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:
mutating the 13 th amino acid residue into arginine; and/or
Mutation of amino acid residue 87 to glycine; and/or
Mutation of amino acid residue 91 to isoleucine; and/or
Mutation of amino acid residue 239 to threonine; 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 170 to threonine; and/or
Mutation of amino acid residue 192 to asparagine; and/or
Amino acid residue 287 is mutated to leucine.
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 application of any of the following in the synthesis of beta-propiolateral:
(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 beta-propiolate dipeptide, which comprises the following steps:
(a) Mixing the raw materials with the mutant to obtain the beta-propiolateral dipeptide; or (b)
(b) Expressing the nucleic acid molecules, and mixing the obtained protein product with raw materials to obtain the beta-propiolateral dipeptide; or (b)
(c) Expressing the expression vector, and mixing the obtained protein product with raw materials to obtain the beta-propiolateral dipeptide; or (b)
(d) Culturing the host cell, mixing the obtained protein product with raw materials to obtain the beta-propiolateral dipeptide; or (b)
(e) Mixing the raw materials with the composition to obtain the beta-propiolate dipeptide;
the raw materials comprise beta-alanine and L-proline.
The beta-alanine 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 with other amino acids, the beta-propiolate dipeptide with high catalytic activity is finally obtained by mutating the catalytic activity pocket of the L-amino acid ligase and amino acid residues at other relevant positions. The method has outstanding advantages in terms of production cost, energy consumption, product quality and green index. Thus the large-scale production of this method would be a preferred option for the production of beta-propiolate dipeptide.
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 beta-propiolate dipeptide 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 L-proline is screened from 6000 mutant libraries, and the actual catalytic process optimization is combined, so that the amplified production of beta-propiolate dipeptide 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 AP 3): 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 AP 3: H13R, N87G, L91I, V170T, E192N, P239T, P287L, Y317M.
Polyphosphate Kinase (PPK): is derived from Thermus rhodochrous (Meiothermus ruber) (Uniprot ID: 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 AP3
|
PPK
|
Unit vitality
|
1100~1500U/mg
|
950~1400U/mg
|
Thermal stability (30 ℃ C.)
|
T 1/2 =60min
|
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 beta-Propriodipeptide (beta-ala-Pro) Using Ligase (Ligase AP 3)
23 g L-proline (200 mM), 17.8 g beta-alanine (220 mM), 116 g adenosine monophosphate monosodium salt (ATP, 220 mM) were added to 1L 100mM Tris-HCl (pH 8.5) solution, and after the reaction system was brought to pH 8.5 by NaOH aqueous solution, the reaction was started by adding the enzyme Ligase AP3 3000U, and after stirring slightly at 30℃and maintaining the reaction system pH between 7.5 and 9.0, the basic reaction of the proline starting material was detected to be complete by HPLC after 4 hours, see 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 onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphoric acid impurities, and finally desalting and concentrating a crude product by using a reverse osmosis membrane and crystallizing (ethanol: water, 1:1, v: v) to obtain 33 g of beta-propiolate dipeptide (yield 89%). The structure was confirmed by mass spectrometry and HPLC, and the results are shown in fig. 2 and 3, respectively.
Example 2: beta-Propriodipeptide (beta-ala-Pro) was prepared using Ligase (Ligase AP 3) and an ATP regenerating system.
To 1L 100mM tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) solution at pH 8.5 was added 23 g L-proline (200 mM), 17.8 g beta-alanine (220 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 8.5, 3000U of polyphosphate kinase PPK enzyme was added, the reaction was started by adding Ligase Ligase AP3 3000U, and the pH of the reaction system was maintained between 7.5 and 9.0 with gentle stirring at 30℃and after 3 hours the proline starting material was detected to be substantially completely reacted 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 onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphoric acid impurities, and finally desalting and concentrating a crude product by using a reverse osmosis membrane and crystallizing (ethanol: water, 1:1, v: v) to obtain 35 g of beta-propiolate dipeptide (yield 95%).
Comparative example: preparation of beta-Propriedipeptide (beta-ala-Pro) using wild ligase (B0 BTG 0), wild ligase mutant (B0 BTG 0-156)
Control group 1:
23 g L-proline (200 mM), 17.8 g beta-alanine (220 mM), 116 g adenosine monophosphate monosodium salt (ATP, 220 mM) were added to 1L 100mM Tris-HCl (pH 8.5) solution, and after the reaction system was brought to pH 8.5 by NaOH aqueous solution, the reaction was started by adding wild type ligase B0BTG0 3000U, stirring slightly at 30℃and maintaining the reaction system pH between 7.5 and 9.0, after 4 hours more than 50% of the proline starting material was still detected by HPLC, the wild type ligase conversion was very low.
Control group 2:
23 g L-proline (200 mM), 17.8 g beta-alanine (220 mM), 116 g adenosine monophosphate monosodium salt (ATP, 220 mM) were added to 1L 100mM Tris-HCl (pH 8.5), and after the reaction system had been brought to pH 8.5 by NaOH aqueous solution, the reaction was started by adding wild type ligase B0BTG0-156 3000U, stirring slightly at 30℃and maintaining the reaction system pH between 7.5 and 9.0, after 4 hours the proline starting material was detected to remain 40% by HPLC, see FIG. 4.
Table 4: comparison summary of mutant B0BTG0-156 and mutant of the invention
The mutant Ligase AP3 is compared with the mutant B0BTG0-156, and the conversion rate is improved by 29%.
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.