;
[0016] The structure of the racemic alkylaniline is shown in the following formula I:
[0017]
[0018] (Formula I)
[0019] The structure of the alkylamide is shown in Formula II below:
[0020]
[0021] (Formula II)
[0022] wherein R is a C1-C9 alkyl group;
[0023] B3. Separate and obtain chiral alkylaniline from the reaction solution.
[0024] Furthermore, in step B2:
[0025] When R-amidase is added, step B3 yields S-alkylaniline;
[0026] When S-amidase is used, Step B3 yields R-alkylaniline.
[0027] Preferably, the catalytic conditions of step B2 are:
[0028] The amount of racemic alkylaniline added is 0.1-100 g / L, the amount of acyl donor added is 0.05-30 g / L, the amount of amidase added is 0.1-20 g / L, the pH is 4-7, the temperature is 25-60° C., and the time is 1-72 h.
[0029] The present invention further provides a method for preparing chiral alkylaniline by enzymatically catalyzing the hydrolysis of racemic alkylamide, comprising the following steps:
[0030] C1. preparing amidase by the above method;
[0031] C2. Under the catalysis of any one or more of the amidases, selectively hydrolyze the racemic alkylamide to generate chiral alkylaniline; wherein the structure of the racemic alkylamide is shown in the following formula II:
[0032]
[0033] (Formula II)
[0034] wherein R is a C1-C9 alkyl group;
[0035] The structure of the alkylaniline is shown in the following formula I:
[0036]
[0037] (Formula I)
[0038] C3. Separate and obtain chiral alkylaniline from the reaction solution.
[0039] Furthermore, in step C2:
[0040] When R-amidase is used, step C3 yields R-alkylaniline;
[0041] When S-amidase is used, step C3 yields S-alkylaniline.
[0042] Preferably, the hydrolysis conditions of step C2 are:
[0043] The amount of racemic alkylamide added is 0.1-150 g / L, the amount of amidase added is 0.1-20 g / L, the pH is 7-11, the temperature is 25-60° C., and the time is 1-72 h.
[0044] The beneficial technical effects of the present invention are:
[0045] 1. The present invention clones the fusion tag coding sequence and the amidase coding sequence into a vector to construct a recombinant expression plasmid. This fusion tag is not a conventional fusion tag, but is specifically used for amidase. It can express about 13 types of amidases and greatly increases the expression level of amidase, which is a first in this field.
[0046] 2. The present invention introduces an amidase recombinant expression plasmid into competent Escherichia coli to construct a recombinant genetically engineered bacterium, and ferments the recombinant genetically engineered bacterium to produce an amidase. The amidase is highly efficient in catalyzing the preparation of chiral alkylanilines from racemic alkylanilines and in catalyzing the hydrolysis of racemic alkylamides to prepare chiral alkylanilines. BRIEF DESCRIPTION OF THE DRAWINGS
[0047] Figure 1 This is a schematic diagram of the construction of the recombinant plasmid in Example 1;
[0048] Figure 2 This is an SDS-PAGE analysis of the fermentation broth of the recombinant strain in Example 2; the meanings represented by the numbers in the figure are: lane M is a protein molecular weight standard, lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 are supernatants of Escherichia coli cell fragments expressing the amidases described in SEQ ID No. 2 to SEQ ID No. 14, respectively, and lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 are resuspensions of precipitates of Escherichia coli cell fragments expressing the amidases described in SEQ ID No. 2 to SEQ ID No. 14, respectively.
[0049] Figure 3 HPLC analysis chart of the alkylaniline enantiomers in Example 3. DETAILED DESCRIPTION
[0050] 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.
[0051] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the materials and reagents used can be purchased from commercial channels.
[0052] The term "racemic alkylaniline" refers to a compound having the following structural formula I:
[0053]
[0054] (Formula I)
[0055] Alkylaniline has a chiral carbon atom and is divided into S-alkylaniline and R-alkylaniline according to the different stereo structures.
[0056] The term "racemic alkylamide" refers to a compound having the following structure:
[0057]
[0058] (Formula II)
[0059] According to the different stereostructures, they are divided into S-alkylamide and R-alkylamide, wherein the R group can be an alkyl group with 1-9 carbon atoms.
[0060] 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 can be selected from Gram-positive or Gram-negative bacteria, such as Rhodococcus sp., Pseudomonas sp., Acinetobacter sp., and Bacillus sp. In a preferred embodiment, the amidases are CamH, TccA, Ppa, Mah, PsaA, NylA, AAM, PamH, AmpA, AmdA, LibA, Oct, and AAA, and the amino acid sequences are shown in SEQ ID No. 2-14. Depending on the substrate and product, an amidase that can selectively hydrolyze R-alkylamide or selectively catalyze the synthesis of R-alkylamide from R-alkylaniline is called R-amidase; an amidase that can selectively hydrolyze S-alkylamide or selectively catalyze the synthesis of S-alkylamide from S-alkylaniline is called S-amidase; and an unselective amidase is called RS-amidase.
[0061] The sources of the different types of amidases involved in the present invention and the corresponding relationships between their amino acids and DNA sequence numbers are shown in Table 1 below.
[0062] Table 1
[0063]
[0064] Example 1: Construction of an Amidase Expression Vector and an Amidase-Expressing Genetically Engineered Bacteria
[0065] The fusion tag (named label1) DNA sequence is shown in SEQ ID No. 1 in the sequence listing and was synthesized by Beijing Qingke Biotechnology Co., Ltd. The synthesized sequence was cloned into the BamHI and SalI sites of the pET-28a plasmid to obtain the first recombinant expression vector pET-label.
[0066] According to the amino acid sequences of the amidases CamH, TccA, Ppa, Mah, PsaA, NylA, AAM, PamH, AmpA, AmdA, LibA, Oct, and AAA (shown in SEQ ID No. 2-14), codon optimization was performed to obtain DNA coding sequences. The DNA sequences are shown in the sequence listings as SEQ ID No. 15 to SEQ ID No. 27, respectively. The DNA sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the SalI and NotI sites of the pET-label vector to obtain the second recombinant expression vector. The plasmid construction schematic is shown in FIG. Figure 1 shown.
[0067] The corresponding second recombinant plasmid was verified by sequencing and then transformed into competent Escherichia coli BL21 (DE3) to obtain 13 recombinant genetically engineered bacteria corresponding to the 13 amidases.
[0068] Example 2: Culture medium preparation, fermentation method and crude enzyme solution preparation
[0069] (1) Prepare the required culture medium:
[0070] ①LB medium: yeast powder 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L.
[0071] ② 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, defoamer 0.5 mL / L.
[0072] ③ Glycerol feed medium: glycerol: 300 g / L, magnesium sulfate heptahydrate: 4 g / L.
[0073] ④ Lactose feed medium: lactose: 200 g / L.
[0074] (2) Fermentation process:
[0075] ① Pick single colonies of 13 recombinant genetically engineered bacteria and place them in test tubes containing 5 mL of LB medium. Culture them at 37°C and 200 rpm overnight. Then transfer them to LB medium containing 1% glucose at a 3% inoculum volume and culture them at 37°C and 200 rpm for 4 hours as seed solution.
[0076] ② Fermentation in a fermenter: 1-3% of the seed solution was inoculated into the fermentation medium. The initial fermentation temperature was 37°C, pH 7.0±0.2, the rotation speed was 750 rpm, and the ventilation volume was 1.5 vvm. After the dissolved oxygen rebounded, glycerol feed medium and lactose feed medium were added (glycerol feed rate was 2-8 g / L / h, lactose feed rate was 2-5 g / L / h). During this period, samples were taken every half hour to measure the OD. 600 The feed rate is adjusted based on the amount of glycerol remaining. During fermentation, dissolved oxygen is controlled in tandem with the rotational speed to maintain a constant level above 30%. Fermentation is terminated after 18-20 hours of induction.
[0077] (3) Preparation of crude enzyme solution:
[0078] The fermentation broth was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. The cells were washed twice with deionized water and then resuspended in appropriate amount of deionized water to make the OD 600 The resuspended cells were disrupted using a high-pressure homogenizer. The conditions for high-pressure homogenization were: three cycles of approximately 900 bar. After centrifugation at 8000 rpm for 10 minutes, the fragments were removed and the supernatant was used as the crude enzyme solution. The pellet was resuspended in an equal volume of deionized water for subsequent analysis.
[0079] The supernatant crude enzyme solution and precipitate reselection solution were diluted 20 times and then subjected to SDS-PAGE detection using FastPAGE protein precast gel (purchased from Beijing Qingke Biotechnology Co., Ltd.). Figure 2 As shown, the odd-numbered lanes are the supernatant and the even-numbered lanes are the precipitate resuspension. The supernatant contains a band of the same theoretical size as the target protein, while the precipitate contains almost no protein bands at the corresponding position or the bands are much smaller than those in the supernatant, indicating that the amidase is expressed in large quantities as a soluble protein.
[0080] Example 3: Detection of Enantiomers of Alkylaniline and Alkylamide by High Performance Liquid Chromatography
[0081] A series of S- and R-alkylaniline standards were prepared at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1 g / L, respectively. The standards were dissolved in acetonitrile and filtered through a 0.45 µm microporous filter membrane before injection.
[0082] The liquid mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A: 1 L of 0.1% phosphoric acid aqueous solution, filtered through a 0.45 µm membrane and then sonicated for half an hour; mobile phase B: 1 L of acetonitrile, filtered through a 0.45 µm membrane and then sonicated for half an hour.
[0083] Liquid chromatography conditions: Mobile phase ratio: 0.1% phosphoric acid in water: acetonitrile = 7:3. Chromatographic column: Lux Cellulose-3 chiral column (4.6 × 250 mm), UV detector, flow rate: 0.4 mL / min, 230 nm, column temperature: 25°C, time: 30 min, injection volume: 10 µL.
[0084] Liquid phase results such as Figure 3 As shown in Figure 3, the results show that S-configuration and R-configuration alkylanilines have different retention times under the above conditions.
[0085] Example 4: Amidase-catalyzed preparation of chiral alkylaniline from racemic alkylaniline
[0086] The protein content in the crude enzyme solution prepared in Example 2 was detected by the BCA method. The crude enzyme solution of amidase was added to a 50 mL conical flask to a final concentration of 10 g / L. 100 g / L of racemic alkylaniline and 30 g / L of acyl donor sodium acetate were added. The catalytic temperature was controlled at 40 ° C and the pH was 6. The reaction was stirred for 20 h. After the reaction was completed, petroleum ether was added to extract the reaction solution, the organic phase was removed, the aqueous phase was denatured by heating and centrifuged to remove protein, and the protein precipitate was thoroughly washed with 95% ethanol. The aqueous phase and ethanol washings were combined, and the solvent was removed by vacuum distillation to obtain chiral alkylaniline. The product alkylaniline was detected by HPLC chiral column, and the yield and ee value were calculated. The results are shown in Table 2.
[0087] Table 2
[0088]
[0089] Example 5: Preparation of chiral alkylanilines by amidase hydrolysis of racemic alkylamides
[0090] The protein content in the crude enzyme solution prepared in Example 2 was detected by the BCA method. The crude enzyme solution of amidase was added to a 50 mL conical flask to a final concentration of 10 g / L. 150 g / L of racemic alkylamide was added, the catalytic temperature was controlled at 45 ° C, the pH was 10, and the reaction was stirred for 10 h. After the reaction, petroleum ether was added to extract the reaction solution, the organic phase was removed, the aqueous phase was denatured by heating and centrifuged to remove protein, and the protein precipitate was thoroughly washed with 95% ethanol. The aqueous phase and ethanol washings were combined, and the solvent was removed by vacuum distillation to obtain alkylaniline. The product alkylaniline was detected by HPLC chiral column, and the yield and ee value were calculated. The results are shown in Table 3.
[0091] Table 3
[0092]
[0093] From the test results in Table 2 and Table 3, it can be seen that the amidase prepared by the present invention has a very high catalytic efficiency, and the ee value of the obtained chiral alkylaniline is basically above 97%, and can reach up to 99.9%.
[0094] 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 expressing amidase and its application in preparing chiral alkylaniline, characterized in that: The method comprises the following steps: A1. Clone a fusion tag encoding sequence for enhancing amidase expression into an Escherichia coli expression vector to obtain a first recombinant expression vector, wherein the fusion tag is encoded by the DNA sequence shown in SEQ ID No. 1; A2. Clone the amidase coding sequence into the first recombinant expression vector obtained in step A1 to construct a second recombinant expression vector, wherein the amidase is fused with the fusion tag and the amidase is located downstream of the fusion tag; A3. Introducing the second recombinant expression vector constructed in step A2 into competent E. coli cells to construct a recombinant genetically engineered bacterium; A4. Fermenting the recombinant genetically engineered bacteria obtained in step A3 to express amidase; The amino acid sequence of the amidase is selected from any one of SEQ ID No. 2 to SEQ ID No.
10.
2. The use according to claim 1, characterized in that The application is to prepare chiral alkylaniline by enzymatic catalysis of racemic alkylaniline, comprising the following steps: B1. preparing amidase by the method according to claim 1; B2. Under the catalysis of any one or more of the amidases, selectively reacting an acyl donor with racemic alkylaniline to form an alkylamide, with the remaining chiral alkylaniline; the acyl donor is a carboxylic acid or a carboxylate containing 2 to 10 carbon atoms; The structure of the racemic alkylaniline is shown in the following formula I: (Formula I) The structure of the alkylamide is shown in Formula II below: (Formula II) wherein R is a C1-C9 alkyl group; B3. Separate and obtain chiral alkylaniline from the reaction solution.
3. The use according to claim 2, characterized in that In step B2: When R-amidase is added, step B3 yields S-alkylaniline; When S-amidase is used, Step B3 yields R-alkylaniline.
4. The use according to claim 2, characterized in that The catalytic conditions of step B2 are: The amount of racemic alkylaniline added is 0.1-100 g / L, the amount of acyl donor added is 0.05-30 g / L, the amount of amidase added is 0.1-20 g / L, the pH is 4-7, the temperature is 25-60° C., and the time is 1-72 h.
5. The use according to claim 1, characterized in that The application is to prepare chiral alkylaniline by enzymatically catalyzing the hydrolysis of racemic alkylamide, comprising the following steps: C1. preparing amidase by the method according to claim 1; C2. Under the catalysis of any one or more of the amidases, selectively hydrolyze the racemic alkylamide to generate chiral alkylaniline; wherein the structure of the racemic alkylamide is shown in the following formula II: (Formula II) wherein R is a C1-C9 alkyl group; The structure of the alkylaniline is shown in the following formula I: (Formula I) C3. Separate and obtain chiral alkylaniline from the reaction solution.
6. The use according to claim 5, characterized in that In step C2: When R-amidase is used, step C3 yields R-alkylaniline; When S-amidase is used, step C3 yields S-alkylaniline.
7. The use according to claim 5, characterized in that The hydrolysis conditions of step C2 are: The amount of racemic alkylamide added is 0.1-150 g / L, the amount of amidase added is 0.1-20 g / L, the pH is 7-11, the temperature is 25-60° C., and the time is 1-72 h.