CN108239664B

CN108239664B - Process for preparing 4-hydroxy-L-threonine

Info

Publication number: CN108239664B
Application number: CN201810060525.2A
Authority: CN
Inventors: 王俊梅; 何遂平; 张新帅; 黄华; 刘建
Original assignee: Shenzhen Reed Forest Biotechnology Co Ltd
Current assignee: Gansu ruidilin biological Co.,Ltd.
Priority date: 2018-01-22
Filing date: 2018-01-22
Publication date: 2020-01-03
Anticipated expiration: 2038-01-22
Also published as: CN108239664A

Abstract

The invention relates to the technical field of biochemistry, and discloses a process for preparing 4-hydroxy-L-threonine by an immobilized enzyme tandem method. The invention utilizes L-threonine activating enzyme to couple L-threonine aldolase to complete conversion, adopts Adk and PaP or fusion protease thereof and cheap polyphosphoric acid to regenerate ATP in a timely and cyclic manner, has simple reaction process, convenient operation and stable product quality, better realizes the continuous conversion of raw materials and the regeneration of coenzyme ATP, can be circularly utilized for a plurality of times by matching with stable immobilized enzyme, thereby greatly reducing the production cost. The process effectively avoids the complicated processes of multi-step group protection, chiral separation and the like in the chemical synthesis technology, simultaneously avoids the influence of metabolites, proteins and other impurities on the purification of later-stage products caused by microbial fermentation, and is very suitable for industrial large-scale production.

Description

Process for preparing 4-hydroxy-L-threonine

Technical Field

The invention relates to the technical field of biochemistry, and particularly relates to a process for preparing 4-hydroxy-L-threonine.

Background

4-hydroxy-L-threonine is a hydroxylated amino acid of formula C₄H₉NO₄And has a molecular weight of 135. L-threonine is an essential amino acid and is mainly used in the fields of medicines, chemical reagents, food enhancers, feed additives and the like. Hydroxylated threonine, on the other hand, is not abundant in nature and is found mainly in the organism pseudomonas (pseudomonas). Many documents report that hydroxylated amino acids can also be widely used as food, feed additives, effective components for treating diabetes, and starting materials of some chemical industries; therefore, the cheap and mass production of the compound is suitable for popularizationThe foundation is laid.

The synthesis of 4-hydroxy-L-threonine mainly comprises a chemical synthesis method, an oxidase synthesis method, a biological fermentation method and the like, wherein the chemical synthesis method commonly used in industry utilizes glyceraldehyde or D-mannitol to prepare through multi-step protection, functionalization and chiral resolution, and has the advantages of complex synthesis process, low final yield and great environmental pollution (chemical routes I and II); a small number of reports have reported that L-threonine is directly hydroxylated to form 4-hydroxy-L-threonine using a specific oxidase, but this method is based on the unstable structure of the oxidase itself, the active center iron (Fe)^II) The complexity of the cycle regeneration and the inhibition effect of the high product concentration on the enzyme activity and other defects seriously restrict the amplification application of the enzyme; several documents also report the use of Pseudomonas aeruginosa (Pseudomonas andropogonis) to metabolize aspartic acid to produce 4-hydroxy-L-threonine, but this method is still limited to theoretical studies due to too low yield, and the need for systematic modification and optimization of the strain to achieve industrial production will necessarily consume a lot of time and resources (chemical scheme III).

Disclosure of Invention

In view of the above, the present invention provides a process for preparing 4-hydroxy-L-threonine, which utilizes multi-enzyme continuous and concerted catalysis to convert cheap raw materials into a specific product, 4-hydroxy-L-threonine, in a very short route at a high yield, and simultaneously realizes continuous conversion of raw materials and regeneration of coenzyme ATP, thereby avoiding complicated processes such as multi-step group protection and chiral resolution in chemical synthesis technology.

In order to achieve the purpose, the invention provides the following technical scheme:

a process for preparing 4-hydroxy-L-threonine comprising:

step 1, producing 4-hydroxy-L-threonine by glycolaldehyde and glycine under the catalysis of L-threonine aldolase;

step 2, the 4-hydroxy-L-threonine in the step 1 generates 4-hydroxy-phosphate-L-threonine and ADP under the catalysis of ATP and threonine activating enzyme;

step 3, generating ATP from the ADP in the step 2 under the catalysis of polyphosphoric acid, adenylate kinase and polyphosphoric acid-AMP phosphotransferase, and entering the step 2 again; the 4-hydroxy-phosphate-L-threonine and sodium salt in the step 2 generate 4-hydroxy-phosphate-L-threonine sodium, and then generate 4-hydroxy-L-threonine under the catalysis of pyrophosphoric acid hydrolase.

Aiming at the problems that the existing 4-hydroxy-L-threonine preparation process is generally complicated and the industrial application efficiency is low, the invention adopts L-threonine aldolase, threonine activating enzyme, adenylate kinase and polyphosphate-AMP phosphotransferase to jointly prepare the 4-hydroxy-L-threonine, and realizes the continuous conversion of raw materials and the regeneration of coenzyme ATP.

The reaction scheme of the process is shown in figure 1, and the L-threonine aldolase (EC4.1.2.5, TA) adopted by the invention can catalyze the condensation of hydroxyacetaldehyde and glycine to generate 4-hydroxy-L-threonine and non-corresponding isomers thereof; the adoption of threonine activating enzyme (EC 2.7.1.-, TK) can selectively phosphorylate 4-hydroxy-L-threonine to 4-hydroxy-L-threonine phosphate, and effectively drag the previous condensation reaction to be complete. Meanwhile, adenosine triphosphate ATP can be efficiently regenerated by using cheap polyphosphoric acid in a circulating manner in a reaction system by adopting adenylate kinase (EC 2.7.4.3, Adk) and polyphosphoric acid-AMP phosphotransferase (EC 2.7.4, Pap), so that the use level of ATP in the reaction system can be effectively controlled, the inhibiting effect of high-concentration Adenosine Diphosphate (ADP) on an enzyme catalyst can be reduced, the yield is improved, and the cost is reduced; finally, 4-hydroxyphosphate-L-threonine is treated with pyrophosphohydrolase (EC3.6.1.1, IPH), and the product, 4-hydroxyphosphate-L-threonine, is finally purified.

In order to facilitate the reaction, in step 1, the invention better realizes the catalytic reaction of L-threonine aldolase by adding pyridoxal phosphate, namely, the hydroxy acetaldehyde and the glycine generate 4-hydroxy-L-threonine under the catalysis of the L-threonine aldolase and the pyridoxal phosphate; the pyridoxal phosphate is preferably sodium pyridoxal phosphate.

Also based on the consideration of promoting the reaction to proceed better, step 2 is: in step 1 4-hydroxy-L-threonine is present in ATP,Threonine activating enzyme, Mg²⁺And K⁺To generate 4-hydroxyphosphate-L-threonine and ADP. Wherein said Mg²⁺And K⁺From Mg capable of liberating free²⁺And K⁺Such as magnesium chloride, potassium chloride, magnesium nitrate, potassium nitrate, magnesium sulfate, potassium sulfate, and the like.

Preferably, in step 3 of the present invention, the process of converting 4-hydroxyphosphate-L-threonine into 4-hydroxyphosphate-L-threonine sodium, and then performing enzymatic hydrolysis to produce 4-hydroxy-L-threonine can be performed as follows:

precipitating the 4-hydroxy-phosphate-L-threonine in the step 2 in a barium salt form, then dissolving the precipitate in a Tris-hydrochloric acid buffer solution again, adding an inorganic sodium salt for reaction, and filtering to remove the barium salt precipitate to obtain 4-hydroxy-phosphate-L-threonine sodium; sodium 4-hydroxy-phosphate-L-threonine is catalyzed by pyrophosphohydrolase to produce 4-hydroxy-L-threonine. In the step, not only can the reaction progress be realized, but also the purification operation of the 4-hydroxyphosphoric acid-L-threonine can be added during the reaction, so that the whole reaction is more efficient, simpler and more convenient, and the subsequent purification operation is also convenient.

Wherein the mode of precipitating the 4-hydroxy-phosphate-L-threonine in the form of barium salt is that soluble barium salt such as barium oxalate is added to form 4-hydroxy-phosphate-L-threonine barium precipitate, and then inorganic sodium salt capable of reacting with barium to form precipitate is added again to generate 4-hydroxy-phosphate-L-threonine sodium. The addition of an inorganic sodium salt capable of reacting with barium to form a precipitate may be carried out according to the barium salt precipitation conventional in the chemical art, such as BaSO₄、BaCO₃、BaSO₃、BaSiO₃、Ba₃(PO4)₂、BaF₂Correspondingly select Na₂SO₄、Na₃PO₄、Na₂CO₃、Na₂SO₃、Na₂SiO₃、NaF。

In a specific embodiment of the invention, the adenylate kinase (EC 2.7.4.3, Adk) and polyphosphate-AMP phosphotransferase (EC 2.7.4-Pap) are fused to form ATP regeneration fusion enzyme Adk-PaP, namely, a fusion enzyme containing two enzymes is expressed in cells after the genes of the two enzymes are assembled, and the fusion enzyme has the functions of both Adk and PaP.

In the process of the invention, Tris-hydrochloric acid buffer solution is preferably used as a reaction solvent for all reactions, and the reaction pH value is 6.0-8.5. Among them, Tris-hydrochloric acid buffer solution of 25mM, pH7.0-7.5 is preferably used.

In the process of the present invention, the polyphosphoric acid is preferably 25 poly, 0.1M phosphoric acid.

Based on the immobilized enzyme technology, the invention also provides that the L-threonine aldolase, the threonine kinase, the adenylate kinase and the polyphosphate-AMP phosphotransferase (including the fusogenic enzyme Adk-PaP) are immobilized on a carrier to form immobilized enzyme for reaction. After the immobilized enzyme is immobilized, the process can recycle the immobilized enzyme participating in the reaction, and the enzyme activity of the reused immobilized enzyme still keeps a higher level.

In addition, the preparation of 4-hydroxy-L-threonine by the process of the invention can finally reach high yield of nearly 80 percent, and is simple and convenient in purification.

According to the technical scheme, the L-threonine aldolase is coupled to the L-threonine aldolase by utilizing the L-threonine activating enzyme to be completely converted, the ATP is regenerated in a timely and cyclic manner by adopting the Adk and the PaP or the fusion protease thereof and the cheap polyphosphoric acid, the reaction process is simple, the operation is convenient, the product quality is stable, the continuous conversion of the raw materials and the regeneration of the coenzyme ATP are better realized, and the stable immobilized enzyme is matched, so that the coenzyme ATP can be recycled for multiple times, and the production cost is greatly reduced. The process effectively avoids the complicated processes of multi-step group protection, chiral separation and the like in the chemical synthesis technology, simultaneously avoids the influence of metabolites, proteins and other impurities on the purification of later-stage products caused by microbial fermentation, and is very suitable for industrial large-scale production.

Drawings

FIG. 1 is a reaction scheme of the process of the present invention;

FIG. 2 shows the nuclear magnetic hydrogen spectrum for identifying 4-hydroxyphosphate-L-threonine¹H-NMR；

FIG. 3 shows identification 4Nuclear magnetic phosphorus spectrum of-hydroxyphosphoric acid-L-threonine³¹P-NMR；

FIG. 4 shows the nuclear magnetic hydrogen spectrum for identifying 4-hydroxy-L-threonine¹H-NMR；

FIG. 5 shows nuclear magnetic carbon spectra for the identification of 4-hydroxy-L-threonine³¹C-NMR。

Detailed Description

The invention discloses a process for preparing 4-hydroxy-L-threonine, which can be realized by appropriately improving process parameters by the technical personnel in the field with reference to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the process described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The steps of the process of the present invention are intended to clearly describe the reaction route at the core, and do not limit the whole reaction to be performed by a one-step method or a multi-step method, for example, step 1 and step 2, and the ATP-producing portion of step 3 of the process of the present invention, and all the reaction raw materials may be put into operation at one time.

In a specific embodiment, the invention employs the fusion enzyme Adk-PaP to carry out the reaction. The adopted L-threonine aldolase, threonine activating enzyme, adenylate kinase, polyphosphate-AMP phosphotransferase and pyrophosphoric acid hydrolase all have corresponding EC numbers, the specific amino acid sequences are sequentially shown as SEQ ID NO. 1-5, and the sequence of the fusion enzyme Adk-PaP is a sequence of SEQ ID NO. 3-4 connected by a connecting short peptide, namely a sequence of SEQ ID NO. 3 + the connecting short peptide + a sequence of SEQ ID NO. 4; in the specific embodiment of the invention, the sequence of the connecting short peptide is shown in SEQ ID NO. 6, and other connecting short peptides commonly used in the field of fusion enzymes can also be used.

The enzymes used in the invention can be artificially synthesized according to sequences, and can also be transformed by constructing recombinant plasmids through respective coding genes, such as:

the TA, TK and IPH gene fragments were amplified by PCR using the extracted DNA of E.coli (Escherichia coli) BL21 strain (available from general-purpose organisms) and the chromosome of Cupriavidus necator ATCC-17699D (available from ATCC) as templates, and then ligated to pET28a plasmid (ATP available from BioFeng) by appropriate enzyme cleavage (e.g., BamHI and XhoI, NdeI and XhoI), wherein the gene encoding the regenerative enzyme Adk-PaP was purchased directly from general-purpose organisms and subcloned into pET28a plasmid. Transferring the four gene sequences into an E.coli BL21(DE3) strain (a general organism), and confirming that correct colonies are cultured in LB culture solution containing 50uM of clarithromycin; when the cells were grown to log phase, 0.2mM isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to induce protein expression for 4 hours, and finally the cells were collected, disrupted, and the protein expression was confirmed by high speed centrifugation of the supernatant on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The seed medium for confirming the protein expression may be inoculated into a 10L culture fermenter and grown to logarithmic phase, and then induced to express with 0.5mM IPTG for 6 hours, and 200g of wet cells are collected. The LB medium is composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate and 5% glycerol.

Aldolase (TA), activating enzyme (TK) and pyrophosphohydrolase (IPH) cell disruption clear solution were precipitated by gradually adding 60% saturated ammonium sulfate, and the solid was slowly dissolved in 25mM Tris buffer solution (pH8.0), desalted by G25 size exclusion column (purchased from Sigma) and then separated by DEAE Seplite FF anion exchange column to obtain primary purified TA, TK and IPH enzymes.

To 500ml of the cell disruption supernatant of the fusion enzyme Adk-PaP was added 100ml of a 0.5M sodium phosphate solution having a pH of 7.0, followed by stepwise addition of dimethoxyethane until the protein precipitation was completed to obtain a purified fusion enzyme Adk-PaP.

When the enzymes of the invention are immobilized, the conventional preparation method of immobilized enzymes in the field can be referred to, and in the specific embodiment of the invention, the invention is carried out according to the following method:

1. aldolase (TA), activating enzyme (TK), pyrophosphate hydrolase (IPH)

1000U of the purified enzyme was dissolved in 1L of 100mM potassium phosphate solution, pH8.0, followed by addition of 60mM phenoxyacetic acid and 200g of LX-1000EP epoxy resin to the buffer, stirring at room temperature for 24 hours, filtering off the immobilized enzyme, finally washing twice each with clean water and 100mM phosphate buffer, pH8.0, and drying at low temperature for use. Immobilized aldolase (TA), activating enzyme (TK), and pyrophosphate hydrolase (IPH) have 20-50% activity as liquid enzymes.

2. Fusion enzyme Adk-PaP

Adding 80ml of 25% glutaraldehyde aqueous solution into the purified fusion enzyme Adk-PaP, slightly stirring the mixed solution at 4 ℃ for 16 hours, and filtering to obtain Adk-PaP enzyme precipitation cross-linked solid CLEAs; to increase the mechanical strength of the cross-linking enzyme, 500ml of methanolic tetramethoxysilane solution was added dropwise to 500ml of stirred CLEAs phosphoric acid solution until clear, and after standing at room temperature for 6 hours, the immobilized Adk-PaP enzyme was filtered. The immobilized Adk-PaP enzyme had 65% activity as the liquid enzyme.

According to the reaction route of the process of the invention, the dosage of each reactant can be adjusted according to actual conditions, and for the maximum efficiency, the invention provides the following mole ratios of each reactant:

glycolaldehyde glycine pyridoxal phosphate adenosine triphosphate polyphosphate aldolase (TA, immobilized), threonine kinase (TK, immobilized), ATP cycle regeneration enzyme (Adk-PaP, immobilized) (100000-;

4-hydroxy-L-threonine sodium pyrophosphate hydrolase (immobilized) (100000-1000000): 5-200);

wherein, the enzyme activity of each enzyme is preferably 1000-50000U, and for balancing efficiency and cost, 1000-20000U can be further preferably adopted, and 1000-10000U or 1000-5000U can be further adopted; in a specific embodiment, the immobilized aldolase (TA) of the present invention is 2000U, the immobilized Threonine Kinase (TK) is 2500U, the immobilized fusion enzyme Adk-PaP is 3000U, and the immobilized pyrophosphate hydrolase is 1000U.

The invention is further illustrated by the following examples.

Example 1: the process of the invention

To 5L of 25mM Tris-HCl (pH 7.5) solution, 30 g of hydroxyacetaldehyde (0.1M),37.5 g of glycine (0.1M), 670 mg of sodium pyridoxal phosphate (0.5mM), 2.5 g of adenosine triphosphate ATP (1mM), 51 g of polyphosphoric acid (Sigma, 25M, 0.1M phosphoric acid), 23.5 g of magnesium chloride (50mM), 9.3 g of potassium chloride (25mM) were added; after the pH value is adjusted to 7.5, 2000U of immobilized aldolase (TA), 2500U of immobilized 4-hydroxy-L-Threonine Kinase (TK) and 3000U of immobilized ATP cyclic regeneration enzyme (Adk-PaP) are sequentially added into a reaction system to start reaction, after the reaction is stirred for 6 hours at room temperature, a solution is taken to quantitatively detect residual hydroxyacetaldehyde by using a M-naphthalenediol method, and the result shows that the reaction is complete (activity unit U represents the enzyme amount required for converting 1 mu M substrate per minute). The products were identified by nuclear magnetic resonance, and the results are shown in FIGS. 2 and 3, and the nuclear magnetic results showed that the target product was 4-hydroxyphosphate-L-threonine.

The immobilized enzyme was recovered from the reaction solution by filtration, and 127.5 g of barium oxalate (0.5mol) was added to the filtrate to precipitate 4-hydroxyphosphoric acid-L-threonine and other phosphoric acid-containing impurities. Then dissolving the precipitated solid in a trihydroxymethylaminomethane buffer solution with the pH value of 1.0, and adding 71 g of anhydrous sodium sulfate (0.5mol) to precipitate insoluble barium sulfate; filtering to remove solid, adjusting pH of filtrate to 7.0, separating with D201 anion resin exchange column (crystal industry), and eluting with gradient ammonium bicarbonate water solution to obtain purified 4-hydroxy sodium phosphate-L-threonine. Finally, desalting by a G25 size exclusion column to obtain 99G of white powder of the 4-hydroxy sodium L-threonine phosphate, wherein the yield reaches 84%. The immobilized enzyme retains 60% of original activity after 12 times of recovery.

50 g of sodium 4-hydroxyphosphate is dissolved in 1L of 25mM Tris-HCl buffer solution with pH7.0, 1.9 g of magnesium chloride and 1000U of immobilized pyrophosphorohydrolase are added and mixed for three hours at room temperature, the immobilized enzyme is recovered by filtration, the filtrate is loaded on a D201 anion resin exchange column, and the product 4-hydroxy-L-threonine is used as loading effluent. The product was concentrated and desalted to give 26 g of a white foamy solid in 78% yield, and the results of nuclear magnetic assay are shown in FIGS. 4 and 5. The immobilized pyrophosphoric acid hydrolase is recycled for 10 times, and the activity is reserved for 80 percent.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg

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Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala

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Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu

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Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly

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Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly

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Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser

195 200 205

Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu

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Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala

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Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala

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Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala

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Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu

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Ala Ser Gln Lys Pro Ser Arg Arg Ala Asp Val Leu Val Ile Asn Thr

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Ala Leu Ser Pro Trp Cys Glu Thr Ser Pro Ala Pro Leu Val Tyr Lys

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Lys Ile Asp Ser Thr Phe Arg Gly Asn Ile Gly Ala Glu Val Thr Ala

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Ala Met Arg Ala Ser Gln Arg Lys Leu Ala Val Ile Ala Ala Ala Ile

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Pro Ala Ala Gly Arg Thr Thr Leu Glu Gly Lys Cys Leu Val Asn Gly

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Val Pro Leu Leu Glu Thr Glu Phe Ala Ser Asp Pro Lys Thr Pro Ile

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Val Ser Ser Arg Ile Ala Glu Ile Val Ala Leu Gln Ser Glu Ile Pro

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Ala Cys Ala Leu Leu Ser Gln His Arg His Thr Ile Leu Arg Thr Ser

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Gly Gly Phe Gly Ser Asp Ser Thr Leu Cys Asp Ala Leu Tyr Tyr Ile

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Glu Glu Met Tyr

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Asp Met Leu Arg Ala Ala Val Lys Ser Gly Ser Glu Leu Gly Lys Gln

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Ala Lys Asp Ile Met Asp Ala Gly Lys Leu Val Thr Asp Glu Leu Val

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Ile Ala Leu Val Lys Glu Arg Ile Ala Gln Glu Asp Cys Arg Asn Gly

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Phe Leu Leu Asp Gly Phe Pro Arg Thr Ile Pro Gln Ala Asp Ala Met

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Lys Glu Ala Gly Ile Asn Val Asp Tyr Val Leu Glu Phe Asp Val Pro

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Asp Glu Leu Ile Val Asp Arg Ile Val Gly Arg Arg Val His Ala Pro

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Glu Glu Thr Val Arg Lys Arg Leu Val Glu Tyr His Gln Met Thr Ala

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Pro Leu Ile Gly Tyr Tyr Ser Lys Glu Ala Glu Ala Gly Asn Thr Lys

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Leu Glu Lys Ile Leu Gly

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Ser Leu Asp Leu Ile Glu Ala Gln Tyr Ala Leu Met Asn Thr Arg Asp

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Ala Gly Lys Gly Glu Ala Val Lys Gln Leu Arg Glu Trp Val Asp Pro

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Arg Phe Leu Tyr Val Lys Ala Asp Pro Pro His Leu Phe Asn Leu Lys

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Gln Pro Phe Trp Gln Pro Tyr Thr Arg Phe Val Pro Ala Glu Gly Gln

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Ile Met Val Trp Phe Gly Asn Trp Tyr Gly Asp Leu Leu Ala Thr Ala

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Met His Ala Ser Lys Pro Leu Asp Asp Thr Leu Phe Asp Glu Tyr Val

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Ser Asn Met Arg Ala Phe Glu Gln Asp Leu Lys Asn Asn Asn Val Asp

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Val Leu Lys Val Trp Phe Asp Leu Ser Trp Lys Ser Leu Gln Lys Arg

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Leu Asp Asp Met Asp Pro Ser Glu Val His Trp His Lys Leu His Gly

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Leu Asp Trp Arg Asn Lys Lys Gln Tyr Asp Thr Leu Gln Lys Leu Arg

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Thr Arg Phe Thr Asp Asp Trp Gln Ile Ile Asp Gly Glu Asp Glu Asp

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Leu Arg Asn His Asn Phe Ala Gln Ala Ile Leu Thr Ala Leu Arg His

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Cys Pro Glu His Glu Lys Lys Ala Ala Leu Lys Trp Gln Gln Ala Pro

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Ile Pro Asp Ile Leu Thr Gln Phe Glu Val Pro Gln Ala Glu Asp Ala

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Asn Tyr Lys Ser Glu Leu Lys Lys Leu Thr Lys Gln Val Ala Asp Ala

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Met Arg Cys Asp Asp Arg Lys Val Val Ile Ala Phe Glu Gly Met Asp

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Ala Ala Gly Lys Gly Gly Ala Ile Lys Arg Ile Val Lys Lys Leu Asp

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Pro Arg Glu Tyr Glu Ile His Thr Ile Ala Ala Pro Glu Lys Tyr Glu

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Leu Arg Arg Pro Tyr Leu Trp Arg Phe Trp Ser Lys Leu Gln Ser Asp

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Asp Ile Thr Ile Phe Asp Arg Thr Trp Tyr Gly Arg Val Leu Val Glu

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Arg Val Glu Gly Phe Ala Thr Glu Val Glu Trp Gln Arg Ala Tyr Ala

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Glu Ile Asn Arg Phe Glu Lys Asn Leu Ser Ser Ser Gln Thr Val Leu

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Ile Lys Phe Trp Leu Ala Ile Asp Lys Asp Glu Gln Ala Ala Arg Phe

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Lys Ala Arg Glu Ser Thr Pro His Lys Arg Phe Lys Ile Thr Glu Glu

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Asp Trp Arg Asn Arg Asp Lys Trp Asp Asp Tyr Leu Lys Ala Ala Ala

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Asp Met Phe Ala His Thr Asp Thr Ser Tyr Ala Pro Trp Tyr Ile Ile

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Ser Thr Asn Asp Lys Gln Gln Ala Arg Ile Glu Val Leu Arg Ala Ile

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Leu Lys Gln Leu Lys Ala Asp Arg Asp Thr Asp

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Ala Met Phe Tyr Pro Cys Asn Tyr Gly Tyr Ile Asn His Thr Leu Ser

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Leu Asp Gly Asp Pro Val Asp Val Leu Val Pro Thr Pro Tyr Pro Leu

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Gln Pro Gly Ser Val Ile Arg Cys Arg Pro Val Gly Val Leu Lys Met

85 90 95

Thr Asp Glu Ala Gly Glu Asp Ala Lys Leu Val Ala Val Pro His Thr

100 105 110

Lys Leu Ser Lys Glu Tyr Asp His Ile Lys Asp Val Asn Asp Leu Pro

115 120 125

Glu Leu Leu Lys Ala Gln Ile Ala His Phe Phe Glu His Tyr Lys Asp

130 135 140

Leu Glu Lys Gly Lys Trp Val Lys Val Glu Gly Trp Asp Asn Ala Glu

145 150 155 160

Ala Ala Lys Ala Glu Ile Ile Ala Ser Phe Glu Arg Ala Ala Lys Lys

165 170 175

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ser Ser Gly Leu Val Pro Arg Gly Ser

1 5

Claims

1. A process for preparing 4-hydroxy-L-threonine, comprising:

step 3, generating ATP from the ADP in the step 2 under the catalysis of polyphosphoric acid, adenylate kinase and polyphosphoric acid-AMP phosphotransferase, and entering the step 2 again; 4-hydroxy-phosphate-L-threonine and sodium salt in the step 2 are used for generating 4-hydroxy-phosphate-L-threonine sodium, and then the 4-hydroxy-L-threonine is generated under the catalysis of pyrophosphoric acid hydrolase;

wherein adenylate kinase and polyphosphate-AMP phosphotransferase participate in the reaction in the form of a fusogenic enzyme, the amino acid sequences of the adopted L-threonine aldolase, threonine activating enzyme, adenylate kinase, polyphosphate-AMP phosphotransferase and pyrophosphate hydrolase are sequentially shown as SEQ ID NO. 1-5, and the sequence of the fusogenic enzyme Adk-PaP is a sequence SEQ ID NO. 3-4 connected by a connecting short peptide.

2. The process of claim 1, wherein step 1 is:

the hydroxyacetaldehyde and glycine generate 4-hydroxy-L-threonine under the catalysis of L-threonine aldolase and pyridoxal phosphate.

3. The process of claim 1, wherein the steps 1-3 use Tris-hydrochloric acid buffer as a reaction solvent.

4. The process of claim 1, wherein the steps 1-3 are carried out at a pH of 6.0-8.5.

5. The process of claim 1, wherein step 2 is:

in step 1, 4-hydroxy-L-threonine is added to ATP, threonine activating enzyme and Mg²⁺And K⁺To generate 4-hydroxyphosphate-L-threonine and ADP.

6. The process of claim 5, wherein the Mg is²⁺And K⁺Provided by an inorganic salt.

7. The process of claim 1, wherein step 3 is:

in the step 2, ADP generates ATP under the catalysis of polyphosphoric acid, adenylate kinase and polyphosphoric acid-AMP phosphotransferase, and the step 2 is entered again;

precipitating the 4-hydroxy-phosphate-L-threonine in the step 2 in a barium salt form, then dissolving the precipitate in a Tris-hydrochloric acid buffer solution again, adding an inorganic sodium salt for reaction, and filtering to remove the barium salt precipitate to obtain 4-hydroxy-phosphate-L-threonine sodium; sodium 4-hydroxy-phosphate-L-threonine is catalyzed by pyrophosphohydrolase to produce 4-hydroxy-L-threonine.

8. The process of claim 1, wherein the L-threonine aldolase, threonine kinase, adenylate kinase, and polyphosphate-AMP phosphotransferase are immobilized enzymes.