Histidine ligase mutant and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to histidine ligase mutants and their applications. The present invention provides a mutant of an amino acid ligase, its application, and a method for preparing a dipeptide. This patent constructs a mutant library by performing site-directed mutagenesis on the enzyme, and finally finds several mutants with high catalytic activity for His-Ala and His-Aib through activity screening, and then combines the actual catalytic process optimization, and finally successfully realizes the amplified production of two application-type dipeptides His-Ala and His-Aib.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a histidine ligase mutant and application thereof. Background Art

[0002] Short peptides containing L-histidine are widely present in nature and have various uses. For example, glucagon-like peptide-1 (GLP-1) in the human body loses its activity under the action of dipeptidyl peptidase (PDD-IV) and releases L-histidine-L-alanine dipeptide (His-Ala). At the same time, His-Ala is also an important raw material for the synthesis of liraglutide, a drug for the treatment of diabetes; the new generation of oral diabetes drug semaglutide modifies the terminal amino acid and replaces alanine Ala with the non-natural amino acid 2-aminoisobutyric acid Aib, thereby greatly improving the stability of the drug in the human body, and thus has gained a huge market; in the preparation of semaglutide, L-histidine-2-aminoisobutyric acid dipeptide (His-Aib) is its important raw material. So far, the main production method of the two commercial dipeptide products His-Ala and His-Aib is still traditional chemical synthesis, that is, by selectively protecting the corresponding amino acids and then chemically coupling and deprotecting. The production process is costly, which directly affects the price of the drugs used. Therefore, there is a huge demand for the development of new and better production processes. The enzymatic preparation of dipeptides has its unique advantages, however, there has been no report on the enzymatic preparation of His-Ala and His-Aib.

[0003] Traditional preparation method of dipeptides:

[0004] 1) The most commonly used method for dipeptides on the market is chemical synthesis. In this preparation process, it is generally necessary to first protect the functional groups that are not desired to participate in the reaction, then chemically couple the two molecules, and finally deprotect them. Therefore, there are many reaction steps and the overall yield is low. At the same time, racemates will inevitably be produced during the chemical coupling process. Since the racemates have properties very similar to the product, they cannot be effectively separated, which greatly increases the purification cost. Therefore, chemical synthesis of dipeptides is not the best choice both economically and in terms of environmental protection (chemical preparation requires a large amount of organic solvents).

[0005] 2) Many literatures have also reported the use of amide bond hydrolases, such as protease, aminopeptidase, etc., to carry out reverse reactions in organic solvents to achieve enzymatic preparation of dipeptides. Compared with the above-mentioned chemical synthesis process, the two amino acid substrates do not need to be protected by corresponding groups, the reaction conditions are relatively mild, and no racemate will be produced; however, the reaction is an equilibrium reaction, and it is difficult to achieve high-yield conversion, and an organic solvent is also required.

[0006] 3) A small number of literatures have reported the use of aminoacid ligase to directly connect amino acids to generate corresponding dipeptides. This method not only does not require the protection of amino acids, but also can be converted with high yield under mild conditions. For example, common glutathione and carnosine. However, the aminoacid ligase reported so far is rarely used in the production and application of dipeptides, which is mainly due to the small number of dipeptidases and the narrow range of catalytic substrates.

[0007] An amino acid ligase (Uniprot: B2DEW2) in Ralstonia solanacearum is reported to have the ability to connect L-histidine with other amino acids to form dipeptides. However, actual tests have found that the catalytic activity of the enzyme is too weak to meet the requirements of industrial applications.

[0008] As mentioned above, the current industrial production method of His-Ala and His-Aib dipeptides is mainly chemical synthesis, but this method has a long preparation route, a large amount of organic solvents, a low final yield and poor product quality. Although some literature reports on the preparation of other dipeptides by enzyme methods, they cannot be used to prepare His-Ala and His-Aib due to the specificity of enzyme catalysts to substrates. Exploring and modifying amino acid ligases to prepare the dipeptides has a relatively large practical application value. Summary of the invention

[0009] In view of this, the present invention provides a histidine ligase mutant and its application, and provides a simple method for preparing His-Ala and His-Aib using an amino acid ligase, which has a very simple route, high yield and good product quality.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a mutant of an amino acid ligase, which:

[0012] The amino acid at position 12 mutated to V, the amino acid at position 83 mutated to I, the amino acid at position 84 mutated to D, the amino acid at position 85 mutated to W, and the amino acid at position 311 mutated to L;

[0013] or

[0014] The amino acid at position 12 mutated to I, the amino acid at position 83 mutated to L, the amino acid at position 84 mutated to E, the amino acid at position 85 mutated to Y, and the amino acid at position 311 mutated to L;

[0015] The Uniprot ID of the amino acid ligase is B2DEW2.

[0016] In some specific embodiments of the present invention, the mutant has:

[0017] (1), the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.3; or

[0018] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0019] (3) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2);

[0020] The plurality is 2 to 130.

[0021] The present invention also provides a nucleic acid molecule encoding the above mutant;

[0022] The nucleic acid molecule has:

[0023] (4) the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6; or

[0024] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or

[0025] (6) a nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5);

[0026] The plurality is from 2 to 400.

[0027] The present invention also provides an expression vector, comprising the above nucleic acid molecule and acceptable gene elements.

[0028] The present invention also provides a host cell, comprising the above nucleic acid molecule or the above expression vector.

[0029] The present invention also provides a composition comprising the mutant and adenosine triphosphate.

[0030] In some specific embodiments of the present invention, the above composition further comprises polyphosphate kinase or polyphosphate;

[0031] The polyphosphoric acid includes phosphoric acid having a polyor ratio of six or more;

[0032] The polyphosphoric acid is hexaphosphoric acid.

[0033] In some specific embodiments of the present invention, the above composition, the polyphosphate kinase has:

[0034] (7) the amino acid sequence shown in SEQ ID NO.1; or

[0035] (8) an amino acid sequence obtained by replacing, deleting or adding one or more residues of the amino acid sequence shown in (7), and having the same or similar function as (7); or

[0036] (9) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (7) or (8);

[0037] The plurality is 2 to 120.

[0038] The present invention also provides the use of any of the following in the synthesis of dipeptides:

[0039] (I) the above mutants;

[0040] (II), the above-mentioned nucleic acid molecule;

[0041] (III) the above-mentioned expression vector;

[0042] (IV), the above-mentioned host cell;

[0043] (V), the above composition;

[0044] The dipeptide includes L-histidine-L-alanine or L-histidine-2-aminoisobutyric acid.

[0045] The present invention also provides a method for preparing the dipeptide, comprising:

[0046] (a), mixing the mutant and amino acids to obtain a dipeptide; or

[0047] (b), expressing the nucleic acid molecule, and mixing the obtained protein product with amino acids to obtain a dipeptide; or

[0048] (c) expressing the above expression vector, mixing the obtained protein product with amino acids to obtain a dipeptide; or

[0049] (d) culturing the above host cell, mixing the obtained protein product with amino acids to obtain a dipeptide; or

[0050] (e), mixing the above composition with amino acids to obtain a dipeptide;

[0051] The dipeptide includes L-histidine-L-alanine or L-histidine-2-aminoisobutyric acid;

[0052] The amino acids include L-histidine, L-alanine and / or 2-aminoisobutyric acid.

[0053] The mutants and methods of the present invention have the following effects:

[0054] The mutant library of amino acid ligases of Ralstonia solanacearum introduced in this patent makes it possible to prepare His-Ala and His-Aib enzymes directly. At the same time, the enzyme preparation method has many advantages, such as simple preparation, high yield, good product quality, good environmental compatibility, low carbon emissions, easy large-scale production, etc.

[0055] The present invention utilizes an amino acid ligase in Ralstonia solanacearum that can catalyze the condensation of histidine and other amino acids, and finally obtains a dipeptide compound that can catalyze the production of L-histidine, L-alanine and 2-aminoisobutyric acid by mutating the amino acid residues in its catalytic activity pocket. The method is much better than the traditional method of chemically synthesizing the dipeptide. It shows outstanding advantages in production cost, energy consumption, product quality and green index. Therefore, the large-scale production of the method will be the best choice for the production of the above dipeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0057] Figure 1 The figure shows the HPLC detection of the reaction process of histidine ligase HA1 catalyzing the formation of His-Ala dipeptide in the absence of ATP regeneration system; wherein, the upper figure is HA1-0h; the lower figure is HA1-2h;

[0058] Figure 2 The figure shows the HPLC detection of the reaction process of histidine ligase HA1 catalyzing the formation of His-Ala dipeptide in the ATP regeneration system; wherein, the upper figure is HA1+PPK-0h; the lower figure is HA1+PPK-3.5h;

[0059] Figure 3 shows the NMR spectrum of His-Ala;

[0060] Figure 4 The mass spectrum of His-Ala is shown (MS (ESI): m / z: 241.1 [M+H]+);

[0061] Figure 5 The figure shows the HPLC detection of the reaction process of histidine ligase HA2 catalyzing the formation of His-Aib dipeptide in the absence of ATP regeneration system; wherein, the upper figure is HA2-0h; the lower figure is HA2-3h;

[0062] Figure 6 The figure shows the HPLC detection of the reaction process of histidine ligase HA2 catalyzing the formation of His-Aib dipeptide in the ATP regeneration system; wherein, the upper figure is HA1+PPK-0h; the lower figure is HA1+PPK-2h;

[0063] Figure 7 Shown is the gel electrophoresis diagram of the enzyme; the three lanes on the left are Marker, HA1, and HA2 respectively; the two lanes on the right are Marker and PPK respectively. DETAILED DESCRIPTION

[0064] The present invention discloses histidine ligase mutants and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0065] On this basis, this patent conducted site-directed mutagenesis on the enzyme, constructed a mutant library, and finally found several mutants with high catalytic activity for His-Ala and His-Aib through activity screening. Combined with the actual catalytic process optimization, it finally successfully achieved the scaled-up production of two application-oriented dipeptides His-Ala and His-Aib.

[0066] The present invention utilizes the corresponding amino acid ligase to directly connect two target amino acid raw materials into the corresponding dipeptide product in one step in a buffer. The adenosine triphosphate (ATP) required in the reaction process can be an equivalent amount or a catalytic amount (by matching the ATP regeneration system, using polyphosphate kinase PPK and hexaphosphate).

[0067] The patented amino acid ligase preparation route:

[0068]

[0069] This route uses cheap, unprotected amino acids as raw materials, and converts them into corresponding dipeptide products with high yield under the action of equivalent adenosine triphosphate (ATP) and corresponding amino acid ligase. To further save costs, the ATP in the reaction system can also be recycled and regenerated to further reduce its usage. Therefore, in general, this preparation route is very streamlined, with high yield and good product quality (no racemic impurities are generated), high green index in production and easy to scale production.

[0070] Enzyme related information:

[0071] Amino acid ligase (Ligase HA1 / HA2): derived from Ralstonia solanacearum (Uniprot ID: B2DEW2, EC 6.3.2.49);

[0072] Polyphosphate kinase (PPK): derived from Paenarthrobacter aurescens (Uniprot ID: A1R8G0)

[0073] The protein electrophoresis results used in the examples are shown in Figure 7 .

[0074] Table 1

[0075]

[0076] Table 2

[0077]

[0078]

[0079] Fermentation production of enzymes:

[0080] The enzymes required for this patent are all synthesized by the company and then constructed on a specific expression plasmid and then produced by E. coli fermentation; it specifically includes the following steps: optimize the sequence of the genes corresponding to the above enzymes and then place an order to General Biological Company for synthesis (Chuzhou, Anhui), then introduce NdeI / XhoI restriction sites and subclone into the pET 28a expression vector. Confirm that the plasmid with the correct sequence is transferred to E. coli (BL21) competent cells for plate culture (Qingke Biology) and monoclonal small-scale liquid culture, and the bacteria with correct protein expression are finally amplified in liquid culture step by step. It specifically includes transferring a single colony into 5mL LB culture medium containing 50μM kanamycin (37℃) for culture, and when the cells grow to the logarithmic phase, they are inoculated into 250mL LB culture medium containing the same antibiotics. When they grow to the logarithmic phase, they are transferred to a 5L culture fermenter for culture and final protein expression. In a 5L fermentation tank, when the cell OD is 20, 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added at 25°C to induce protein expression for 6 hours, and finally the cells are collected by high-speed centrifugation (4000rpm, 20min) to obtain 25-50g of enzyme overexpression wet cells. A small amount of cells is first mixed evenly with tris-hydroxymethylaminomethane hydrochloric acid (Tris.HCl) buffer (50mM, pH 8.0) on an ice basin, and then the cells are broken by freeze-thaw method, and the supernatant is run on SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. The bacterial cells with correct protein expression were used for the next catalytic experiment. Specifically, the remaining cells were mixed with Tris.HCl buffer (50mM, pH8.0) at low temperature (~10g wet cells: 200mL buffer), and then the cell wall was broken by low temperature and high pressure. After the cell wall was removed by high-speed centrifugation (16000rpm, 45min), the enzyme-containing clear solution was obtained for use (the enzyme activity obtained was 200-600U / mL, U is the amount of enzyme required to convert 1μmol of substrate in one minute at room temperature). LB medium is composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0081] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0082] The present invention will be further described below in conjunction with embodiments:

[0083] Example 1: Preparation of L-histidine-L-alanine His-Ala using ligase (ligase HA1)

[0084]

[0085] 23.2 g of L-histidine (150 mM), 14 g of L-alanine (157 mM), and 83.3 g of adenosine triphosphate monosodium salt (ATP, 157 mM) were added to 1 L of 100 mM pH 8.0 tris(hydroxymethylaminomethane) hydrochloric acid (Tris.HCl) solution, and then the pH value of the reaction system was adjusted to 8.0 by NaOH aqueous solution, and then ligase HA1 2000 U was added to start the reaction, and the reaction system was slightly stirred at 30°C and the pH of the reaction system was maintained between 7.0 and 9.0. After 2 hours, the raw material conversion rate was detected by HPLC at 80-85% ( Figure 1 ). Then, the pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and the protein solid was removed by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 3:1, v:v) to obtain 30.8 grams of white L-histidine-L-alanine (His-Ala) dipeptide (yield 91%). The crystallized and purified product His-Ala was sent for nuclear magnetic resonance and mass spectrometry, and the correct structure was confirmed, see Figure 3 , 4 .

[0086] Signal attribution of the nuclear magnetic spectrum of His-Ala: 1H NMR (400MHz, Deuterium Oxide) δ8.71 (d, J = 1.4 Hz, 1H), 7.47 (d, J = 1.4 Hz, 1H), 4.44 (q, J = 7.3 Hz, 1H), 4.32 (dd, J = 7.7, 5.8 Hz, 1H), 3.48–3.39 (m, 2H), 1.43 (d, J = 7.4 Hz, 3H).

[0087] Example 2: Preparation of L-histidine-2-aminoisobutyric acid His-Aib using ligase (HA2)

[0088]

[0089] Similar to the above-mentioned His-Ala preparation method, 15.5 g of L-histidine (100 mM), 11.3 g of 2-aminoisobutyric acid (110 mM), and 55.5 g of adenosine triphosphate monosodium salt (ATP, 105 mM) were added to 1 L of 100 mM pH 8.0 tris hydroxymethylaminomethane hydrochloric acid (Tris.HCl) solution, and then the pH value of the reaction system was adjusted to 8.0 by NaOH aqueous solution, and then ligase HA2 2000U was added to start the reaction. The reaction system was gently stirred at 30°C and the pH of the reaction system was maintained between 7.0 and 9.0. After 3 hours, the raw material conversion rate was detected by HPLC to reach 80-85% ( Figure 5 ). Then, the pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and the protein solid was removed by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 2:1, v:v) to obtain 18 grams of white L-histidine-2-aminoisobutyric acid (His-Aib) dipeptide (yield 75%). The crystallized and purified product His-Aib was sent for nuclear magnetic resonance, which confirmed the correct structure. The nuclear magnetic resonance data obtained after the nuclear magnetic resonance was as follows:

[0090] Signal attribution of His-Aib NMR spectrum: 1 H NMR (400 MHz, D 2 O)δ8.65(s,1H),7.74(s,1H),3.90-3.82(m,1H),3.88-3.89(m,1H),3.40–3.14(m,1H),1.70(s,6H).

[0091] Example 3: Preparation of L-histidine-L-alanine His-Ala using ligase HA1 and ATP regeneration system

[0092]

[0093] Similarly, 23.2 g of L-histidine (150 mM), 14 g of L-alanine (157 mM), 2.7 g of adenosine triphosphate monosodium salt (ATP, 5 mM), and 24.1 g of sodium hexaphosphate (39.4 mM) were added to 1 L of 100 mM pH 7.5 tris(hydroxymethylaminomethane) hydrochloric acid (Tris.HCl) solution. After adjusting the pH value of the solution to 7.5, 3500 U of polyphosphate kinase PPK enzyme and 12500 U of ligase HA were added. The reaction system was gently stirred at room temperature (25°C) and the pH value was maintained between 7.0 and 8.5. After 3.5 hours, it was detected by HPLC that the raw materials were basically reacted completely. Then, the pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and the protein solid was removed by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 3:1, v:v) to obtain 29.4 g of white L-histidine-L-alanine (His-Ala) dipeptide (yield 87%). Figure 2 ).

[0094] Example 4: Preparation of L-histidine-2-aminoisobutyric acid His-Aib using ligase HA2 and ATP regeneration system

[0095]

[0096] Similarly, 15.5 g of L-histidine (100 mM), 11.3 g of 2-aminoisobutyric acid (110 mM), 2.7 g of adenosine triphosphate monosodium salt (ATP, 5 mM), and 16.1 g of sodium hexaphosphate (26.3 mM) were added to 1 L of 100 mM pH 7.5 tris(hydroxymethylaminomethane) hydrochloric acid (Tris.HCl) solution. After adjusting the pH value of the solution to 7.5, 3000 U of polyphosphate kinase PPK enzyme and 3000 U of ligase HA2 were added. The reaction system was gently stirred at room temperature (25°C) and the pH value was maintained between 7.0 and 8.5. After 2 hours, it was detected by HPLC that the raw materials were basically reacted completely. Then, the pH was adjusted to 2.0 with HCl aqueous solution to denature the enzyme in the reaction system and precipitate it, and the protein solid was removed by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 2:1, v:v) to obtain 17.1 g of L-histidine-2-aminoisobutyric acid (His-Aib) dipeptide (yield 71%). Figure 6 ).

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant of an amino acid ligase, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that Encoding the mutant according to claim 1; The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.5 or SEQ ID NO.

6.

3. An expression vector, characterized in that It comprises the nucleic acid molecule as claimed in claim 2, and acceptable genetic elements.

4. A host cell, characterized in that Comprising the nucleic acid molecule according to claim 2 or the expression vector according to claim 3.

5. A composition, characterized in that It comprises the mutant as claimed in claim 1 and adenosine triphosphate.

6. The composition according to claim 5, characterized in that Also included are polyphosphate kinases or polyphosphates; The polyphosphoric acid includes hexaphosphoric acid.

7. The composition according to claim 6, characterized in that The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO.

1.

8. Use of any of the following in the synthesis of dipeptides: (I) The mutant according to claim 1; (II), the nucleic acid molecule according to claim 2; (III) the expression vector according to claim 3; (IV) The host cell according to claim 4; (V) A composition according to any one of claims 5 to 7; The dipeptide includes L-histidine-L-alanine or L-histidine-2-aminoisobutyric acid.

9. A method for preparing a dipeptide, characterized in that: include: (a) mixing the mutant according to claim 1 with an amino acid to obtain a dipeptide; or (b) expressing the nucleic acid molecule of claim 2, and mixing the obtained protein product with amino acids to obtain a dipeptide; or (c) expressing the expression vector as claimed in claim 3, and mixing the obtained protein product with amino acids to obtain a dipeptide; or (d) culturing the host cell according to claim 4, mixing the obtained protein product with amino acids to obtain a dipeptide; or (e) mixing the composition according to any one of claims 5 to 7 with an amino acid to obtain a dipeptide; The dipeptide includes L-histidine-L-alanine or L-histidine-2-aminoisobutyric acid; The amino acids include L-histidine, L-alanine and / or 2-aminoisobutyric acid; The preparation method requires synthesizing the dipeptide under the action of adenosine triphosphate.

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