Threonine aldolase, its preparation method and uses

Abstract

This invention relates to threonine aldolase, its preparation method, and its uses. Specifically, this invention relates to the functional identification and in vitro directed evolution of a threonine aldolase gene-encoded protein. By amplifying the threonine aldolase gene from *Neptunomonas marine*, and through rational design and directed evolutionary modification, a threonine enzyme mutant N18S / Q39R / Y319L with significantly enhanced diastereoselectivity for L-threo-methanesulfonylphenylserine was obtained. This mutant can be used for the in vitro enzymatic synthesis of pharmaceutical intermediates such as L-threo-methanesulfonylphenylserine. This invention confirms the function of the gene-encoded protein and its potential in the synthesis of pharmaceutical intermediates such as L-threo-methanesulfonylphenylserine.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and biocatalysis, specifically to a threonine aldolase, its preparation method and uses, and particularly to the application of a threonine aldolase and its mutants in the synthesis of pharmaceutical intermediates such as L-threo-methanesulfonylphenylserine (L-threo-MPTS). Background Technology

[0002] β-hydroxy-α-amino acids are an important class of chiral compounds used in chemical, pharmaceutical, and other fields. In recent years, biocatalysis has emerged as a new tool in chemical industrial production, particularly for the chiral building blocks required in the production of fine chemicals and pharmaceuticals. Biocatalysis, with its mild reaction conditions, high stereoselectivity, and lack of the need for any protecting groups, has become a more attractive approach combining classical chemistry and chemicatalysis.

[0003] Threonine aldolases have attracted widespread attention for their ability to catalyze the formation or cleavage of carbon-carbon bonds, producing stereochemically pure chiral products. Threonine aldolases are ubiquitous in nature, found in vertebrates, plants, bacteria, yeast, and fungi. They are a type I folding enzyme dependent on pyridoxal 5-phosphate (PLP), catalyzing the cleavage of L-threonine or L-allo-threonine into glycine and acetaldehyde in the glycine biosynthesis pathway. They can also catalyze the condensation of aldehydes and glycine via the reverse reaction to produce amino acid derivatives with two chiral centers.

[0004] To date, reported threonine aldolases suffer from poor stereoselectivity, low activity, and narrow substrate range in aldol condensation reactions, limiting their application in the synthesis of β-hydroxy-α-amino acids. Therefore, discovering and modifying new aldolases with good stereoselectivity, high activity, and broad substrate range is crucial research, and could further be applied to the efficient and green synthesis of various chiral amino acid drugs and their precursors.

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop a novel threonine aldolase (L-TAnem) for in vitro enzymatic conversion and synthesis of a single diastereomeric product L-thero-methanesulfonylphenylserine and L-threo-phenylserine derivatives. Summary of the Invention

[0006] The purpose of this invention is to provide a threonine aldolase and its mutant.

[0007] Another objective of this invention is to provide the application of threonine aldolase and its mutants in the synthesis of pharmaceutical intermediates such as L-threo-methanesulfonylphenylserine.

[0008] In a first aspect of the invention, a threonine aldolase is provided, said threonine aldolase being selected from the group consisting of:

[0009] (a) A polypeptide having the amino acid sequence shown in SEQ ID NO:2;

[0010] (b) A derivative polypeptide having threonine aldolase activity formed by substituting, deleting or adding one or more amino acid residues of a polypeptide with the amino acid sequence shown in SEQ ID NO:2.

[0011] (c) A derivative polypeptide having ≥85% (preferably ≥95%) homology with the amino acid sequence shown in SEQ ID NO:2 and possessing threonine aldolase activity.

[0012] In another preferred embodiment, the amino acid sequence of the threonine aldolase is shown in SEQ ID NO:2.

[0013] In another preferred embodiment, the amino acid sequence of the threonine aldolase is mutated at one or more sites corresponding to the amino acid sequence shown in SEQ ID NO:2 at amino acid residues selected from the group consisting of: position 18, position 39, and position 319.

[0014] In another preferred embodiment, the threonine aldolase has the following mutation: N18S / Q39R / Y319L.

[0015] In a second aspect of the invention, a separated polynucleotide is provided, said polynucleotide encoding the threonine aldolase described in the first aspect of the invention.

[0016] In another preferred embodiment, the isolated polynucleotide is a sequence selected from the group consisting of:

[0017] (a) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO:2;

[0018] (b) The nucleotide sequence shown in SEQ ID NO:1;

[0019] (c) A nucleotide sequence that is ≥95% homology (preferably ≥98%) to the sequence shown in SEQ ID NO:1;

[0020] (d) A nucleotide sequence formed by truncating or adding 1-60 (preferably 1-30, more preferably 1-10) nucleotides to the 5' end and / or 3' end of the nucleotide sequence shown in SEQ ID NO:1;

[0021] (e) A nucleotide sequence that is complementary (preferably perfectly complementary) to any of the nucleotide sequences described in (a)-(d).

[0022] In another preferred embodiment, the sequence of the nucleotide is shown in SEQ ID NO:1.

[0023] In another preferred embodiment, the polynucleotide sequence shown in SEQ ID NO:1 encodes a threonine aldolase with an amino acid sequence shown in SEQ ID NO:2.

[0024] In a third aspect of the invention, a carrier is provided, wherein the carrier contains the polynucleotide described in the second aspect of the invention.

[0025] In another preferred embodiment, the vector includes a recombinant vector, an expression vector, and an integration vector.

[0026] In a fourth aspect of the invention, a genetically engineered host cell is provided, the host cell containing the vector described in the third aspect of the invention, or having an exogenous polynucleotide described in the second aspect of the invention integrated into its genome.

[0027] In another preferred embodiment, the host cells include prokaryotic cells and eukaryotic cells.

[0028] In another preferred embodiment, the host cell is Escherichia coli.

[0029] In a fifth aspect of the invention, a method for preparing the threonine aldolase described in the first aspect of the invention is provided, the method comprising:

[0030] (a) Under suitable expression conditions, the host cells described in the fourth aspect of the present invention are cultured to express the threonine aldolase described in the first aspect of the present invention; and

[0031] (b) Isolate the expression product to obtain the threonine aldolase.

[0032] In a sixth aspect of the invention, the use of the threonine aldolase described in the first aspect of the invention is provided for catalyzing aldol condensation reactions or for preparing catalytic reagents for aldol condensation reactions.

[0033] In another preferred embodiment, the aldol condensation reaction is as follows:

[0034]

[0035] in,

[0036] R is one or more groups selected from the group consisting of halogen, nitro, methyl, and methanesulfonyl, located at the ortho, meta, or para position.

[0037] PLP stands for pyridoxal 5-phosphate.

[0038] In a seventh aspect of the invention, a catalytic method for the in vitro preparation of a compound of formula (I) is provided, comprising the steps of:

[0039] In the presence of threonine aldolase, glycine and benzaldehyde derivatives are used as substrates for an aldol condensation reaction to form the amino acid product shown in Formula I:

[0040]

[0041]

[0042] in,

[0043] R is one or more groups selected from the group consisting of the following groups: halogen, nitro, methyl, and methanesulfonyl, located in the ortho, meta, or para position.

[0044] PLP stands for pyridoxal 5-phosphate.

[0045] In another preferred embodiment, the molar ratio of the benzaldehyde derivative to glycine is 0.5–2:1–100.

[0046] In another preferred embodiment, the reaction is carried out in a hydrophilic organic solvent.

[0047] In another preferred embodiment, the hydrophilic organic solvent is N,N dimethylamide (DMF).

[0048] In another preferred embodiment, the method further includes providing an additive to the reaction system for regulating enzyme activity.

[0049] In another preferred embodiment, the additive used to regulate enzyme activity is an additive that increases or inhibits enzyme activity.

[0050] In another preferred embodiment, the additive for regulating enzyme activity is selected from the group consisting of: Mn 2+ K + Ba 2+ Zn 2+ Mg 2+ NH4 + Ca 2+ Fe 2+ or Fe 3+ , or a combination thereof.

[0051] In another preferred embodiment, the concentration of pyridoxal 5-phosphate (PLP) in the method is 1 μM to 100 μM, preferably 20 μM to 50 μM.

[0052] In another preferred embodiment, the pH conditions of the method are: pH 5.0 to 10.0, more preferably pH 7.0 to 9.0, and even more preferably pH 8.0.

[0053] In another preferred embodiment, the temperature conditions of the method are: 20℃-50℃, more preferably 25℃~30℃, and even more preferably 30℃.

[0054] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0055] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0056] Figure 1 The image shows the SDS-PAGE electrophoresis results of purified threonine aldolase L-TAnem.

[0057] Figure 2 The graph shows the optimal pH required for the L-TAnem reaction of threonine aldolase.

[0058] Figure 3 The graph shows the optimal temperature required for the L-TAnem reaction of threonine aldolase.

[0059] Figure 4 The graph shows the optimal concentration of pyridoxal 5-phosphate (PLP) required for the L-TAnem reaction of threonine aldolase.

[0060] Figure 5 The graph shows the effect of metal ions on the activity of threonine aldolase L-TAnem.

[0061] Figure 6 The results of the temperature tolerance of threonine aldolase L-TAnem are shown in the figure.

[0062] Figure 7 The results for the natural substrate of threonine aldolase L-TAnem are shown.

[0063] Figure 8 The diagram shows the relative activity and diastereoselectivity of L-threo-methanesulfonylphenylserine synthesized by saturated mutants at S16, N18, Q39 and Y319 sites in the threonine aldolase L-TAnem.

[0064] Figure 9 The diagram shows the relative activity and diastereoselectivity of L-threo-methanesulfonylphenylserine synthesized by multi-point combinatorial mutants of threonine aldolase L-TAnem.

[0065] Figure 10The image shows the amplified results of the synthesis of L-threo-methanesulfonylphenylserine by threonine aldolase L-TAnem and its mutant N18S / Q39R / Y319L.

[0066] Figure 11 The results show the activity and diastereoselectivity of threonine aldolase L-TAnem and its mutant N18S / Q39R / Y319L in synthesizing different β-hydroxy-α amino acids using non-natural benzaldehyde derivatives and glycine as substrates.

[0067] Figure 12 The HPLC and MS spectra of L-threo-methanesulfonylphenylserine synthesized by the L-TAnem mutant threonine aldolase are shown. Detailed Implementation

[0068] Through extensive and in-depth research, the inventors have, for the first time, provided a threonine aldolase, its preparation method, and its uses. Specifically, by amplifying the threonine aldolase gene from *Neptunomonas marine*, and through rational design and directed evolutionary modification, a threonine enzyme mutant with significantly enhanced diastereoselectivity for L-threo-methanesulfonylphenylserine was obtained. This mutant can be used for the in vitro enzymatic synthesis of pharmaceutical intermediates such as L-threo-methanesulfonylphenylserine. This invention is based on this foundation.

[0069] the term

[0070] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0071] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0072] Threonine aldolase (L-TAnem)

[0073] Threonine aldolase L-TAnem is a type I folding enzyme dependent on pyridoxal 5-phosphate, which can further catalyze the condensation of aldehydes and glycine to produce β-hydroxy-α amino acid derivatives with two chiral centers.

[0074] In this invention, using the reported sequences and structures of threonine aldolases, a number of candidate enzyme genes were screened through non-redundant searches in databases such as NCBI, based on principles such as protein structural similarity, conserved site analysis, and diverse host origins. The screened genes were functionally expressed in an *E. coli* expression system, and purified to obtain pure enzymes. The preferred threonine aldolase is L-TAnem, derived from *Neptunomonas marine*, which has a broad substrate spectrum and can catalyze the formation of corresponding β-hydroxy-α-amino acid compounds from a series of aldehydes and α-amino acid substrates.

[0075] The wild-type L-TAnem nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0076] SEQ ID NO:1:

[0077]

[0078] SEQ ID NO:2:

[0079] MASNDSCIEDTVSFTSDNIAAAAPEIVQAMAQACQGNAQPYGGDALTQNVEAQLKAIFECDLQLFLVPTGSAANAISLAALTPPWGAILCHQESHINNDECGAPEFFTAGAKLIAVAGTHGKLDPQALTQAARNKRGDVHSVEPTTVSITQATEVGSIYALDELNEIGQICRNEGLKL HMDGARFANALSALGCTPAEMTWKAGVDVLSFGATKNGSLCAEAIILFDKSYAQEIAFRRKRGGHLLSKMRFLSAQMHAYLADDLWLTNARHANLMAARLAAGLSALSRVSLIAPTESNIIFCRMPTKMIAALQQQGFQFYHDRWGDGIVRLVTSFATTQAQVDTFIAAAAQLNQNTD

[0080] As used herein, the terms “active polypeptide,” “polypeptide of the present invention,” “enzyme of the present invention,” “threonine aldolase,” “threonine aldolase of the present invention,” or “L-TAnem” are used interchangeably and all refer to the threonine aldolase described in the first aspect of the present invention.

[0081] As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify the polypeptide using standard protein purification techniques. A substantially pure polypeptide will produce a single master band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using its amino acid sequence.

[0082] In this invention, mutations were made at specific sites in the wild-type threonine aldolase, as shown in SEQ ID NO:1, to obtain a corresponding mutant with significantly enhanced activity. As used herein, the terms "mutant protein" and "mutant" are used interchangeably and refer to the threonine aldolase mutant.

[0083] As used herein, when describing mutations, the term "N18S / Q39R / Y319L" is used as an example, referring to a mutation based on the sequence shown in SEQ ID NO:1 (wild type), where N at position 18 is mutated to S, Q at position 39 is mutated to R, and Y at position 319 is mutated to L. Other mutations are described in a similar manner.

[0084] The active polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the polypeptides of the present invention can be glycosylated or non-glycosylated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0085] The present invention also includes fragments, derivatives, and analogs of the said polypeptide. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the said polypeptide.

[0086] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify the polypeptide or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0087] The active polypeptide of the present invention has glycosyltransferase activity and is capable of catalyzing the following reaction:

[0088]

[0089] The definitions of R and PLP are as described above.

[0090] The preferred sequence of the polypeptide is the polypeptide shown in SEQ ID NO:2. This term also includes variations of the sequence of SEQ ID NO:2 having the same function as the polypeptide shown. These variations include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of human EGFRvA protein. The present invention also provides analogs of the polypeptide. These analogs may differ from the natural human EGFRvA polypeptide in amino acid sequence differences, differences in modifications that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0091] A preferred class of active derivatives refers to polypeptides formed by replacing up to 5, more preferably up to 3, more preferably up to 2, and most preferably 1 amino acid with an amino acid of similar or analogous properties, compared to the amino acid sequence shown in SEQ ID NO.:2. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0092] Table A

[0093]

[0094]

[0095] Encoding nucleic acids and their combinations

[0096] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO:1. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding the protein having SEQ ID NO:2 but differing from the coding region sequence shown in SEQ ID NO:1.

[0097] The polynucleotide encoding the mature polypeptide of SEQ ID NO:2 includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0098] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0099] This invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0100] The polypeptides and polynucleotides in this invention are preferably provided in isolated form and are more preferably purified to homogenization.

[0101] The full-length L-TAnem nucleotide sequence or fragments thereof of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0102] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0103] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0104] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0105] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0106] carrier

[0107] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or L-TAnem protein coding sequences, and methods for generating the polypeptides of the present invention via recombinant technology.

[0108] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce recombinant threonine aldolase L-TAnem. Generally, the following steps are involved:

[0109] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding threonine aldolase of the present invention, or using a recombinant expression vector containing the polynucleotide.

[0110] (2) Host cells cultured in a suitable culture medium;

[0111] (3) Isolate and purify proteins from culture media or cells.

[0112] In this invention, the L-TAnem polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0113] Methods well known to those skilled in the art can be used to construct expression vectors containing L-TAnem-coding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0114] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0115] host cells

[0116] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0117] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; plant cells; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0118] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0119] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0120] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0121] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0122] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0123] application

[0124] The uses of the threonine aldolase and its mutants involved in this invention include (but are not limited to): specifically and efficiently catalyzing the conversion of the substrate 4-methanesulfonylbenzaldehyde or benzaldehyde derivatives with glycine to synthesize L-threo-methanesulfonylphenylserine and its L-threo-phenylserine derivatives.

[0125] The present invention provides an industrial catalytic method comprising: converting a compound of formula (I) into a compound of formula (II) using the threonine aldolase of the present invention in the presence of a threonine aldolase, wherein the enzyme is preferably an active polypeptide having the amino acid sequence shown in SEQ ID NO:2 and an amino-selective sequence with single or multiple mutations therein.

[0126] In the method, enzyme activity additives (additives that increase or inhibit enzyme activity) may also be added. The enzyme activity additives may be selected from the group consisting of Mn. 2+ K + Ba 2+ Zn2+ Mg 2+ NH4 + Ca 2+ Fe 2+ or Fe 3+ , or a combination thereof.

[0127] The pH conditions for the method are: pH 5.0 to 10.0, preferably pH 7.0 to 9.0, and more preferably pH 8.0.

[0128] The temperature conditions for the method are: 20℃~50℃, preferably 25℃~30℃, and more preferably 30℃.

[0129] The PLP concentration conditions for the method are: 1μM to 100μM, preferably 20μM to 50μM.

[0130] The present invention also provides a composition containing an effective amount of the active polypeptide or threonine aldolase L-TAnem of the present invention, and a food- or industrially acceptable carrier or excipient. Such carriers include (but are not limited to): water, buffer solutions, glucose, glycerol, ethanol, and combinations thereof.

[0131] The composition may also contain substances that regulate the activity of the threonine aldolase of the present invention. Any substance that enhances enzyme activity is acceptable.

[0132] After obtaining the threonine aldolase of the present invention, those skilled in the art can conveniently use the enzyme to perform in vitro enzymatic synthesis, especially for the conversion of substrates 4-methanesulfonylbenzaldehyde and glycine.

[0133] As a preferred embodiment of the present invention, a method for forming L-threo-methanesulfonylphenylserine is also provided, the method comprising: treating a substrate to be converted with the threonine aldolase described in this invention, the substrate comprising 4-methanesulfonylbenzaldehyde and glycine. Preferably, the substrate to be converted is treated with the threonine aldolase at a pH of 5.0-10. Preferably, the substrate to be converted is treated with the threonine aldolase at a temperature of 25-50°C.

[0134] The main advantages of this invention are:

[0135] 1. The threonine aldolase described in this invention can be used to synthesize L-threo-methanesulfonylphenylserine via in vitro enzymatic method, achieving a conversion rate of up to 80% for the substrates 4-methanesulfonylbenzaldehyde and glycine. Under the same conditions, the mutant exhibits 1.4-1.5 times the activity of the wild type in catalyzing the conversion of 4-methanesulfonylbenzaldehyde and glycine to L-threo-methanesulfonylphenylserine, with the de value increased to 99%.

[0136] 2. Using the threonine aldolase mutant described in this invention, a single enantiopure L-threo-methanesulfonylphenylserine was successfully synthesized in vitro via enzymatic method.

[0137] 3. The threonine aldolase and its mutants obtained in this invention can be used for the in vitro enzymatic synthesis of L-threo-methanesulfonylphenylserine and L-threo-phenylserine derivatives. This is a new production method that is different from existing production technologies. The production process is simplified, has low consumption, and is green and environmentally friendly.

[0138] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0139] General description of the source of the biological materials described in this invention:

[0140] 1. Primer synthesis: All primers used in this invention were synthesized and prepared by Qingke Biotechnology Co., Ltd.

[0141] 2. The Primer STAR Max DNA polymerase used in the experiment was purchased from TakaRa; the DNA gel extraction kit and plasmid miniprep kit were purchased from Axygen.

[0142] Example 1: Screening of threonine aldolase

[0143] Using the reported sequences and structures of threonine aldolases, a non-redundant search was conducted in databases such as NCBI. Based on principles such as protein structural similarity, conserved site analysis, and diverse host origins, the threonine aldolase gene L-TAnem from Neptunomonas marine was screened. After codon optimization, the gene was synthesized in its entirety and cloned into the pET22b(+) plasmid using NdeⅠ and XhoⅠ restriction sites.

[0144] Example 2: Expression and purification of threonine aldolase

[0145] The recombinant expression plasmids containing the selected genes were heat-shock transformed into *E. coli* BL21(DE3) competent cells for gene expression and protein purification. When the recombinant bacterial concentration reached an OD600 of 0.6-0.9, IPTG was added to a final concentration of 0.5 mM, and the cells were induced and cultured overnight at 25°C and 220 rpm. The cells were collected by centrifugation and resuspended in 50 mM HEPES buffer (pH 8.0, 500 mM NaCl, 50 mM imidazole). 250 mL of cultured cells were resuspended in 40 mL of buffer and disrupted using a high-pressure cell disruptor (4–6 °C, 700 Pa). The cell disruption buffer was then centrifuged at 12,000 rpm for 30 min (4 °C). The supernatant was collected and centrifuged again at 12,000 rpm for 30 min (4 °C). The supernatant was collected and the protein was purified using a Ni-NTA column affinity purification method. Impurities were eluted with 50 mM HEPES buffer (pH 8.0, 500 mM NaCl, 50 mM imidazole). Finally, the target protein was eluted with 50 mM HEPES buffer (pH 8.0, 500 mM NaCl, 250 mM imidazole). The eluted protein was concentrated and desalted to obtain the purified protein. The purified protein was stored in 100 mM Tris-HCl buffer (pH 8.0), and the purified protein was detected by electrophoresis using 12% SDS-PAGE. The protein concentration was determined using the Bradford Protein Assay Kit (Shanghai Sangon Biotech).

[0146] The results are as follows Figure 1 As shown in the figure. The results indicate that a clear band was obtained at 41.9 kDa, suggesting that the target protein L-TAnem has been purified.

[0147] Example 3: Determination of the enzymatic properties of threonine aldolase L-TAnem

[0148] The activity of threonine aldolase L-TAnem was determined using coupled alcohol dehydrogenase (ADH). The specific steps are as follows:

[0149] 1. Determination of optimal pH, optimal temperature, optimal PLP concentration, and different metal ions for threonine aldolase L-TAnem:

[0150] The enzyme activity of L-TAnem was investigated at different pH values ​​(5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0). The reaction system consisted of 10 μg / mL L-TAnem pure enzyme, 10 U ADH (Sigma), and 0.2 mM NAD. + 50 mM threonine. The final reaction volume was brought to 1000 μL using 50 mM HEPES buffer. The OD was monitored at 25 °C. 340The activity was calculated by observing the changes in pH. The relative enzyme activity at different pH values ​​was also calculated.

[0151] The results are as follows Figure 2 As shown in the figure. The results indicate that L-TAnem exhibits high activity at pH 8.0.

[0152] The enzyme activity of L-TAnem was investigated at different temperatures (20℃, 25℃, 30℃, and 37℃). The reaction system consisted of 10 μg / mL L-TAnem pure enzyme, 10 U ADH (Sigma), and 0.2 mM NADH. + 50 mM threonine. The final reaction volume was brought to 1000 μL using 50 mM HEPES buffer. The OD was monitored at 25 °C. 340 The changes in enzyme activity were used to calculate the relative enzyme activity under different temperature reaction conditions.

[0153] The results are as follows Figure 3 As shown in the figure. The results indicate that L-TAnem exhibits high activity at 30℃.

[0154] The enzyme activity of L-TAnem was investigated at different PLP concentrations (1 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM). The reaction system consisted of 10 μg / mL L-TAnem pure enzyme, 10 U ADH (Sigma), and 0.2 mM NADH. + 50 mM threonine. The final reaction volume was brought to 1000 μL using 50 mM HEPES buffer. The OD was monitored at 25 °C. 340 The changes in enzyme activity were used to calculate the relative enzyme activity under different temperature reaction conditions.

[0155] The results are as follows Figure 4 As shown in the figure. The results indicate that L-TAnem exhibits high activity at a concentration of 40 μM.

[0156] At different metal ion concentrations (Mn) at 1 mM 2+ K + Ba 2+ Ni 2+ Zn 2+ Cu 2+ Mg 2+ NH4 + Ca 2+ Fe 2+ Fe 3+ The enzyme activity of L-TAnem was investigated using the following reaction system: 10 μg / mL L-TAnem pure enzyme, 10 U ADH (Sigma), and 0.2 mM NADPH. +50 mM threonine. The final reaction volume was brought to 1000 μL using 50 mM HEPES buffer. The OD was monitored at 25 °C. 340 The changes in enzyme activity were used to calculate the relative enzyme activity under different temperature reaction conditions.

[0157] The results are as follows Figure 5 As shown. The results indicate that Ni + and Cu 2+ It has a significant inhibitory effect on the activity of L-TAnem, while other metal ions have little effect on the activity of L-TAnem.

[0158] 2. Determination of the natural substrate profile of threonine aldolase L-TAnem:

[0159] The activities of L-TAnem on D-threonine and L-threonine were investigated separately. The reaction system consisted of 10 μg / mL L-TAnem pure enzyme, 10 U ADH (Sigma), and 0.2 mM NADH. + 50 mM threonine, 25 °C, and the final reaction volume was adjusted to 1000 μL using 50 mM HEPES (pH 8.0) buffer. OD was monitored. 340 The change in activity is used to calculate the activity.

[0160] The results are as follows Figure 7 As shown. The results indicate that L-TA nem It exhibits good activity towards L-threonine but no activity towards D-threonine.

[0161] 3. Threonine aldolase L-TA nem Thermal stability determination:

[0162] The enzyme activity of L-TAnem was investigated after incubation at 30℃, 40℃, 50℃, and 60℃ for different times (0 min, 5 min, 10 min, 15 min, 30 min, and 60 min). The reaction system consisted of 10 μg / mL L-TAnem pure enzyme, 5 U ADH (Sigma), and 0.2 mM NADH. + 50 mM threonine. Bring the final reaction volume to 1000 μL using 50 mM HEPES buffer.

[0163] The results are as follows Figure 6 As shown in the figure. The results indicate that L-TAnem exhibits good thermal stability, retaining approximately 85% of its activity after treatment at 50℃ for 30 min.

[0164] Example 4 Construction and screening of L-TAnem mutants of threonine aldolase

[0165] Based on the activity of threonine aldolase L-TAnem in catalyzing the synthesis of L-threo-methanesulfonylphenylserine, and through rational design and site selection based on the crystal structure and molecular docking analysis of L-TAnem, the enzyme was subjected to directed evolutionary modification to obtain a mutant that can catalyze the synthesis of a single diastereomer of L-threo-methanesulfonylphenylserine.

[0166] 4.1 Using the recombinant plasmid pET22b-L-TAnem as a template, and a pair of complementary oligonucleotides with a degenerate (NNK) mutation site as primers, full-plasmid PCR amplification was performed using Primestar high-fidelity enzyme to obtain the recombinant plasmid with the specific mutation site. The primer sequences are as follows:

[0167] The mutant corresponding to the 16th serine in SEQ NO:2 being replaced by 19 other amino acids:

[0168] S16-F:AGCTTCACCNNKGACAACATCGCGGCTGCG(SEQ ID NO:3)

[0169] S16-R:GATGTTGTCMNNGGTGAAGCTCACTGTGTC(SEQ ID NO:4)

[0170] The mutant corresponding to the one in SEQ NO:2 where asparagine at position 18 is replaced by 19 other amino acids:

[0171] N18-F:ACCTCCGACNNKATCGCGGCTGCGGCTCCG(SEQ ID NO:5)

[0172] N18-R:AGCCGCGATMNNGTCGGAGGTGAAGCTCAC(SEQ ID NO:6)

[0173] The mutant corresponding to the one in SEQ NO:2 where glutamine at position 39 is replaced by 19 other amino acids:

[0174] Q39-F:GGCAATGCGNKCCGTACGGCGGAGACGCG(SEQ ID NO:7)

[0175] Q39-R:GCCGTACGGMNNCGCATTGCCCTGACACGC(SEQ ID NO:8)

[0176] The mutant corresponding to the one in SEQ NO:2 where histidine at position 140 is replaced by 19 other amino acids:

[0177] H140-F:GGCGACGTTNNKAGCGTCGAGCCGACCACC(SEQ ID NO:9)

[0178] H140-R:CTCGACCGMNNAACGTCGCCGCGTTTATT(SEQ ID NO:10)

[0179] The mutant corresponding to the substitution of tyrosine at position 319 in SEQ NO:2 with 19 other amino acids:

[0180] Y319-F:TTTCAGTTTNNKCACGATCGTTGGGGCGAC(SEQ ID NO:11)

[0181] Y319-R:ACGATCGTGMNNAAACTGAAAACCCTGTTG(SEQ ID NO:12)

[0182] The amplification system consisted of: 1 ng of recombinant plasmid template, 2 μL each of primers (10 μM), 25 μL of PrimeSTAR Max DNA polymerase, and double-distilled water to a final volume of 50 μL.

[0183] The amplification conditions were: 94℃ pre-denaturation for 1 minute, 94℃ denaturation for 10 seconds, 56℃ annealing for 35 seconds, and 72℃ extension for 1 minute, for a total of 30 cycles.

[0184] After the reaction, the amplified products were detected by 1% agarose gel electrophoresis. The products were purified and recovered using a PCR product purification kit, and then digested with DpnI enzyme (NEB) at 37°C for 2 hours to degrade the initial template. The digested products were transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Positive clones were screened, and sequencing verification yielded a saturated mutant recombinant bacterium with the specified site of threonine aldolase L-TAnem.

[0185] The purified protein of the saturated mutant at the specified site of threonine aldolase L-TAnem was obtained according to the method in Example 2.

[0186] The reaction system for the mutant enzyme activity assay consisted of 20 μg / mL L-TAnem purified enzyme, 100 mM aldehyde, 1 M glycine, 50 μM PLP, and 10% (v / v) DMF. The final reaction volume was brought to 500 μL using HEPES (pH 8.0) buffer. The reaction was incubated at 25°C with shaking at 250 rpm for 0.5 hours. Then, 50 μL of the reaction solution was added to 950 μL of acetone to terminate the reaction. The supernatant was collected by centrifugation and analyzed by HPLC.

[0187] result

[0188] The results are as follows Figure 8 As shown. Approximately 100 clones were screened from the library, and 12 positive mutants were obtained, namely S16G, S16A, N18T, N18S, Q39A, Q39K, Q39L, Q39R, Y319L, Y319S, and Y319D.

[0189] For the S16 and N18 sites, the activity of all mutants was reduced. Figure 8 Regarding stereoselectivity, only S16A, S16G, N18S, and N18T showed increased de values ​​compared to the wild type, with de values ​​of 94.3%, 94.1%, 92.5%, and 91.1%, respectively. However, compared to L-TAnem-WT, their activities decreased by 54.7%, 74.9%, 24.4%, and 33.3%, respectively. Figure 8 ).

[0190] For the Q39 site, five mutants (Q39A, Q39R, Q39K, Q39H, and Q39L) showed increased de values ​​while retaining most of their activity. Among them, Q39R and Q39K showed increased activity compared to L-TAnem-WT by 3.5 U / mg and 8 U / mg, respectively, reaching 68.3 U / mg and 72.8 U / mg. Figure 8 ).

[0191] For the Y319 site, Y319D, Y319L, and Y319S all had a positive effect on the de value, reaching 94.0%, 93.7%, and 92.7%, respectively. In terms of activity, Y319D was only 27.5% of L-TAnem-WT. The activity of Y319S was 1.4 U / mg higher than that of L-TAnem-WT. Figure 8 ).

[0192] 4.2 To further improve the stereoselectivity of L-TAnem, multi-site combination mutagenesis was performed. Using the 12 positive mutants obtained earlier as templates, primers were designed, and full-plasmid PCR amplification was performed using Primestar high-fidelity enzyme to obtain recombinant plasmids with specific mutation sites. The primer sequences are as follows:

[0193] The mutant corresponding to the one in SEQ NO:2 where tyrosine at position 319 is replaced by leucine and serine at position 16 is replaced by alanine:

[0194] S16A-F:AGCTTCACCGCTGACAACATCGCGGCTGCG(SEQ ID NO:13)

[0195] S16A-R:GATGTTGTCAGCGGTGAAGCTCACTGTGTC(SEQ ID NO:14)

[0196] The mutant corresponding to the one in SEQ NO:2 where tyrosine at position 319 is replaced by leucine and serine at position 16 is replaced by glycine:

[0197] S16G-F:AGCTTCACCGGCGACAACATCGCGGCTGCG(SEQ ID NO:15)

[0198] S16G-R:GATGTTGTCGCCGGTGAAGCTCACTGTGTC(SEQ ID NO:16)

[0199] The mutant corresponding to the substitution of leucine for tyrosine at position 319 and serine for asparagine at position 18 in SEQ NO:2:

[0200] N18S-F:ACCTCCGACTCAATCGCGGCTGCGGCTCCG(SEQ ID NO:17)

[0201] N18S-R:AGCCGCGATTGAGTCGGAGGTGAAGCTCAC(SEQ ID NO:18)

[0202] The mutant corresponding to the one in SEQ NO:2 where tyrosine at position 319 is replaced by leucine and glutamine at position 39 is replaced by arginine:

[0203] Q39R-F:GGCAATGCGCGTCCGTACGGCGGAGACGCG(SEQ ID NO:19)

[0204] Q39R-R:GCCGTACGGACGCGCATTGCCCTGACACGC(SEQ ID NO:20)

[0205] The mutant corresponding to the substitution of aspartic acid for tyrosine at position 319 and arginine for glutamine at position 39 in SEQ NO:2:

[0206] Q39R-F:GGCAATGCGCGTCCGTACGGCGGAGACGCG(SEQ ID NO:21)

[0207] Q39R-R:GCCGTACGGACGCGCATTGCCCTGACACGC(SEQ ID NO:22)

[0208] The mutant corresponding to the one in SEQ NO:2 where glutamine at position 39 is replaced by arginine and serine at position 16 is replaced by alanine:

[0209] S16A-F: AGCTTCACCGCTGACAACATCGCGGCTGCG (SEQ ID NO: 23)

[0210] S16A-R:GATGTTGTCAGCGGTGAAGCTCACTGTGTC(SEQ ID NO:24)

[0211] The mutant corresponding to the substitution of arginine for glutamine at position 39 and leucine for tyrosine at position 319 in SEQ NO:2, while the substitution of serine for asparagine at position 18:

[0212] N18S-F:ACCTCCGACTCAATCGCGGCTGCGGCTCCG(SEQ ID NO:25)

[0213] N18S-R:AGCCGCGATTGAGTCGGAGGTGAAGCTCAC(SEQ ID NO:26)

[0214] The mutant corresponding to the substitution of arginine for glutamine at position 39 and leucine for tyrosine at position 319 in SEQ NO:2, while the substitution of threonine for asparagine at position 18:

[0215] N18T-F:ACCTCCGACACATCGCGGCTGCGGCTCCG(SEQ ID NO:27)

[0216] N18T-R:AGCCGCGATGGTGTCGGAGGTGAAGCTCAC(SEQ ID NO:28)

[0217] The mutant corresponding to the substitution of arginine for glutamine at position 39 and aspartic acid for tyrosine at position 319 in SEQ NO:2, while the substitution of serine for asparagine at position 18:

[0218] N18S-F:ACCTCCGACTCAATCGCGGCTGCGGCTCCG(SEQ ID NO:29)

[0219] N18S-R:AGCCGCGATTGAGTCGGAGGTGAAGCTCAC(SEQ ID NO:30)

[0220] The purified protein of the saturated mutant at the specified site of threonine aldolase L-TAnem was obtained according to the method in Example 2.

[0221] The reaction system for the mutant enzyme activity assay consisted of 20 μg / mL L-TAnem mutant, 100 mM aldehyde, 1 M glycine, 50 μM PLP, and 10% (v / v) DMF. The final reaction volume was brought to 500 μL using HEPES (pH 8.0) buffer. The reaction was incubated at 25°C with shaking at 250 rpm for 0.5 h. Then, 50 μL of the reaction solution was added to 950 μL of acetone to terminate the reaction. The supernatant was collected by centrifugation and analyzed by HPLC.

[0222] result

[0223] The results are as follows Figure 9 As shown in Figure A, a total of 6 positive double-point mutants were obtained, namely S16G / Y319L, Q39R / Y319L, N18S / Y319L, S16A / Q39R, Q39R / Y319D and S16A / Y319L.

[0224] Among them, Q39R / Y319L exhibited the best stereoselectivity, with a de value of 97.6%, an 8% improvement over WT. The specific activity of Q39R / Y319L was 2.0 times that of WT, reaching 133 U / mg. Meanwhile, the other five double mutants also showed further improvements in de values ​​compared to single mutants. Specifically, N18S / Y319L achieved a de value of 96.7% and a specific activity of 64.4 U / mg. S16A / Y319L, S16A / Q39R, Q39R / Y319D, and S16G / Y319L also showed improved stereoselectivity, with de values ​​of 95.9%, 95.2%, 96.3%, and 96.2%, respectively.

[0225] Based on the double mutation, further combinations were used to improve the diastereoselectivity of L-TAnem. Three highly efficient mutants were identified: N18T / Q39R / Y319L, N18S / Q39R / Y319L, and N18S / Q39R / Y319D. Their de values ​​were 98.6%, 99.3%, and 98.5%, respectively. Figure 9 A). The specific activity of N18S / Q39R / Y319L reached 95.7 U / mg, significantly higher than that of wild type (64.8 U / mg). Figure 9 B).

[0226] Example 5: Synthesis of L-threo-methanesulfonylphenylserine from threonine aldolase L-TAnem and its mutants.

[0227] Scale-up of the reaction system for the synthesis of L-threo-methanesulfonylphenylserine was performed. The reaction conditions were: 15 mg L-TAnem and the mutant, 100 mM aldehyde, 1 M glycine, 50 μM PLP, and 10% (v / v) DMF. The final reaction volume was brought to 1 L using HEPES (pH 8.0) buffer. The reaction was incubated at 25 °C with shaking at 250 rpm for 2.5 h. At different time points, 50 μL of the reaction solution was taken and added to 950 μL of acetone to terminate the reaction. The supernatant was collected by centrifugation and analyzed by HPLC.

[0228] The results are as follows Figure 10 As shown in the figure. The results indicate that after 140 min of reaction, the conversion rate of L-threo-methanesulfonylphenylserine synthesized by the threonine aldolase L-TAnem reached 54.8%, with a de value of 89.1%; the conversion rate of L-threo-methanesulfonylphenylserine synthesized by the mutant N18S / Q39R / Y319L reached 80.1%, with a de value >99%.

[0229] Example 6: Synthesis of L-threo-phenylserine derivatives from threonine aldolase L-TAnem and its mutant N18S / Q39R / Y319L

[0230] The reaction conditions were: 1.5 U L-TAnem and its mutant, 100 mM aldehyde, 1 M glycine, 50 μM PLP, and 10% (v / v) DMF. The final reaction volume was brought to 1 L using HEPES (pH 8.0) buffer. The reaction was incubated at 25°C with shaking at 250 rpm for 2 hours. At different time points, 50 μL of the reaction solution was taken and added to 950 μL of acetone to terminate the reaction. The supernatant was collected by centrifugation and analyzed by HPLC.

[0231] The results are as follows Figure 10 As shown in the figure. The results indicate that this threonine aldolase exhibits good activity and selectivity for various aromatic aldehyde substrates. For para-substituted substrates, the mutant showed better diastereomeric stereoselectivity. It also exhibited good activity for disubstituted substrates.

[0232] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A threonine aldolase, characterized in that, The threonine aldolase is obtained by mutating any of the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 2: Q39A, Q39R, Q39K, Q39H, Q39L, Q39R / Y319L, N18S / Q39R / Y319L, or N18S / Q39R / Y319D.

2. The threonine aldolase as described in claim 1, characterized in that, The threonine aldolase is obtained by mutating any one of the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 2: Q39A, Q39R, Q39K, Q39H or Q39L.

3. The threonine aldolase as described in claim 1, characterized in that, The threonine aldolase described herein is obtained by the following amino acid residue mutation in the amino acid sequence shown in SEQ ID NO: 2: Q39R / Y319L.

4. The threonine aldolase as described in claim 1, characterized in that, The threonine aldolase is obtained by mutating any one of the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 2: N18S / Q39R / Y319L or N18S / Q39R / Y319D.

5. The threonine aldolase as described in claim 1, characterized in that, The threonine aldolase described herein is obtained by the following amino acid residue mutation in the amino acid sequence shown in SEQ ID NO: 2: N18S / Q39R / Y319L.

6. An isolated polynucleotide, characterized in that, The polynucleotide encodes the threonine aldolase of claim 1.

7. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 6.

8. The carrier as described in claim 7, characterized in that, The carrier includes a recombinant carrier.

9. The carrier as described in claim 7, characterized in that, The carriers mentioned include expression carriers or integration carriers.

10. A genetically engineered host cell, characterized in that, The host cell contains the vector of claim 7 or its genome integrated with the exogenous polynucleotide of claim 6.

11. The host cell as described in claim 10, characterized in that, The host cells mentioned include prokaryotic cells or eukaryotic cells.

12. The host cell as described in claim 10, characterized in that, The host cell is Escherichia coli.

13. A method for preparing the threonine aldolase according to claim 1, characterized in that, The method includes: (a) Culturing the host cells of claim 10 under suitable expression conditions to express the threonine aldolase of claim 1; and (b) Isolate the expression product to obtain the threonine aldolase.

14. The use of a threonine aldolase, characterized in that, Used for catalyzing aldol condensation reactions or for preparing catalytic reagents for aldol condensation reactions; the aldol condensation reaction is shown below: in, R is one of the following groups: 2-F, 2-Cl, 2-Br, 2-NO2, 3-F, 3-Cl, 3-Br, 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3 or 4-methanesulfonyl; PLP is pyridoxal 5-phosphate; L-TAnem is a threonine aldolase; The threonine aldolase described herein is obtained by the following amino acid residue mutation in the amino acid sequence shown in SEQ ID NO: 2: N18S / Q39R / Y319L.

15. A catalytic method for the in vitro preparation of a compound of formula (I), characterized in that, Including the following steps: In the presence of threonine aldolase, glycine and benzaldehyde derivatives are used as substrates for an aldol condensation reaction to form the amino acid product shown in formula (I): in, R is one of the following groups: 2-F, 2-Cl, 2-Br, 2-NO2, 3-F, 3-Cl, 3-Br, 4-F, 4-Cl, 4-Br, 4-NO2, 4-CH3 or 4-methanesulfonyl; PLP is pyridoxal 5-phosphate; L-TAnem is a threonine aldolase; The threonine aldolase described herein is obtained by the following amino acid residue mutation in the amino acid sequence shown in SEQ ID NO: 2: N18S / Q39R / Y319L.

16. The method as described in claim 15, characterized in that, The molar ratio of the benzaldehyde derivative to glycine is 0.5~2:1~100.

17. The method as described in claim 15, characterized in that, The reaction is carried out in a hydrophilic organic solvent.

18. The method as described in claim 17, characterized in that, The hydrophilic organic solvent is N,N dimethylamide.

19. The method as described in claim 15, characterized in that, The method further includes: providing an additive for regulating enzyme activity to the reaction system, wherein the additive for regulating enzyme activity is selected from the group consisting of Mn 2+ K + Ba 2+ Mg 2+ NH4 + or Ca 2+ .

20. The method as described in claim 15, characterized in that, The concentration conditions for pyridoxal 5-phosphate in the method are: 1 μM ~ 100 μM.

21. The method as described in claim 15, characterized in that, The concentration conditions for pyridoxal 5-phosphate in the method are: 20 μM to 50 μM.

22. The method as described in claim 15, characterized in that, The pH conditions for the method are: pH 7.0 ~ pH 9.

0.

23. The method as described in claim 15, characterized in that, The temperature conditions for the method are 20 ℃-50 ℃.

24. The method as described in claim 15, characterized in that, The temperature conditions for the method are 25 ℃~30 ℃.

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