CN116445429A - Beta-alanine ligase mutant and application thereof - Google Patents

Beta-alanine ligase mutant and application thereof Download PDF

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Publication number
CN116445429A
CN116445429A CN202310444653.8A CN202310444653A CN116445429A CN 116445429 A CN116445429 A CN 116445429A CN 202310444653 A CN202310444653 A CN 202310444653A CN 116445429 A CN116445429 A CN 116445429A
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amino acid
acid residue
mutation
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赵弘
丁颖
段晓伟
丁小妹
陈淋转
于铁妹
潘俊锋
刘建
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Zhuhai Ruidelin Biology Co ltd
Shenzhen Readline Biotechnology Co ltd
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Shenzhen Readline Biotechnology Co ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)

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Abstract

The invention relates to the field of biotechnology, in particular to a beta-alanine ligase mutant and application thereof. The invention provides a ligase mutant, the amino acid sequence of which is shown as SEQ ID NO. 2. According to the invention, by utilizing the characteristic that one L-amino acid ligase in actinobacillus can catalyze and connect beta-alanine with other amino acids, the high catalytic activity production decarboxylation carnosine is finally obtained by mutating the amino acid residues of the catalytic activity pocket and other relevant parts. The method is greatly superior to a chemical synthesis preparation process, and has outstanding advantages in production cost, energy consumption, product quality and green index. Thus, large scale production of this method would be a preferred option for decarboxylation carnosine production.

Description

Beta-alanine ligase mutant and application thereof
Technical Field
The invention relates to the field of biotechnology, in particular to a beta-alanine ligase mutant and application thereof.
Background
Beta-alanine is a naturally occurring beta amino acid whose amino group is attached to the beta-carbon of alanine, rather than the more common alpha-carbon. Beta-alanine is commonly used as a motor supplement and endurance aid that enhances motor performance and promotes overall health, while beta-alanine is also a component of many other short peptides such as decarboxylated carnosine. Decarboxylated carnosine (Decarboxy Carnosine) is a biomimetic peptide with antioxidant capacity and anti-aging effects.
The preparation method of decarboxylated carnosine reported on the market at present is mainly a chemical synthesis method.
The traditional chemical synthesis method is a main production process of the dipeptide product at present, and the preparation process is to utilize beta-alanine protected by Boc and histamine protected by Boc for condensation, and finally remove Boc protecting group, or to use chloropropionyl chloride and histamine hydrochloride as raw materials for distributed synthesis preparation. Both schemes need a large amount of organic solvents, have more production steps and do not meet the requirements of green production.
In summary, the preparation of decarboxylated carnosine by chemical synthesis has the problems of long preparation route, large organic solvent consumption, low final yield and poor product quality. The preparation of the dipeptide by exploring and modifying the L-amino acid connection has great competitive advantage and practical application value, and the development of a new and better production process is a relatively realistic requirement for producing the dipeptide product at present.
Disclosure of Invention
In view of the above, the beta-alanine ligase mutant and the application thereof provided by the invention have the advantages of simple preparation route, high yield, good product quality, high green index in production and easy mass production.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a mutant of Uniprot ID B0BTG0 amino acid ligase, wherein mutation sites comprise:
mutation of amino acid residue 13 to threonine; and/or
Mutation of amino acid residue 14 to phenylalanine; and/or
Mutation of amino acid residue 87 to glutamine; and/or
The amino acid residue at position 91 is mutated to alanine; and/or
Mutation of amino acid residue 239 to asparagine; and/or
Amino acid residue 287 is mutated to valine; and/or
The 291 amino acid residue is mutated to tryptophan; and/or
Amino acid residue 317 is mutated to methionine.
In some embodiments of the invention, the mutation sites of the above mutants further comprise:
mutation of amino acid residue 107 to asparagine; and/or
The amino acid residue at position 170 is mutated to cysteine; and/or
Mutation of amino acid residue 192 to proline; and/or
Amino acid residue 243 is mutated to isoleucine.
In some embodiments of the invention, the mutant has:
(1) An amino acid sequence shown as SEQ ID NO. 2; or (b)
(2) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (1), and having the same or similar functions as those of (1); or (b)
(3) An amino acid sequence having at least 70% homology with the amino acid sequence as set forth in (1) or (2);
the plurality is 2 to 60.
The invention also provides a nucleic acid molecule encoding the mutant, which has:
(4) A nucleotide sequence shown as SEQ ID NO. 4; or (b)
(5) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (4), and having the same or similar function as (4); or (b)
(6) A nucleotide sequence having at least 80% homology with the nucleotide sequence as set forth in (4) or (5);
the plurality is 2 to 200.
The invention also provides expression vectors comprising the above nucleic acid molecules, as well as acceptable genetic elements.
The invention also provides a host cell comprising the above nucleic acid molecule or the above expression vector.
The invention also provides a composition comprising the mutant and adenosine triphosphate.
In some embodiments of the invention, the above composition further comprises a polyphosphate kinase and a polyphosphate;
the polyphosphoric acid includes metaphosphoric acid.
In some embodiments of the invention, the polyphosphate kinase in the above composition has:
(7) An amino acid sequence shown as SEQ ID NO. 1; or (b)
(8) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (7), and having the same or similar functions as those of (7); or (b)
(9) An amino acid sequence having at least 90% homology with the amino acid sequence as set forth in (7) or (8);
the plurality is 2 to 30.
The invention also provides the use of any of the following in decarboxylation carnosine synthesis:
(i) The mutant;
(ii) The nucleic acid molecules described above;
(iii) The expression vector;
(iv) The host cell;
(v) And the composition.
The invention also provides a preparation method of the decarboxylated carnosine, which comprises the following steps:
(a) Mixing the raw materials with the mutant to obtain the decarboxylated carnosine; ###
(b) Expressing the nucleic acid molecules, and mixing the obtained protein product with raw materials to obtain the decarboxylated carnosine; or (b)
(c) Expressing the expression vector, and mixing the obtained protein product with raw materials to obtain the decarboxylated carnosine; or (b)
(d) Culturing the host cell, and mixing the obtained protein product with a raw material to obtain the decarboxylated carnosine; or (b)
(e) Mixing the raw materials with the composition to obtain the decarboxylated carnosine;
the raw materials include beta-alanine and histamine.
The ligase mutant and the application thereof have the following effects:
according to the invention, by utilizing the characteristic that one L-amino acid ligase in actinobacillus can catalyze and connect beta-alanine and other amino acids, the high catalytic activity production decarboxylation carnosine is finally obtained by mutating the catalytic activity pocket of the L-amino acid ligase and other amino acid residues at relevant positions. The method is greatly superior to a chemical synthesis preparation process, and has outstanding advantages in production cost, energy consumption, product quality and green index. Thus, large scale production of this method would be a preferred option for decarboxylation carnosine production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows an HPLC plot of a Ligase ADH2 reaction for 3 hours;
FIG. 2 shows a nuclear magnetic spectrum of the product decarboxylated carnosine of example 1;
FIG. 3 shows a mass spectrum of the product decarboxylated carnosine in example 1;
FIG. 4 shows an HPLC chromatogram of the modification of wild B0BTG0 to the intermediate mutant B0BTG0-130 enzyme reaction for 3 hours.
Detailed Description
The invention discloses a beta-alanine ligase mutant and application thereof, and a person skilled in the art can refer to the content of the specification to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The patent discloses a novel and simple scheme for preparing decarboxylated carnosine at low cost and high efficiency by adopting beta-alanine ligase.
Screening results by a large number of amino acid ligase catalytic substrate activities showed that one L-amino acid ligase (Uniprot: B0BTG 0) present in actinobacillus (Actinobacillus pleuropneumoniae) has the ability to ligate beta-alanine with other various amino acids such as beta-alanine, L-cysteine, L-histidine, etc. However, the catalytic activity is still too weak, and the catalytic substrate is too narrow, so that the catalyst cannot be directly applied to industrial production. The enzyme is used as a reconstruction template, rational site-directed mutagenesis and irrational random mutagenesis are combined, finally, the ligase with high catalytic activity on beta-alanine and histamine is screened from 6000 mutant libraries, and the actual catalytic process optimization is combined, so that the amplified production of the decarboxylated carnosine is finally successfully realized.
The amino acid ligase method of the patent comprises the following steps:
this route utilizes inexpensive amino acids without protecting groups as starting materials for high yield conversion to the corresponding dipeptide product under the action of equivalent amounts of Adenosine Triphosphate (ATP) and the corresponding amino acid ligase. In order to further save the cost, the ATP recycling system is introduced into the reaction system, so that the ATP usage amount can be further reduced. Therefore, the preparation method has the advantages of simple preparation route, high yield, good product quality, high green index in production and easy mass production.
The invention can directly connect and convert two target amino acid raw materials into corresponding dipeptide products in one step in buffer solution by using corresponding amino acid ligase. The Adenosine Triphosphate (ATP) required during the reaction may be equivalent or catalytic (by being complexed with an ATP regeneration system, the polyphosphate kinase PPK and metaphosphoric acid are employed).
Enzyme related information:
amino acid Ligase (Ligase ADH 2): the template gene is derived from an L-amino acid ligase (Uniprot: B0BTG 0) existing in the actinobacillus (Actinobacillus pleuropneu moniae) body; and (5) taking the modified material as a template for transformation. Specific mutation site information for Ligase ADH 2: H13T, F14L, N87Q, L91A, K107N, V170C, E192P, P239N, S243I, P287V, T291W, Y317M.
Polyphosphate Kinase (PPK): is derived from Thermus rhodochrous (Meiothermus ruber) (uniprotID: M9XB 82).
The sequence information of the enzymes is shown in tables 1 and 2.
TABLE 1
TABLE 2
Fermentation production of accessory enzyme: the enzyme required by the invention is prepared by constructing a specific expression plasmid after synthesizing corresponding genes by commercial companies and then producing the corresponding genes by escherichia coli fermentation, and specifically comprises the following steps: after sequence optimization, the genes corresponding to the enzymes are ordered to be synthesized by general biological company (Chuzhou of Anhui), and NdeI/XhoI restriction sites are introduced and subcloned into a pET-28a expression vector; plasmid with correct sequence was confirmed to be transferred into E.coli (BL 21) competent cells plate culture (of the species Prinsepia) and monoclonal miniculture, the bacteria with correct protein expression are finally amplified and cultured step by step. Specifically, single colony is transferred into 5ml LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, when cells grow to the logarithmic phase, the cells are inoculated into 250ml LB culture solution containing the same antibiotics, and when the cells grow to the logarithmic phase, the cells are transferred into a 5L culture fermentation tank for culture and final protein expression is carried out. In 5L fermentation tank culture, 0.5mM isopropyl-beta-D-thiopyran galactoside (IPTG) is added at 25 ℃ to induce protein expression for 6 hours when the cells OD-20, and finally the cells are collected by high-speed centrifugation (4000 rpm,20 min) to obtain 25-50 g of wet cells with over-expressed enzyme. A small amount of cells are firstly mixed with a buffer solution (50 mM, pH 8.0) of tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) on an ice basin uniformly, then the cells are broken by a freeze thawing method, and clear liquid is subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) after cell walls are removed by high-speed centrifugation to determine protein expression. Cells with correct protein expression were used for the next catalytic experiment, specifically, the remaining cells were mixed with Tris.HCl buffer (50 mM, pH 8.0) at low temperature (200 ml buffer mixing with 10 g wet cells), then crushed cell walls at low temperature Gao Yapo, and the cell walls were removed by high speed centrifugation (16000 rpm,45 min) to obtain enzyme-containing supernatant (the enzyme activity obtained was 500-1000U/ml, U was the amount of enzyme required for converting 1. Mu. Mol of substrate in one minute at room temperature). LB medium consisted of: 1% tryptone, 0.5% yeast powder, 1% NaCl,1% dipotassium hydrogen phosphate and 5% glycerol.
The table of the attached enzyme activity statistics is shown in Table 3.
TABLE 3 Table 3
Properties of (C) Ligase ADH2 PPK
Unit vitality 850～1000U/mg 950～1400U/mg
Thermal stability (30 ℃ C.) T 1/2 ＝35min T 1/2 ＝80min
Enzyme expression level mg pure protein/liter culture solution 400～600 700～900
Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all commercially available and commercially available.
The invention is further illustrated by the following examples:
example 1: preparation of decarboxylated carnosine (Decarboxy Carnosine) Using Ligase (Ligase ADH 2)
To 1L 100mM tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) solution at pH 7.5 was added 16.6 g of histamine (150 mM), 16 g of beta-alanine (180 mM), 87.5 g of adenosine triphosphate monosodium salt (ATP, 165 mM), and after the pH of the reaction system was adjusted to 7.5 by NaOH aqueous solution, the reaction was started by adding Ligase Ligase ADH2 2000U, and after stirring slightly at 30℃and maintaining the pH of the reaction system between 6.5 and 8.5, the reaction of histamine starting material was detected to be substantially complete by HPLC after 3 hours, and FIG. 1 shows an HPLC chart. However, the method is thatThen regulating pH to 2.0 with HCl aqueous solution, carrying out enzyme denaturation precipitation in the reaction system, centrifuging to remove protein solids, regulating pH of the reaction solution to 7.0, directly loading the reaction solution into a D201 anion exchange resin purification column, removing adenosine diphosphate and free phosphoric acid impurities, and finally desalting the crude product by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 3:1, v: v) to obtain 24.8 g of decarboxylated carnosine (yield 91%). And (3) confirming that the product is decarboxylated carnosine through sample feeding nuclear magnetism and mass spectrum. The nuclear magnetic resonance spectrum and mass spectrum result are respectively shown in figure 2 fig. 3. 1 H NMR(400MHz,D 2 O)δ8.66(s,1H),7.34(s,1H),3.56(t,J＝6.5Hz,2H),3.28(t,J＝6.5Hz,2H),3.00(t,J＝6.5Hz,2H),2.70(t,J＝6.5Hz,2H).ESI+:[m+H] + :183.1；[2m+H] + :365.3。
Example 2: preparation of decarboxylated carnosine (Decarboxy Carnosine) Using Ligase (Ligase ADH 2) and an ATP regenerating System
To 1L 100mM tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) solution at pH 7.5 was added 16.6 g histamine (150 mM), 16 g beta-alanine (180 mM), 2.7 g adenosine triphosphate monosodium salt (ATP, 5 mM), 33.6 g sodium metaphosphate (55 mM), after adjusting the pH of the solution to 7.5, 3000U of polyphosphate kinase PPK enzyme was added, and the reaction was started by adding Ligase Ligase ADH2 2000U, slightly stirred at 30℃and maintained at pH of the reaction system between 6.5 and 8.5, after 3 hours the reaction of the histamine starting material was detected to be substantially complete by HPLC. Then regulating pH to 2.0 by using an HCl aqueous solution, carrying out enzyme denaturation precipitation in a reaction system, centrifuging to remove protein solids, regulating the pH of the reaction solution to 7.0, directly loading the reaction solution into a D201 anion exchange resin purification column, removing adenosine diphosphate and free phosphoric acid impurities, and finally desalting a crude product by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 3:1, v: v) to obtain 25.9 g of decarboxylated carnosine (yield 95%).
Comparative example: preparation of decarboxylated carnosine (Decarboxy Carnosine) Using wild-type ligase (B0 BTG 0), wild-type ligase mutant (B0 BTG 0-130)
Control group 1:
to 1L 100mM tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) solution at pH 7.5 was added 16.6 g of histamine (150 mM), 16 g of beta-alanine (180 mM), 87.5 g of adenosine triphosphate monosodium salt (ATP, 165 mM), and after the pH of the reaction system was brought to 7.5 by NaOH aqueous solution, the reaction was started by adding wild type ligase B0BTG0 2000U, and after stirring slightly at 30℃and maintaining the pH of the reaction system between 6.5 and 8.5, 60% of histamine starting material was still left by HPLC after 3 hours, and the reaction was very incomplete.
Control group 2:
to 1L 100mM tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) solution at pH 7.5 was added 16.6 g of histamine (150 mM), 16 g of beta-alanine (180 mM), 87.5 g of adenosine triphosphate monosodium salt (ATP, 165 mM), and after the pH of the reaction system was brought to 7.5 by NaOH aqueous solution, the reaction was started by adding wild type ligase B0BTG0-130 2000U, and after stirring slightly at 30℃and maintaining the pH of the reaction system between 6.5 and 8.5, 30% of histamine starting material was still left by HPLC after 3 hours, which was shown in FIG. 4.
Table 4: comparison summary of mutant B0BTG0-130 and the mutant of the invention
The mutant Ligase ADH2 and the mutant B0BTG0-130 are compared, and the conversion rate is improved by 21%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

  1. A mutant of an amino acid ligase having a uniprot ID of B0BTG0, characterized in that the mutation site comprises:
    mutation of amino acid residue 13 to threonine; and/or
    Mutation of amino acid residue 14 to phenylalanine; and/or
    Mutation of amino acid residue 87 to glutamine; and/or
    The amino acid residue at position 91 is mutated to alanine; and/or
    Mutation of amino acid residue 239 to asparagine; and/or
    Amino acid residue 287 is mutated to valine; and/or
    The 291 amino acid residue is mutated to tryptophan; and/or
    Amino acid residue 317 is mutated to methionine.
  2. 2. The mutant of claim 1, wherein the mutation site further comprises:
    mutation of amino acid residue 107 to asparagine; and/or
    The amino acid residue at position 170 is mutated to cysteine; and/or
    Mutation of amino acid residue 192 to proline; and/or
    Amino acid residue 243 is mutated to isoleucine.
  3. 3. The mutant according to claim 2, which has:
    (1) An amino acid sequence shown as SEQ ID NO. 2; or (b)
    (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (1), and having the same or similar functions as those of (1); or (b)
    (3) An amino acid sequence having at least 70% homology with the amino acid sequence as set forth in (1) or (2);
    the plurality is 2 to 60.
  4. 4. A nucleic acid molecule encoding the mutant according to claim 2 or 3, which has:
    (4) A nucleotide sequence shown as SEQ ID NO. 4; or (b)
    (5) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (4), and having the same or similar function as (4); or (b)
    (6) A nucleotide sequence having at least 80% homology with the nucleotide sequence as set forth in (4) or (5);
    the plurality is 2 to 200.
  5. 5. An expression vector comprising the nucleic acid molecule of claim 4, and an acceptable genetic element.
  6. 6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.
  7. 7. A composition comprising a mutant according to any one of claims 1 to 3 and adenosine triphosphate.
  8. 8. The composition of claim 7, further comprising a polyphosphate kinase and a polyphosphate;
    the polyphosphoric acid includes metaphosphoric acid.
  9. 9. Use in the synthesis of decarboxylated carnosine of any of the following:
    (i) A mutant according to any one of claims 1 to 3;
    (ii) The nucleic acid molecule of claim 4;
    (iii) The expression vector of claim 5;
    (iv) The host cell of claim 6;
    (v) A composition according to claim 7 or 8.
  10. 10. A method for preparing decarboxylated carnosine, comprising:
    (a) Mixing a raw material with the mutant according to any one of claims 1 to 3 to obtain the decarboxylated carnosine; or (b)
    (b) Expressing the nucleic acid molecule of claim 4, mixing the obtained protein product with a starting material to obtain said decarboxylated carnosine; or (b)
    (c) Expressing the expression vector according to claim 5, mixing the obtained protein product with a raw material to obtain the decarboxylated carnosine; or (b)
    (d) Culturing the host cell of claim 6, mixing the obtained protein product with a starting material to obtain the decarboxylated carnosine; or (b)
    (e) Mixing the raw materials with the composition according to claim 7 or 8 to obtain the decarboxylated carnosine;
    the raw materials include beta-alanine and histamine.
CN202310444653.8A 2023-04-14 2023-04-14 Beta-alanine ligase mutant and application thereof Pending CN116445429A (en)

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