WO2019157823A1 - Preparation method for l-alanyl-l-glutamine, enzyme for l-alanyl-l-glutamine preparation, and application - Google Patents

Abstract

Provided is a preparation method for L-alanyl-L-glutamine, said method comprising: in the presence of α-amino acid ester acyltransferase, using L-alanine methyl ester hydrochloride and L-glutamine as substrates to prepare a reaction solution, adjusting the pH of the reaction solution to 7.0-9.0, and after reacting at a constant temperature of 20-40°C, collecting L-alanyl-L-glutamine. The α-amino acid ester acyltransferase is derived from elizabethkingia meningoseptica, and the amino acid sequence of the α-amino acid ester acyltransferase comprises the amino acid sequence in any one of SEQ ID NO: 1 – SEQ ID NO: 6. The preparation method is highly effective, simple, low-cost and environmentally friendly, has a high conversion rate, and may be widely applied in industrial scale production. Also provided are an enzyme for L-alanyl-L-glutamine preparation, and an application.

Description

Method for preparing proglycol dipeptide, enzyme for preparing proglycol dipeptide and application thereof Technical field

The invention relates to the technical field of biomedicine, in particular to a preparation method of propargedopeptide, an enzyme for preparing proglycol dipeptide and an application thereof.

Background technique

Proglycol dipeptide, also known as L-Alanyl-L-Glutamine (Ala-Gln), is a biologically active dipeptide composed of alanine and glutamine residues. A dipeptide molecule that is stable in nature and readily soluble in water. Studies have shown that proglycol dipeptide has a variety of pharmacological activities, such as can promote muscle protein synthesis, improve clinical and biochemical indicators of critically ill patients; maintain intestinal function, maintain the body's nitrogen balance; enhance the role of the immune system. At present, proglycol dipeptide has been widely used in the treatment of serious infections, trauma, major surgery, extensive burns and malignant tumors.

The existing process mainly synthesizes proglycol dipeptide by chemical synthesis. However, these chemical synthesis methods usually have complicated synthesis processes, many intermediate links, easy formation of by-products, difficulty in purification of the target product, harsh synthesis conditions, and often use some toxic and harmful substances.

Therefore, it is necessary to develop a preparation method of propionol dipeptide which is simple in process, short in time, low in cost, low in environmental pollution, high in yield, and environmentally friendly.

Summary of the invention

In order to solve the above technical problems, the present invention provides a method for preparing a tragadipeptide, an enzyme for preparing a propanol dipeptide, and an application thereof; and the preparation method of the invention adopts a biological enzymatic method, which has the advantages of simple process, low cost, high yield and environmental protection.

In a first aspect, the present invention provides a method for preparing a proglycol dipeptide, comprising:

The reaction solution is prepared by using L-alanine methyl ester hydrochloride and L-glutamine as a substrate in the presence of α-amino acid ester acyltransferase (XPD), and the pH of the reaction solution is adjusted to 7.0-9.0. After reacting at a constant temperature of 20-40 ° C, the proglycol dipeptide is collected; the α-amino acid ester acyltransferase is derived from Elizabethkingia meningoseptica; the amino acid sequence of the α-amino acid ester acyltransferase includes The amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6.

Alternatively, the gene coding sequence of the α-amino acid ester acyltransferase includes the nucleotide sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12. Preferably, the α-amino acid ester acyltransferase has outstanding biological activity and strong specificity, and is capable of efficiently catalyzing the conversion of L-alanine methyl ester hydrochloride and L-glutamine to propionol dipeptide.

Alternatively, the gene encoding the amino acid sequence shown by SEQ ID NO: 1 includes the nucleotide sequence shown in SEQ ID NO: 7. The gene encoding the amino acid sequence shown by SEQ ID NO: 2 includes the nucleotide sequence shown in SEQ ID NO: 8. By analogy, the coding gene of the amino acid sequence shown in SEQ ID NO: 3 - SEQ ID NO: 6 includes the nucleotide sequence shown in SEQ ID NO: 9 - SEQ ID NO: 12, respectively.

Alternatively, the gene encoding the α-amino acid ester acyltransferase should consider a degenerate base, ie, the coding gene of the amino acid sequence shown in SEQ ID NO: 1 includes the nucleoside as shown in SEQ ID NO: 2. The acid sequence, the protection range should also protect the nucleotide sequence having the base degeneracy of SEQ ID NO: 2, and the amino acid sequence corresponding to these nucleotide sequences is still SEQ ID NO: 1. The gene encoding the amino acid sequence shown in SEQ ID NO: 2 - SEQ ID NO: 6 should also consider degenerate bases.

In the present invention, the specific process route of the preparation method of the proglycol dipeptide is as shown in the formula (1):

Wherein the L-alanine methyl ester hydrochloride has the molecular formula C ₄ H ₉ NO ₂ HCl, and the chemical structure is as shown in Formula I; the L-glutamine has a molecular formula of C ₅ H ₁₀ N ₂ O ₃ , the chemical structure is shown in Formula II; the proglycol dipeptide has a molecular formula of C ₈ H ₁₅ N ₃ O ₄ , and the chemical structure is as shown in Formula III. The preparation method of the present invention adopts a biological enzymatic method, and the L-alanine methyl ester hydrochloride and the L-glutamine form a proglycol dipeptide under the catalysis of an α-amino acid ester acyltransferase.

Optionally, in the preparation method of the present invention, the pH of the reaction solution is adjusted to be 7.0-9.0. Further optionally, the pH of the reaction solution is adjusted to be 8.0 to 9.0. For example, the pH of the reaction solution is adjusted to 8.2, or 8.5, or 9.0. Optionally, in the preparation method of the present invention, the reaction temperature of the reaction solution is constant at 20-40 °C. Further optionally, the reaction temperature of the reaction liquid is constant at 20 to 30 °C. For example, the reaction temperature of the reaction solution is constant at 20 ° C, or 23 ° C, or 25 ° C, or 30 ° C, or 35 ° C.

Alternatively, the reaction time of the reaction is from 20 to 120 min. Further optionally, the stirring time of the stirring reaction is 20-30 min. In the preparation process of the preparation method of the present invention, the content of proglycol dipeptide can be monitored by using a detection means, and the reaction is stopped after the propionate dipeptide is no longer increased; the detection means includes a liquid chromatography detection method.

Optionally, the method for preparing the L-alanine methyl ester hydrochloride comprises: adding the thionyl chloride to the reaction kettle containing methanol at a temperature of 0-10 ° C; and then in the reaction kettle Adding L-alanine to the stirring reaction, the temperature is slowly raised to 25-30 ° C during the stirring reaction, and after the reaction is 0.5-1.0 hours, the temperature is further increased to 45-50 ° C and stirred. 1.0-2.0 hours; after completion of the reaction, the L-alanine methyl ester hydrochloride was collected.

Alternatively, the stirring reaction is stirred at a rate of 200-300 rpm. Alternatively, the collection process of collecting the L-alanine methyl ester hydrochloride after the end of the reaction comprises obtaining the L-alanine methyl ester hydrochloride by a method of crystallization under reduced pressure.

Alternatively, the α-amino acid ester acyltransferase is added to the reaction solution in the form of an expression host cell or an enzyme powder. The expression host cell refers to a host cell which contains a nucleotide sequence in which an α-amino acid ester acyltransferase is expressed, and which stably expresses the α-amino acid ester acyltransferase.

Alternatively, the gene is knocked out of the protease gene and/or peptidase gene expressing the host cell; the peptidase gene includes one or more of an aminopeptidase gene and a carboxypeptidase gene. Further, optionally, the gene knocks out one of the peptidase A (pepA) gene, the peptidase B (pepB) gene, the peptidase D (pepD) gene, and the peptidase N (pepN) gene of the expression host cell. Kind or more. The pepA, pepB, pepD or pepN have strong hydrolysis effects on short peptides.

Alternatively, the process of knocking out the protease gene and/or peptidase gene of the host cell comprises: designing a primer, genetically recombining the expression host cell by gene editing technology, and knocking out the protease gene And/or the peptidase gene. Alternatively, the primer comprises the nucleotide sequence set forth in SEQ ID NO: 13 - SEQ ID NO: 36.

Further, optionally, the gene knocking out the protease gene and/or peptidase gene expressing the host cell comprises: designing a primer, and performing genetic recombination on the expression host cell by using CRISPR/Cas9 gene editing technology, The protease gene and/or the peptidase gene is knocked out, and the primer includes the nucleotide sequence shown as SEQ ID NO: 13 - SEQ ID NO: 36.

Optionally, the expression host cell comprises one or more of E. coli and yeast. Further, optionally, the Escherichia coli may be Escherichia coli JM109 (DE3) or Escherichia coli BL21 (DE3). The present invention preferably has an E. coli expression system with a short culture period, low preparation cost, and high enzyme yield. The expression host cell engineered by gene knockout has a more stable and more efficient expression of the α-amino acid ester acyltransferase; at the same time, the expression host cell can also release the α-amino acid having high biological activity in a larger amount. Ester acyltransferase.

Alternatively, the process of collecting the proglycol dipeptide comprises separating the reaction solution and then obtaining the proglycol dipeptide. Alternatively, when the α-amino acid ester acyltransferase is added to the reaction solution in the form of an expression host cell, most of the L-alanine methyl ester hydrochloride and the L-glutamine enter To the intracellular of the expression host cell, the proglycol dipeptide is obtained under the catalysis of the intracellular α-amino acid ester acyltransferase and released into the extracellular reaction solution, and a small portion of the L-alanine The proteyl dipeptide is obtained by catalyzing the acid methyl ester hydrochloride and the L-glutamine in the reaction liquid in the reaction of the α-amino acid ester acyltransferase. Alternatively, in the method for preparing the proglycol dipeptide, the reaction may be stopped by isolating the expression host cell; the isolated expression host cell may be cyclically used for the preparation of the proglycol dipeptide.

Since the proglycol dipeptide is a short peptide product, it is often accompanied by decomposition of proglycol dipeptide when catalyzed by a biological enzyme prepared by a conventional prior art; since the biological enzyme system contains a large amount of protease and/or The peptidase, which is decomposed by the proteoglycan and/or peptidase, will greatly affect the yield of proglycol dipeptide. The α-amino acid ester acyltransferase prepared by knocking out the expression gene of the protease gene and/or peptidase gene of the present invention can efficiently catalyze the production of proglycol dipeptide; meanwhile, the expression host cell does not produce protease and/or peptidase Does not decompose intracellular or extracellular proglycol dipeptide. Furthermore, the efficiency of the intracellular α-amino acid ester acyltransferase to catalyze the production of proglycol dipeptide is higher than the efficiency of the catalyzed production of proglycol dipeptide by the α-amino acid ester acyltransferase released by the host cell in the reaction solution.

Alternatively, the expression host cells have a mass fraction in the reaction solution of 1% to 5%. Further, optionally, the expression host cells have a mass fraction in the reaction solution of 1% to 3%.

Alternatively, the expression host cell can be, but is not limited to, added to the reaction solution as an expression host cell solution. Optionally, the expression host cell solution further comprises a buffer, and the buffer comprises any one or more of a phosphate buffer solution, a borate buffer solution, and a Tris-HCl buffer solution. Optionally, the buffer also includes other types of buffers. Alternatively, the concentration of the buffer is 10-500 mmol/L. Preferably, the concentration of the buffer is from 200 to 500 mmol/L. For example, the concentration of the buffer is 100 mmol/L, or 200 mmol/L, or 500 mmol/L.

Optionally, the mass fraction of the L-alanine methyl ester hydrochloride in the reaction solution is 3%-15%; the L-alanine methyl ester hydrochloride and the L-Valley The mass ratio of aminoamide is 1: (0.3-2). Further, optionally, the mass fraction of the L-alanine methyl ester hydrochloride in the reaction liquid is 10% to 15%. For example, the mass fraction of the L-alanine methyl ester hydrochloride in the reaction liquid is 5%, or 10%, or 15%, or 20%. Further, optionally, the mass ratio of the L-alanine methyl ester hydrochloride to the L-glutamine is 1: (0.5-1.5). For example, the mass ratio of the L-alanine methyl ester hydrochloride to the L-glutamine is either 1:0.8, or 1:1, or 1:1.2.

Alternatively, the mass ratio of the L-glutamine to the α-amino acid ester acyltransferase is 1: (0.1-2). Further, optionally, the mass ratio of the L-glutamine to the α-amino acid ester acyltransferase is 1: (0.5-1).

The preparation method of the proglycol dipeptide provided by the first aspect of the invention adopts the biological enzymatic method, the whole process is simple and efficient, the green is safe, the cost is low, and the time is short; the proglycol dipeptide obtained by the preparation method has extremely high Yield.

In a second aspect, the present invention provides an enzyme for preparing a propanol dipeptide, the preparation enzyme comprising an α-amino acid ester acyltransferase derived from Escherichia coli; The amino acid sequence of the α-amino acid ester acyltransferase includes the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6.

Alternatively, the gene coding sequence of the α-amino acid ester acyltransferase includes the nucleotide sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12.

Alternatively, the α-amino acid ester acyltransferase is expressed in an expression host cell by constructing a recombinant plasmid, and the vector plasmid of the recombinant plasmid is a pET28a(+) vector plasmid. Inserting the gene coding sequence of the α-amino acid ester acyltransferase into the pET28a(+) vector plasmid to obtain a recombinant plasmid, which can be efficiently and efficiently produced in heterologous expression in an expression host cell. Alpha-amino acid ester acyltransferase.

Alternatively, the gene coding sequence of the α-amino acid ester acyltransferase is inserted into the multiple cloning site region of the pET28a(+) vector plasmid. For example, the gene coding sequence for the alpha-amino acid ester acyltransferase can be, but is not limited to, inserted between the BamH I and Hind III restriction sites of the pET28a(+) vector plasmid. When the gene coding sequence of the α-amino acid ester acyltransferase is inserted into the pET28a(+) vector plasmid, the 5' end of the gene coding sequence of the α-amino acid ester acyltransferase may be added with a start codon (such as ATG). Linked to the BamHI restriction site in the pET28a(+) vector plasmid, the 3' end can be ligated with a stop codon (such as TAA) and a Hind III restriction site in the pET28a(+) vector plasmid.

Alternatively, the nucleotide sequence of the His-tag (histidine tag) is added to the gene coding sequence of the α-amino acid ester acyltransferase, so that the expressed protein can be tagged with His tag, and the His tag is favorable for expression. Isolation and purification of proteins, and analysis and tracking in experiments, such as analysis used in immunoblot experiments.

Due to the differences in the enzymatic properties of α-amino acid ester acyltransferases from different species, including the specific activity of the enzyme, the substrate range of the enzyme, the optimum pH, the optimum temperature, the action time and the stability of the enzyme. . The enzyme-α-amino acid ester acyltransferase prepared by the second aspect of the present invention has good biological activity and high purity; and the preferred α-amino acid ester acyltransferase of the present invention has more than the conventional method. High yield, short time, and more biological activity and specificity.

Alternatively, the alpha-amino acid ester acyltransferase is prepared by expressing a host cell; the expression host cell comprises one or more of E. coli and yeast.

Alternatively, the gene is knocked out of the protease gene and/or peptidase gene expressing the host cell; the peptidase gene includes one or more of an aminopeptidase gene and a carboxypeptidase gene.

Alternatively, the process of knocking out the protease gene and/or peptidase gene of the host cell comprises: designing a primer, and performing genetic recombination on the expression host cell by using CRISPR/Cas9 gene editing technology, and knocking out The protease gene and/or the peptidase gene, the primer comprising the nucleotide sequence set forth in SEQ ID NO: 13 - SEQ ID NO: 36.

The third aspect of the present invention provides a biocatalytic use of an α-amino acid ester acyltransferase derived from a meninges of a gene containing the α-amino acid ester acyltransferase derived from the meninges An alpha-amino acid ester acyltransferase gene encoding an E. aureus strain, the gene coding sequence of the α-amino acid ester acyltransferase comprising the nucleoside as shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12. An acid sequence; the alpha-amino acid ester acylase catalyzes the conversion of L-alanine methyl ester hydrochloride and L-glutamine to form proglycol dipeptide.

The beneficial effects of the present invention include the following aspects:

1. The preparation method of the invention adopts the biological enzymatic method, has high conversion rate, low cost and green safety, and can be widely applied to industrial scale production;

2. The preparation enzyme-α-amino acid ester acyltransferase used in the preparation method of the present invention has outstanding biological activity and strong specificity, and can efficiently catalyze L-alanine methyl ester hydrochloride and L. - conversion of glutamine to proglycol dipeptide;

3. The preparation method according to the invention, the final concentration of the substrate can reach 3%-15%, which is much higher than the final concentration of the traditional process substrate;

4. The expression host cell modified by gene knockout can more efficiently and efficiently express α-amino acid ester acyltransferase in the cell and release the reaction solution, which can greatly improve the reaction conversion rate, and the prepared proglycol dipeptide yield is higher and purity. better.

DRAWINGS

1 is a plasmid map of a recombinant plasmid pET28a-XPD02 according to an embodiment of the present invention;

2 is a view showing a nuclear magnetic resonance spectrum of proglycol dipeptide according to an embodiment of the present invention;

FIG. 3 is a diagram showing a propionol dipeptide nuclear magnetic carbon spectrum according to an embodiment of the present invention.

Detailed ways

The following are the preferred embodiments of the embodiments of the present invention, and it should be noted that those skilled in the art can make some improvements and refinements without departing from the principles of the embodiments of the present invention. And retouching is also considered to be the scope of protection of the embodiments of the present invention.

Unless otherwise stated, the raw materials and other chemical reagents used in the examples of the present invention are all commercially available.

1. Expression of α-amino acid ester acyltransferase

(1) Nucleotide sequence of α-amino acid ester acyltransferase

The α-amino acid ester acyltransferase XPD-01 is obtained by experimental screening, and the nucleotide sequence of the α-amino acid ester acyltransferase XPD01 is the nucleotide sequence shown in SEQ ID NO: 37;

(2) According to the upstream and downstream primers designed in Table 1, the α-amino acid ester acyltransferase XPD-01 was genetically modified by reverse PCR to prepare α-amino acid ester acyltransferase XPD-02 to XPD- 07; wherein the nucleotide and amino acid sequences corresponding to the α-amino acid ester acyltransferases XPD-02 to XPD-07 are shown in Table 2:

Table 1. Mutant primer sequences

The experimental parameters of the PCR amplification system are as follows:

The PCR amplification procedure was: predenaturation at 98 ° C for 2 min; denaturation at 98 ° C for 10 s; annealing at 55-65 ° C for 30 s; extension at 72 ° C for 7 min; after 30 cycles, extension at 72 ° C for 10 min. The PCR product was purified by a gel recovery kit, and then digested with restriction endonucleases respectively, and digested and ligated to plasmid pET28a (+) with T4 ligase. The plasmid was extracted, and the recombinant plasmid obtained after successful sequencing was obtained. For example, using the α-amino acid ester acyltransferase XPD-02 as an example, an upstream primer and a downstream primer are provided, and a gene coding sequence of an α-amino acid ester acyltransferase (XPD-02) obtained by an experiment; the α-amino acid The gene coding sequence of the ester acyltransferase XPD-02 comprises the nucleotide sequence shown as SEQ ID NO: 1;

The gene coding sequence of XPD-02 was inserted between the BamH I and Hind III restriction sites of the pET28a(+) vector plasmid. When the gene coding sequence of XPD-02 is inserted into the pET28a(+) vector plasmid, the 5' end of the XPD gene coding sequence is added with a start codon (such as ATG) and the pET28a (+) vector plasmid is digested with BamHI. The sites are ligated, and a stop codon (such as TAA) is added to the 3' end to be ligated to the Hind III restriction site in the pET28a(+) vector plasmid. Then, the E. coli competent cell DH5α was transformed into a positive clone PCR and sequenced to identify a recombinant plasmid pET28a-XPD-02, such as the plasmid map of the recombinant plasmid pET28a-XPD-02 shown in FIG. The other α-amino acid ester acyltransferase XPD-01, and the recombinant plasmid of XPD-03 to XPD-07 can also be obtained by referring to the above method.

An expression host cell is selected, the expression host cell comprising one or more of E. coli and yeast. The prepared recombinant plasmid was transferred to an expression host cell, Escherichia coli, to express the corresponding α-amino acid ester acyltransferase XPD-01 to XPD-07.

Table 2. Alpha-Amino Acid Ester Acyltransferase Data Sheet

2. Determination of α-amino acid ester acyltransferase activity

(1) Take 0.5 mL of bacterial solution, add 500 μL of 200 mM glutamine, 200 mM L-alanine methyl ester hydrochloride, 0.1 M boric acid-sodium hydroxide buffer at pH=9, mix and set at 25 °C. The reaction was carried out for 5 min in a constant temperature water bath. The reaction was stopped by adding an equal volume of 1.7% phosphoric acid (v/v) solution, and centrifuged at 12000 r/min for 5 min. The supernatant of the reaction solution was determined by high performance liquid chromatography (HPLC) to determine the concentration of proglycol dipeptide. An enzyme unit is defined as the amount of enzyme required to produce 1 μmol of product in 1 min; the experimental test parameters are shown in the table below (Remarks: by volume ratio):

Column	月旭UItimate XB-NH2 5μm×250×4.6mm
Mobile phase	0.05mol/L potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid) - acetonitrile (35:65)
Instrument model	Agilent1260
Detector	UV215nm
Flow rate	1.0mL/min
Column temperature	30 ° C
Injection volume	20μL
Sample processing	Dilute with mobile phase
Product concentration	About 0.5mg/mL

Standard curve drawing: set the injection volume and injection degree to 2μL, 4μL, 5μL, 6μL, 8μL, respectively, and draw a standard curve; calculate the enzyme activity of α-amino acid ester acyltransferase XPD-01 to XPD-07 Data, and calculate the growth rate of the enzyme activity of the α-amino acid ester acyltransferase XPD-02 to XPD-07 obtained by genetic modification compared to the α-amino acid ester acyltransferase XPD-01, as shown in Table 3 below:

Table 3. Enzyme activity data sheets of α-amino acid ester acyltransferase XPD-01 to XPD-07

The experimental test results show that the α-amino acid ester acyltransferases XPD-02 to XPD-07 described in the present application have outstanding enzyme activities, and have higher enzyme activities than the α-amino acid ester acyltransferase XPD-01. The growth rate and pH stability also have a large range of growth, especially the growth rate of the enzyme activity of the α-amino acid ester acyltransferase XPD-04 is higher than 50%.

3. Expression of α-amino acid ester acyltransferase on recombinant host cell after expression transformation

(1) An expression host cell is selected, the expression host cell comprising one or more of Escherichia coli and yeast. The Escherichia coli may be Escherichia coli JM109 (DE3) or Escherichia coli BL21 (DE3). Recombination of the expression host cells using the CRISPR/Cas9 gene editing technique includes the following steps:

a) gene cloning

The primers were designed to use pSGF/PASGR, PBSGF/PBSGR, PDSGF/PDSGR and PNSGF/PNSGR primer pairs, and pTargetF was used as a template to PCR-approve pTargetF which recognizes pepA, pepB, pepD and pepN genes. The sgRNA complementary to the 20 bp base of the target fragment in the genome is then designated as sgRNA-pepA, sgRNA-pepB, sgRNA-pepD and sgRNA-pepN, respectively; then the amplified PCR product is transformed into E. coli DH5α, respectively, and utilized The above primers were used to identify positive clones and sequenced, and the positive clones with the correct sequencing were named pTargetF-pepA, pTargetF-pepB, pTargetF-pepD and pTargetF-pepN, respectively.

Amplification of pepA, pepB, pepD and pepN genes for homologous recombination with primers PA01/PA02 and PA03/PA04, PB01/PB02 and PB03/PB04, PD01/PD02 and PD03/PD04, PN01/PN02 and PN03/PN04, respectively Upstream and downstream homology arm fragments pepA up/down, pepB up/down, pepD up/down and pepN up/down, then pepA up/down, pepB up/down, pepD up/down by overlap PCR (Overlap PCR) Connected to the top/bottom of pepN respectively, the correct sequencing of the sequencing strips is named pepA, pepB, pepD and pepN.

Wherein the PA01 comprises the nucleotide sequence set forth in SEQ ID NO: 13; the PA02 comprises the nucleotide sequence set forth in SEQ ID NO: 14; and the PA03 comprises as set forth in SEQ ID NO: 15. a nucleotide sequence; the PA04 comprises a nucleotide sequence as set forth in SEQ ID NO: 16; the PASGF comprises a nucleotide sequence as set forth in SEQ ID NO: 17; and the PASGR comprises as SEQ ID NO : nucleotide sequence shown in 18. The PB01 comprises a nucleotide sequence as set forth in SEQ ID NO: 19; the PB02 comprises a nucleotide sequence as set forth in SEQ ID NO: 20; the PB03 comprises a nucleus as set forth in SEQ ID NO: a nucleotide sequence; the PB04 comprising the nucleotide sequence set forth in SEQ ID NO: 22; the PBSGF comprising the nucleotide sequence set forth in SEQ ID NO: 23; the PBSGR comprising SEQ ID NO: 24 The nucleotide sequence shown. The PD01 comprises a nucleotide sequence as set forth in SEQ ID NO: 25; the PD02 comprises a nucleotide sequence as set forth in SEQ ID NO: 26; and the PD03 comprises a nucleus as set forth in SEQ ID NO: a nucleotide sequence; the PD04 comprises a nucleotide sequence as set forth in SEQ ID NO: 28; the PDSGF comprises a nucleotide sequence as set forth in SEQ ID NO: 29; and the PDSGR comprises SEQ ID NO: 30 The nucleotide sequence shown. The PN01 comprises a nucleotide sequence as set forth in SEQ ID NO: 31; the PN02 comprises a nucleotide sequence as set forth in SEQ ID NO: 32; and the PN03 comprises a nucleus as set forth in SEQ ID NO: a nucleotide sequence; the PN04 comprises a nucleotide sequence as set forth in SEQ ID NO: 34; the PNSGF comprises a nucleotide sequence as set forth in SEQ ID NO: 35; and the PNSGR comprises as SEQ ID NO: 36 The nucleotide sequence shown.

b) gene knockout

The pCas was transformed into the expression host cell, then the monoclonal containing pCas was picked, and then added to the LB medium of 50 mg/L kanamycin, and cultured at 30 ° C, 200 rpm until the OD ₆₀₀ was 0.2, and added to the shake flask. Arabinosine at a final concentration of 10 mM induced expression of the λ-Red protein on the pCas vector, then was shaken to an OD ₆₀₀ of 0.6 to recover the expression host cells and prepare for electroporation. On electroporation, 100 ng of pTargetF plasmid (including one or more of pTargetF-pepA, pTargetF-pepB, pTargetF-pepD and pTargetF-pepN) and 400 ng of homologous arm DNA were added to 100 μL of the prepared competent cells. Fragments (including one or more of pepA, pepB, pepD and pepN), gently mixed, added to a pre-cooled 0.2cm electric rotor, placed in an electrorotator at 2.2kv (Bio- Rad), after electro-transfer, quickly add 1mL of LB medium, resuscitate at 30°C, 180rpm for 1h, apply LB double-antiplate containing 50mg/L kanamycin and 50mg/L spectinomycin, culture at 30°C. Pick a single clone and eliminate pTargetF and pCas after PCR verification is correct.

The step of eliminating pTargetF and pCas comprises: culturing the recombinant expression host cell (containing pTargetF and pCas) in 50 mg/L kanamycin LB medium, and adding the final concentration to 0.5 mM IPTG to induce culture overnight, and It was applied to a plate containing 50 mg/L kanamycin for elimination of pTargetF, and a monoclonal was grown on the plate, and the monoclonal was picked up and cultured in liquid LB containing 50 mg/L spectinomycin overnight, not long. That is, the recombinant expression host cell depleted of pTargetF, and then the recombinant expression host cell was picked and cultured overnight at 42 ° C to eliminate pCas.

(2) Expression of α-amino acid ester acyltransferase

Taking the α-amino acid ester acyltransferase XPD-02 as an example, the constructed recombinant plasmid pET28a-XPD-02 was transferred into the recombinant expression host cell, and inoculated into 4 mL of LB medium at a dose of 1%. Maintain a constant shaking temperature of 37 ° C, 200 rpm, after overnight culture, transfer the expression host cells to a flask containing 1 L of LB medium (50 μg / mL kanamycin) at 1% inoculation, continue at 37 ° C The OD600 value in the culture medium was adjusted to about 0.6 at a constant temperature, and the inducer IPTG was added to a final concentration of 0.5 mM, and cultured at 30 ° C for 16-20 hours, and then the expression host cells were collected by centrifugation. The expression host cells were resuspended in 100 mM borax-boric acid buffer (pH = 8.0) and stored to obtain an expression host cell solution. Through the parameter adjustment, the expression host cell solution with different mass fractions can be prepared; during the experiment, the expression host cell solution with a mass fraction of 5%-50% is usually added.

A portion of the expression host cell solution was taken, and the XPD-02 was collected by sonication and centrifugation; and identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular sizes of XPD and XPD obtained by the expression in this embodiment are similar to the theoretical calculation values of the corresponding proteins, wherein the theoretical molecular weight of XPD-02 is 66.8 kDa. In addition, the collected XPD-02 was further purified to obtain XPD-02 enzyme powder. Other α-amino acid ester acyltransferases XPD-03 to XPD-07 can be prepared by the above preparation methods, and the present embodiment will not be discussed too much.

Example 1

A method for preparing a propanol dipeptide, comprising:

180g of methanol was added to the 1L reactor and cooled to 15 ° C; 78.6 g of thionyl chloride was added dropwise to the reaction vessel at 10 ° C; L-alanine was added to the reaction vessel after the dropwise addition of the thionyl chloride The mixture was stirred for 30 minutes, and then slowly heated to 25 ° C, stirred for 1 hour, and then heated to 50 ° C for 2 hours; distilled at 50 ° C under reduced pressure to obtain 82 g of L-alanine methyl ester hydrochloride.

The reaction substrate L-alanine methyl ester hydrochloride 20 g and glutamine 21 g were dissolved in pure water (400 mL), and 100 mL XPD-02 cell solution was slowly added to the reaction substrate, wherein XPD-02 cells were throughout. The mass percentage in the reaction solution was 1.5%, and the pH of the reaction solution was monitored in real time, and the pH was adjusted to 8 with a 5 M sodium hydroxide solution, and the temperature was maintained at 25 °C. During the reaction, the amount of proglycol dipeptide was determined by liquid chromatography after sampling and dilution. The reaction is stopped when the proglycol dipeptide begins to decompose. After the reaction solution was centrifuged, the supernatant was added to a final concentration of 20% methanol in a water bath at 60 ° C for 30 min, 0.5% activated carbon was stirred for 30 min, and centrifuged to obtain 625 mL of the supernatant reaction solution to obtain a propionol dipeptide content of 59.9 g/L, and a conversion rate of 92.0%.

The prepared propionol dipeptide was sampled and tested to obtain a propionol dipeptide nuclear magnetic resonance spectrum (Fig. 2) and a propionol dipeptide nuclear magnetic carbon spectrum (Fig. 3).

Example 2

A method for preparing a propanol dipeptide, comprising:

180g of methanol was added to the 1L reactor and cooled to 10 ° C; 78.6 g of thionyl chloride was added dropwise to the reaction vessel at 0 ° C; L-alanine was added to the reaction vessel after the dropwise addition of the thionyl chloride The mixture was stirred for 60 minutes, and then slowly heated to 30 ° C, stirred for 2 hours, and then heated to 45 ° C for 2 hours; distilled at 50 ° C under reduced pressure to obtain 82.7 g of L-alanine methyl ester hydrochloride.

The reaction substrate L-alanine methyl ester hydrochloride 20 g and glutamine 21 g were dissolved in pure water (400 mL), and 100 mL XPD-02 cell solution was slowly added to the reaction substrate, wherein XPD-02 cells were throughout. The mass percentage in the reaction solution was 5.0%, and the pH of the reaction solution was monitored in real time, and the pH was adjusted to 9 with a 5 M sodium hydroxide solution, and the temperature was maintained at 40 °C. During the reaction, the amount of proglycol dipeptide was determined by liquid chromatography after sampling and dilution. The reaction is stopped when the proglycol dipeptide begins to decompose. After the reaction solution was centrifuged, the supernatant was added to a final concentration of 20% methanol at 60 ° C for 30 min, 0.5% activated carbon was stirred for 30 min, and centrifuged to obtain 625 mL of the supernatant, and the content of proglycol dipeptide was 60.6 g/L, and the conversion rate was 93.0%.

Example 3

A method for preparing a propanol dipeptide, comprising:

180g of methanol was added to the 1L reactor and cooled to 15 ° C; 78.6 g of thionyl chloride was added dropwise to the reaction vessel at 5 ° C; L-alanine was added to the reaction vessel after the dropwise addition of the thionyl chloride The mixture was stirred for 60 min, and then slowly heated to 30 ° C, stirred for 1.5 h, and then heated to 48 ° C for 2 h; distilled at 50 ° C under reduced pressure to obtain 82.5 g of L-alanine methyl ester hydrochloride.

The reaction substrate L-alanine methyl ester hydrochloride 20 g and glutamine 21 g were dissolved in pure water (400 mL), and 100 mL of XPD-03 cell solution was slowly added to the reaction substrate, wherein XPD-03 cells were throughout. The mass percentage in the reaction solution was 1.5%, and the pH of the reaction solution was monitored in real time, and the pH was adjusted to 8 with a 5 M sodium hydroxide solution, and the temperature was maintained at 25 °C. During the reaction, the amount of proglycol dipeptide was determined by liquid chromatography after sampling and dilution. The reaction is stopped when the proglycol dipeptide begins to decompose. After the reaction solution was centrifuged, the supernatant was added to a final concentration of 20% methanol at 60 ° C for 30 min, 0.5% activated carbon was stirred for 30 min, and the supernatant was centrifuged to obtain 625 mL of the supernatant, the content of proglycol dipeptide was 60.4 g/L, and the conversion rate was 92.8%.

Example 4

A method for preparing a propanol dipeptide, comprising:

The reaction substrate L-alanine methyl ester hydrochloride 20 g and glutamine 21 g were dissolved in pure water (400 mL), and 80 mL of XPD-04 cell solution was slowly added to the reaction substrate, wherein XPD-04 cells were throughout. The mass percentage in the reaction solution was 1.5%, and the pH of the reaction solution was monitored in real time, and the pH was adjusted to 8 with a 5 M sodium hydroxide solution, and the temperature was maintained at 25 °C. During the reaction, the amount of proglycol dipeptide was determined by liquid chromatography after sampling and dilution. The reaction is stopped when the proglycol dipeptide begins to decompose. After centrifugation of the reaction mixture, the supernatant was added to a final concentration of 20% methanol in a water bath at 60 ° C for 30 min, 0.5% activated carbon was stirred for 30 min, and the supernatant was centrifuged to obtain 600 mL of the supernatant, the content of proglycol dipeptide was 61.5 g/L, and the conversion rate was 94.5%.

Example 5

A method for preparing a propanol dipeptide, comprising:

The reaction substrate L-alanine methyl ester hydrochloride 20 g and glutamine 21 g were dissolved in pure water (400 mL), and 100 mL of XPD-05 cell solution was slowly added to the reaction substrate, wherein XPD-05 cells were throughout the reaction. The mass percentage in the liquid was 1.5%, the pH of the reaction solution was monitored in real time, the pH was adjusted to 8, and the temperature was maintained at 25 °C. During the reaction, the amount of proglycol dipeptide was determined by liquid chromatography after sampling and dilution. The reaction is stopped when the proglycol dipeptide begins to decompose. After the reaction solution was centrifuged, the supernatant was added to a final concentration of 20% methanol in a water bath at 60 ° C for 30 min, 0.5% activated carbon was stirred for 30 min, and centrifuged to obtain 625 mL of the supernatant reaction solution, the content of proglycol dipeptide was 55.8 g/L, and the conversion rate was 85.7%.

Example 6

A method for preparing a propanol dipeptide, comprising:

The reaction substrate L-alanine methyl ester hydrochloride 20 g and glutamine 21 g were dissolved in pure water (400 mL), and 90 mL of XPD-06 cell solution was slowly added to the reaction substrate, wherein XPD-05 cells were throughout. The mass percentage in the reaction solution was 1.5%, and the pH of the reaction solution was monitored in real time, and the pH was adjusted to 8 with a 5 M sodium hydroxide solution, and the temperature was maintained at 25 °C. During the reaction, the amount of proglycol dipeptide was determined by liquid chromatography after sampling and dilution. The reaction is stopped when the proglycol dipeptide begins to decompose. After the reaction solution was centrifuged, the supernatant was added to a final concentration of 20% methanol at 60 ° C for 30 min, 0.5% activated carbon was stirred for 30 min, and the supernatant was subjected to centrifugation to obtain 612.5 mL of the supernatant, and the content of proglycol dipeptide was 60.7 g/L, and the conversion rate was 93.2%.

Example 7

A method for preparing a propanol dipeptide, comprising:

The reaction substrate L-alanine methyl ester hydrochloride 20 g and glutamine 21 g were dissolved in pure water (400 mL), and 50 mL of XPD-07 cell solution was slowly added to the reaction substrate, wherein XPD-07 cells were throughout. The mass percentage in the reaction solution was 1.5%, and the pH of the reaction solution was monitored in real time, and the pH was adjusted to 8 with a 5 M sodium hydroxide solution, and the temperature was maintained at 25 °C. During the reaction, the amount of proglycol dipeptide was determined by liquid chromatography after sampling and dilution. The reaction is stopped when the proglycol dipeptide begins to decompose. After the reaction solution was centrifuged, the supernatant was added to a final concentration of 20% methanol at 60 ° C for 30 min, 0.5% activated carbon was stirred for 30 min, and the supernatant was centrifuged to obtain 562.5 mL of the supernatant, the content of proglycidide was 60.9 g/L, and the conversion rate was 93.5%.

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (15)

1. A method for preparing a proglycol dipeptide, which comprises:

   In the presence of α-amino acid ester acyltransferase, the reaction solution is prepared by using L-alanine methyl ester hydrochloride and L-glutamine as a substrate, and the pH of the reaction solution is adjusted to 7.0-9.0 at a constant temperature. After the reaction at 20-40 ° C, the proglycol dipeptide is collected; the α-amino acid ester acyltransferase is derived from Escherichia coli; the amino acid sequence of the α-amino acid ester acylase includes SEQ ID NO: 1 - SEQ ID NO: The amino acid sequence shown in any one of 6.
2. The production method according to claim 1, wherein the gene coding sequence of the α-amino acid ester acyltransferase comprises the nucleotide sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12.
3. The production method according to claim 1, wherein the α-amino acid ester acyltransferase is added to the reaction solution in the form of an expression host cell or an enzyme powder.
4. The preparation method according to claim 1, wherein the method for preparing the L-alanine methyl ester hydrochloride comprises: dropwise adding chlorine disulfoxide to a reaction kettle containing methanol at a temperature of 0 to 10 ° C. And then adding L-alanine to the reaction vessel to carry out a stirring reaction, during which the temperature is slowly raised to 25-30 ° C, and after 0.5-1.0 hours of reaction, the The temperature was raised to 45-50 ° C and stirred for 1.0-2.0 hours; after the reaction was over, the L-alanine methyl ester hydrochloride was collected.
5. The production method according to claim 1, wherein the mass fraction of the L-alanine methyl ester hydrochloride in the reaction liquid is from 3% to 15%; the L-alanine methyl ester salt The mass ratio of the acid salt to the L-glutamine is 1: (0.3-2).
6. The production method according to claim 1, wherein the mass ratio of the L-glutamine to the α-amino acid ester acyltransferase is 1: (0.1-2).
7. The production method according to claim 3, wherein the expression host cell has a mass fraction of 1% to 5% in the reaction solution.
8. The production method according to claim 3, wherein the gene knocks out the protease gene and/or peptidase gene expressing the host cell; the peptidase gene includes one of an aminopeptidase gene and a carboxypeptidase gene or A variety.
9. The production method according to claim 8, wherein the gene knocking out the protease gene and/or peptidase gene expressing the host cell comprises: designing a primer for the expression host using a CRISPR/Cas9 gene editing technique The cell is subjected to genetic recombination, and the protease gene and/or the peptidase gene is knocked out, and the primer includes a nucleotide sequence as shown in SEQ ID NO: 13 to SEQ ID NO: 36.
10. An enzyme for preparing a propanol dipeptide, wherein the preparation enzyme comprises an α-amino acid ester acyltransferase derived from Escherichia coli; the α-amino acid ester acyltransferase The amino acid sequence includes the amino acid sequence set forth in any one of SEQ ID NO: 1 - SEQ ID NO: 6.
11. The enzyme for preparing a proglycol dipeptide according to claim 10, wherein the gene coding sequence of the α-amino acid ester acyltransferase comprises the nucleoside as shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12. Acid sequence.
12. The enzyme for preparing a proglycol dipeptide according to claim 10, wherein the α-amino acid ester acyltransferase is produced by expressing a host cell; and the expression host cell comprises one or more of Escherichia coli and yeast.
13. The enzyme for preparing proacetin dipeptide according to claim 12, wherein the gene knocks out a protease gene and/or a peptidase gene expressing the host cell; and the peptidase gene includes an aminopeptidase gene and a carboxypeptidase gene. One or more.
14. The production method according to claim 13, wherein the gene knocking out the protease gene and/or peptidase gene expressing the host cell comprises: designing a primer for the expression host using a CRISPR/Cas9 gene editing technique The cell is subjected to genetic recombination, and the protease gene and/or the peptidase gene is knocked out, and the primer includes a nucleotide sequence as shown in SEQ ID NO: 13 to SEQ ID NO: 36.
15. Use of a microbial strain of an α-amino acid ester acyltransferase and a gene containing the α-amino acid ester acyltransferase in biocatalysis, wherein the α-amino acid ester acyltransferase is derived from Escherichia coli An α-amino acid ester acyltransferase gene encoding a gene coding sequence comprising the nucleotide sequence set forth in any one of SEQ ID NO: 7 to SEQ ID NO: 12; The α-amino acid ester acyltransferase catalyzes the conversion of L-alanine methyl ester hydrochloride and L-glutamine to form propional dipeptide.

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