CN116410938A

CN116410938A - Beta-alanine ligase mutant and application thereof

Info

Publication number: CN116410938A
Application number: CN202310421142.4A
Authority: CN
Inventors: 赵弘; 丁颖; 段晓伟; 丁小妹; 陈淋转; 于铁妹; 潘俊锋; 刘建
Original assignee: Zhuhai Ruidelin Biology Co ltd; Shenzhen Readline Biotechnology Co ltd
Current assignee: Zhuhai Ruidelin Biology Co ltd; Shenzhen Readline Biotechnology Co ltd
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-07-11
Anticipated expiration: 2043-04-14
Also published as: CN116410938B

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 carnosine is finally obtained by mutating the amino acid residues in 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 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 carnosine. Carnosine (L-Carnosine) is a molecule that helps to buffer muscle acids, and has effects of regulating immunity, maintaining endocrine balance in human body, and nourishing body.

The methods for preparing carnosine reported on the market at present are chemical synthesis methods and biological methods.

The traditional chemical synthesis method is a main production process of the dipeptide product at present, and the preparation process comprises the steps of protecting beta-alanine amino with phthalic anhydride, activating carboxyl with thionyl chloride, condensing with L-histidine protected by trimethylchlorosilane to obtain protected carnosine, and deprotecting. The chemical synthesis method generally needs complicated steps of chemical protection/deprotection of functional groups, and has the disadvantages of high production cost, low overall yield and easy racemization in the process due to long synthesis route, thereby greatly restricting the further popularization and application of the product.

The existing biological method is used for preparing carnosine, the main production process is an enzymatic method, the preparation process adopts aminopeptidase to catalyze beta-alanine methyl ester and L-histidine to synthesize carnosine, the method needs esterified beta-alanine, and meanwhile, the aminopeptidase reaction is a reversible reaction, so that the conversion is incomplete, the overall yield is directly low, and the final product is difficult to separate and purify; meanwhile, a method of culturing and fermenting by utilizing separated active microorganisms and then separating and purifying fermentation liquor is reported, but the final separation and purification are difficult due to low product concentration and complex components of cell culture liquor.

In summary, the preparation of carnosine (L-carnosine) by chemical synthesis has the problems of long preparation route, large amount of organic solvent, low final yield and poor product quality; the method for preparing carnosine (L-carnosine) by using an enzyme method has the problems of incomplete conversion, low overall yield, difficult separation and purification and the like in the industrial amplification process. Therefore, the research and transformation of L-amino acid connection to prepare the carnosine 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 carnosine products at present.

Disclosure of Invention

In view of the above, the beta-alanine ligase mutant and the application thereof provided by the invention are superior to chemical synthesis preparation process, and all the advantages in the aspects of production cost, energy consumption, product quality and green index are shown.

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:

mutating the 13 th amino acid residue into lysine; and/or

Mutation of amino acid residue 14 to threonine; and/or

Amino acid residue 87 is mutated to serine; and/or

The 291 amino acid residue is mutated to glutamine; 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 91 to methionine; and/or

Mutation of amino acid residue 239 to asparagine; and/or

Amino acid residue 287 is mutated to leucine.

In some embodiments of the invention, the amino acid sequence of the above-mentioned beta-alanine ligase mutant is shown in SEQ ID NO. 2.

In some embodiments of the invention, the above-described β -alanine ligase mutant has:

(1) An amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in SEQ ID NO. 2, and the functions are the same or similar; or (b)

(2) An amino acid sequence having at least 70% homology with the amino acid sequence as shown in (1);

the plurality is 2 to 60.

The invention also provides a nucleic acid molecule for encoding the beta-alanine ligase mutant, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 4.

In some embodiments of the invention, the nucleic acid molecules described above have:

(3) A nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in SEQ ID NO. 4 and has the same or similar functions; or (b)

(4) A nucleotide sequence having at least 80% homology with the nucleotide sequence as shown in (3);

the plurality is 2 to 200.

The invention also provides an expression vector comprising the nucleic acid molecule.

The invention also provides a host cell comprising the above nucleic acid molecule or the above expression vector.

The invention also provides compositions that are adenosine triphosphates and the above-described beta-alanine ligase mutants.

In some embodiments of the invention, the above-described compositions are adenosine triphosphate, polyphosphate kinase, metaphosphoric acid, and the above-described beta-alanine ligase mutants.

In some embodiments of the invention, the amino acid sequence of the polyphosphate kinase in the above composition is shown in SEQ ID NO. 1.

In some embodiments of the invention, the polyphosphate kinase in the above composition has:

(5) An amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in SEQ ID NO.1, and the functions are the same or similar; or (b)

(6) An amino acid sequence having at least 70% homology with the amino acid sequence as set forth in (5) or (6);

the plurality is 2 to 80.

In some embodiments of the invention, the use of the above-described β -alanine ligase mutants in the synthesis of carnosine.

The invention also provides the application of any of the following in the synthesis of carnosine:

(I) The nucleic acid molecules described above;

(II) the expression vector;

(III) the host cell;

(IV) the above composition.

The invention also provides a synthesis method of the carnosine, which is obtained by mixing the beta-alanine ligase mutant with beta-alanine and L-histidine for reaction.

In some embodiments of the present invention, the above-described synthetic method provides for the above-described composition to be mixed with β -alanine, L-histidine, adenosine triphosphate, polyphosphate kinase, and metaphosphoric acid to obtain the carnosine.

In some embodiments of the present invention, the above-mentioned method for synthesizing carnosine further comprises:

(a) Expressing the nucleic acid molecules, and mixing the obtained protein product with amino acid to obtain carnosine; or (b)

(b) Expressing the expression vector, and mixing the obtained protein product with amino acid to obtain carnosine; or (b)

(c) Culturing the host cell, and mixing the obtained protein product with amino acid to obtain carnosine;

the amino acids include beta-alanine and/or L-histidine.

The mutant has 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 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 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 the results of HPLC liquid phase detection of Ligase (Ligase AH 1) catalyzed carnosine synthesis reaction for 4 hours;

FIG. 2 shows a mass spectrum of the reaction product of example 1;

FIG. 3 shows the HPLC liquid phase detection result of the wild-type ligase (B0 BTG 0) synthesized carnosine;

FIG. 4 shows HPLC liquid phase detection results of the preparation of carnosine by the ligase mutant (B0 BTG 0-101).

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 invention discloses a novel and simple beta-alanine ligase scheme for preparing carnosine at low cost and high efficiency.

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 L-histidine is screened from 6000 mutant libraries, and then, the actual catalytic process optimization is combined, so that the amplified production of carnosine is finally successfully realized.

The amino acid ligase method of the invention has the following preparation route:

this route uses inexpensive amino acids without protecting groups as starting materials for high yields of conversion to carnosine 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 carnosine in one step in buffer solution by utilizing 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 AH 1): 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 of Ligase AH 1: H13K, F14T, N87S, L91M, P239N, P287L, T291Q, 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 AH1	PPK
			Unit vitality	1200～1800U/mg	950～1400U/mg
Thermal stability (30 ℃ C.)	T _1/2 ＝50min	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 carnosine (L-carnosine) Using Ligase (Ligase AH 1)

To 1L 100mM tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) solution at pH 8.0 was added 23.2 g L-histidine (150 mM), 14.7 g beta-alanine (165 mM), 87.5 g adenosine triphosphate monosodium salt (ATP, 165 mM), and after adjusting the pH of the reaction system to 8.0 by NaOH aqueous solution, the reaction was started by adding Ligase Ligase AH1 2000U, and after slightly stirring at 30℃and maintaining the pH of the reaction system between 7.0 and 9.0, and after 4 hours the reaction was detected to be substantially complete by HPLC. The HPLC liquid phase detection reaction is shown in FIG. 1. 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, 2:1, v: v) to obtain 31.8 g of white carnosine solids (yield 94%). The product was confirmed to be carnosine by sample-feeding mass spectrometry. The mass spectrum results are shown in FIG. 2.

Example 2: preparation of Carnosine (L-Carnosine) using Ligase (Ligase AH 1) and ATP regenerating System

23.2 g L-histidine (150 mM), 14.7 g L-alanine (165 mM), 2.7 g adenosine monophosphate monosodium salt (ATP, 5 mM), 33.6 g sodium metaphosphate (55 mM) were also added to 1L 100mM tris (hydroxymethyl) aminomethane HCl (Tris.HCl) solution at pH 7.5, after adjusting the pH of the solution to 7.5, 3000U of polyphosphate kinase PPK enzyme and 2000U of Ligase Ligase AH were added, the reaction system was stirred slightly at 30℃and maintained at pH between 7.0 and 8.5, and after 3 hours the histidine 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 the 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 the crude product by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 2:1, v: v) to obtain 32.2 g of white carnosine (yield 95%).

Comparative example

Control group 1-preparation of carnosine (L-carnosine) Using wild-type ligase (B0 BTG 0)

23.2 g L-histidine (150 mM), 14.7 g beta-alanine (165 mM), 87.5 g adenosine triphosphate monosodium salt (ATP, 165 mM) was added to 1L 100mM Tris-aminomethane hydrochloride (Tris-HCl) solution at pH 8.0, then after adjusting the pH of the reaction system to 8.0 by NaOH aqueous solution, the reaction was started by adding wild-type ligase B0BTG0 2000U, slightly stirring at 30℃and maintaining the pH of the reaction system between 7.0 and 9.0, after 2 hours the reaction was stopped, a lot of histidine starting material remained as detected by HPLC, about 37% conversion, and the HPLC liquid phase results are shown in FIG. 3.

(II) control group 2-preparation of carnosine (L-carnosine) Using the modified ligase (B0 BTG 0-101)

23.2 g L-histidine (150 mM), 14.7 g beta-alanine (165 mM), 87.5 g adenosine triphosphate monosodium salt (ATP, 165 mM) was added to 1L 100mM Tris-aminomethane HCl (Tris.HCl) solution at pH 8.0, then after adjusting the pH of the reaction system to 8.0 by NaOH aqueous solution, the reaction was started by adding wild-type ligase B0BTG0-101 2000U, slightly stirring at 30℃and maintaining the pH of the reaction system between 7.0 and 9.0, after 2 hours the reaction was stopped, a lot of histidine starting material was detected to remain by HPLC, about 57% conversion was found, and the HPLC liquid phase results are shown in FIG. 4.

Table 4: comparison summary of wild ligase B0BTG0, mutant B0BTG0-101 and mutant enzymes of the patent

The conversion rate of the mutant 101 is improved by 20% compared with that of the wild type enzyme. The conversion rate of mutant Ligase AH1 is improved by 57%.

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

A mutant of an amino acid ligase having a uniprot ID of B0BTG0, characterized in that the mutation site comprises:

mutating the 13 th amino acid residue into lysine; and/or

Mutation of amino acid residue 14 to threonine; and/or

Amino acid residue 87 is mutated to serine; and/or

The 291 amino acid residue is mutated to glutamine; and/or

Amino acid residue 317 is mutated to methionine.
2. The mutant of claim 1, wherein the mutation site further comprises:

mutation of amino acid residue 91 to methionine; and/or

Mutation of amino acid residue 239 to asparagine; and/or

Amino acid residue 287 is mutated to leucine.
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. 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. An expression vector comprising the nucleic acid molecule of claim 4, and an acceptable genetic element.
6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.
7. A composition comprising a mutant according to any one of claims 1 to 3 and adenosine triphosphate.
8. The composition of claim 7, further comprising a polyphosphate kinase and a polyphosphate;

the polyphosphoric acid includes metaphosphoric acid.
9. Use in carnosine synthesis 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. A method for producing carnosine, comprising:

(a) Mixing a raw material with the mutant according to any one of claims 1 to 3 to obtain the carnosine; or (b)

(b) Expressing the nucleic acid molecule of claim 4, mixing the obtained protein product with a starting material to obtain the carnosine; or (b)

(c) Expressing the expression vector according to claim 5, mixing the obtained protein product with a raw material to obtain the carnosine; or (b)

(d) Culturing the host cell of claim 6, and mixing the obtained protein product with a raw material to obtain the carnosine; or (b)

(e) Mixing the raw materials with the composition according to claim 7 or 8 to obtain the carnosine;

the starting materials include beta-alanine and L-histidine.