Preparation method of kyotomorphin and its derivatives

Abstract

The present invention relates to the field of biotechnology, and in particular to methods for preparing kyotomorphin and its derivatives. The present invention provides mutants of amino acid ligases and the use of such mutants in the synthesis of kyotomorphin and / or kyotomorphin derivatives. The methods for preparing kyotomorphin and its derivatives provided by the present invention are simple to operate, have high yields, provide high product quality, exhibit good environmental compatibility, have low carbon emissions, and are easily scalable.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method of kyotomorphin and its derivatives. Background Art

[0002] Kyotorphin (KTP) is a naturally occurring endogenous dipeptide, L-tyrosine-L-arginine (L-Tyr-L-Arg-OH). This compound is structurally similar to morphine and exhibits analgesic effects similar to those of morphine, with an analgesic potency 4.2 times greater than that of endogenous opioid peptides. It also functions as a nociceptive neurotransmitter / neuromodulator in the central nervous system, inhibiting cell proliferation and acting as an anti-hibernation regulator. Kyotorphin was first isolated from bovine brain in 1979 by Takagi et al. at Kyoto University in Japan, and has subsequently been found in the human brain and spinal cord. Kyotorphin amide (L-Tyr-L-Arg-NH2, KTP-NH2) is a derivative of kyotorphin with enhanced anesthetic properties. It triggers analgesia through an indirect opioid mechanism and exhibits excellent analgesic efficacy, making it a potential alternative to current opioids. While opium and its alkaloids (such as morphine) are widely used to relieve severe acute and chronic pain, they can also cause side effects such as constipation, respiratory depression, confusion, motor disturbances, and addiction. Long-term use of opioids can lead to serious side effects. KTP and KTP-NH2 have analgesic activity comparable to morphine, yet they do not produce the side effects associated with opioids.

[0003] Preparation method of Kyotomorphin (KTP) and Kyotomorphin amide (KTP-NH2):

[0004] Chemical synthesis method:

[0005] To date, the preparation method for kyotomorphin / kyotomorphin amide is a chemical synthesis method. During the preparation process, the tyrosine functional group must first be selectively protected to obtain BOC-Tyr(tBU)-OH, then chemically condensed with arginine amide (Arg-NH2), and finally the protecting groups must be removed separately. This preparation method has many reaction steps and low overall yield (<50%). The reaction requires a large amount of organic solvents (such as HOBt, DMF, dichloromethane, etc.) and chemical reagents (such as BOP, NMM, TFA, etc.), which makes the preparation process environmentally costly. At the same time, the chemical coupling process inevitably produces racemic dipeptides, which greatly increases the purification cost of the subsequent products. Therefore, both from economic and environmental perspectives, this chemical preparation method has significant drawbacks. Its synthesis route is shown below:

[0006]

[0007] Bacillus subtilis contains an amino acid ligase (Uniprot: C0STU2), which is reported to have the ability to connect multiple amino acid carboxyl groups to the arginine amino group to form Xaa-Arg dipeptides. However, experimental tests have found that this enzyme mainly catalyzes the connection of valine (Val), leucine (Leu), and isoleucine (Ile) with arginine. The wild amino acid ligase (C0STU2) does not have the ability to connect tyrosine and arginine to generate the target dipeptide, and its activity cannot meet the requirements of industrial applications.

[0008] As mentioned above, the primary method for producing Tyr-Arg-OH (KTP) and Tyr-Arg-NH2 (KTP-NH2) dipeptides today is chemical synthesis, which has disadvantages such as long preparation routes, low overall yields, high organic solvent usage, and poor final product quality. Biosynthesis holds promise, but a suitable ligase has yet to be found to synthesize Tyr-Arg-OH (KTP) and Tyr-Arg-NH2 (KTP-NH2) dipeptides. Summary of the Invention

[0009] In view of this, the present invention provides a method for preparing kyotomorphin and its derivatives, which has simple operation, high yield, good product quality, good environmental compatibility, low carbon emissions, and is easy to scale up production.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a mutant of amino acid ligase, comprising:

[0012] Asparagine at position 7 is mutated to serine or glutamine; and / or

[0013] Lysine 8 is mutated to methionine or phenylalanine; and / or

[0014] Serine 77 is mutated to leucine or tryptophan; and / or

[0015] Tyrosine 79 is mutated to tryptophan or alanine; and / or

[0016] Mutation of glutamic acid at position 217 to tryptophan; and / or

[0017] Phenylalanine at position 220 is mutated to proline or threonine; and / or

[0018] Glycine at position 279 is mutated to threonine or asparagine; and / or

[0019] Isoleucine at position 281 was mutated to leucine or valine;

[0020] The Uniprot ID of the amino acid ligase is COSTU2.

[0021] In some specific embodiments of the present invention, the mutant further comprises:

[0022] The amino acid ligase has asparagine at position 7 mutated to serine, lysine at position 8 mutated to methionine, serine at position 77 mutated to leucine, tyrosine at position 79 mutated to tryptophan, glutamic acid at position 217 mutated to tryptophan, phenylalanine at position 220 mutated to proline, glycine at position 279 mutated to threonine, and isoleucine at position 281 mutated to leucine;

[0023] or

[0024] The asparagine at position 7 of the amino acid ligase is mutated to glutamine, the lysine at position 8 is mutated to phenylalanine, the serine at position 77 is mutated to tryptophan, the tyrosine at position 79 is mutated to alanine, the glutamic acid at position 217 is mutated to tryptophan, the phenylalanine at position 220 is mutated to threonine, the glycine at position 279 is mutated to asparagine, and the isoleucine at position 281 is mutated to valine.

[0025] In some specific embodiments of the present invention, the mutant has:

[0026] (1) the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3; or

[0027] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0028] (3) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2);

[0029] The plurality is 2 to 120.

[0030] The present invention also provides a nucleic acid molecule encoding the above mutant;

[0031] or with:

[0032] (4) the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6; or

[0033] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or

[0034] (6) a nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5);

[0035] The plurality is 2 to 360.

[0036] The present invention also provides an expression vector comprising the above nucleic acid molecule and an acceptable gene element;

[0037] The backbone of the expression vector includes pET-28a.

[0038] The present invention also provides a host cell, which comprises the above nucleic acid molecule or the above expression vector;

[0039] The host cell includes Escherichia coli BL21.

[0040] The present invention also provides a composition comprising the mutant and adenosine triphosphate.

[0041] In some specific embodiments of the present invention, the above composition further comprises polyphosphate kinase and polyphosphate;

[0042] The polyphosphoric acid includes hexaphosphate.

[0043] In some specific embodiments of the present invention, the polyphosphate kinase in the above composition has:

[0044] (7) the amino acid sequence shown in SEQ ID NO.1; or

[0045] (8) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (7), and having the same or similar function as (7); or

[0046] (9) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (7) or (8);

[0047] The plurality is 2 to 120.

[0048] The present invention also provides the use of any of the following in the synthesis of kyotomorphin and / or kyotomorphin derivatives:

[0049] (I), the above mutants;

[0050] (II), the above-mentioned nucleic acid molecule;

[0051] (III) the above-mentioned expression vector;

[0052] (IV), the above-mentioned host cell;

[0053] (V), the above composition;

[0054] The kyotomorphin derivatives include kyotomorphin amide.

[0055] The present invention also provides a method for preparing kyotomorphin or a kyotomorphin derivative, comprising:

[0056] (a) mixing the mutant and amino acids to obtain the kyotomorphin or the kyotomorphin derivative; or

[0057] (b) expressing the nucleic acid molecule, and mixing the obtained protein product with amino acids to obtain the kyotomorphin or the kyotomorphin derivative; or

[0058] (c) expressing the above expression vector, and mixing the obtained protein product with amino acids to obtain the kyotomorphin or the kyotomorphin derivative; or

[0059] (d) culturing the host cell, mixing the obtained protein product with amino acids to obtain the kyotomorphin or the kyotomorphin derivative; or

[0060] (e) mixing the above composition with amino acids to obtain the kyotomorphin or the kyotomorphin derivative;

[0061] The kyotomorphin derivatives include kyotomorphin amide;

[0062] The amino acids include L-tyrosine and / or L-arginine.

[0063] The preparation method and mutant of the Kyotomorphin of the present invention have the following effects:

[0064] The present invention utilizes inexpensive amino acids as raw materials to produce kyotomorphin and kyotomorphin amide in high yields under the catalytic action of equivalent amounts of adenosine triphosphate (ATP) and the corresponding amino acid ligase. Furthermore, to further save costs and reduce the amount of ATP used in the reaction system, an ATP recycling system can be employed. Overall, compared to traditional chemical preparation processes, the present invention offers a shorter preparation route, higher yields, better product quality, a high green index, and ease of large-scale production and application.

[0065] The present invention utilizes the corresponding amino acid ligase to directly connect two target amino acid raw materials and convert them into the corresponding dipeptide product in a single step in a buffer. The required adenosine triphosphate (ATP) can be an equivalent amount or a catalytic amount (via the use of an ATP regeneration system, such as polyphosphate kinase (PPK) and hexaphosphate). BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0067] Figure 1 The liquid phase detection HPLC of KTP (Tyr-Arg) produced by the reaction of Example 1 is shown; wherein, the upper diagram is 0h; the lower diagram is 2h;

[0068] Figure 2 shows the mass spectrum of KTP (Tyr-Arg);

[0069] Figure 3 Shows the KTP (Tyr-Arg) NMR spectrum;

[0070] Figure 4 Liquid chromatography HPLC of KTP-NH2 produced by the reaction of Example 2 is shown;

[0071] Figure 5 Shown is the mass spectrum of Tyr-Arg-NH2;

[0072] Figure 6 The liquid chromatography HPLC of the reaction to generate KTP-NH2 in Example 3 is shown;

[0073] Figure 7 The liquid chromatography HPLC of KTP-NH2 produced by the reaction of Example 4 is shown;

[0074] Figure 8 Shows the electrophoresis diagram of PPK, KTP-LigA and KTP-LigB; the leftmost lane is the marker. DETAILED DESCRIPTION

[0075] The present invention discloses methods for preparing kyotomorphin and its derivatives. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0076] The present invention utilizes the amino acid ligase in Bacillus subtilis as a template, and after mutational modification, it is able to ligate amino acids in one step to produce KTP and KTP-NH2. This enzyme preparation method offers numerous advantages in practical applications, including ease of operation, high yield, high product quality, good environmental compatibility, low carbon emissions, and ease of large-scale production.

[0077] Based on the information in the background art, the present invention uses COSTU2 as a template to provide two mutants with multiple mutations, LigA and LigB. These two mutants have high catalytic synthesis activity for Tyr-Arg-OH and Tyr-Arg-NH2. Through catalytic process optimization, the scaled-up production of the dipeptide was successfully achieved. The enzymatic preparation route of kyotomorphin (KTP) and kyotomorphin amide (KTP-NH2) of the present invention is as follows:

[0078]

[0079] This route utilizes inexpensive amino acids as raw materials, producing kyotomorphin and kyotomorphin amide in high yields under the catalytic action of equivalent amounts of adenosine triphosphate (ATP) and the corresponding amino acid ligase. Furthermore, to further save costs and reduce the amount of ATP used in the reaction system, an ATP recycling system can be employed. Overall, compared to traditional chemical preparation processes, this method offers a shorter preparation route, higher yields, better product quality, a high green index, and ease of scalable production and application.

[0080] The present invention utilizes the corresponding amino acid ligase to directly connect two target amino acid raw materials and convert them into the corresponding dipeptide product in a single step in a buffer. The required adenosine triphosphate (ATP) can be an equivalent amount or a catalytic amount (via the use of an ATP regeneration system, such as polyphosphate kinase (PPK) and hexaphosphate).

[0081] Related information about the enzymes involved in the present invention:

[0082] Amino acid ligase (KTP-LigA / KTP-LigB): both derived from Bacillus subtilis (Uniprot ID: COSTU2) parent;

[0083] The KTP-LigA enzyme undergoes mutations at the following amino acid positions in the parent enzyme:

[0084] N7S, K8M, S77L, Y79W, E217W, F220P, G279T, I281L.

[0085] The KTP-LigB enzyme was mutated at the following amino acid positions in the parent enzyme:

[0086] N7Q, K8F, S77W, Y79A, E217W, F220T, G279N, I281V.

[0087] Polyphosphate kinase (PPK): derived from Pseudomonas aeruginosa (Uniprot ID: Q9HYF1, EC 2.7.4.33).

[0088] The sequence information involved in the present invention is shown in Table 1 and Table 2:

[0089] Table 1

[0090]

[0091]

[0092] Table 2

[0093]

[0094]

[0095] Appendix: Enzyme fermentation production and enzyme immobilization methods:

[0096] Fermentation production of enzymes:

[0097] The enzymes required by the present invention are all synthesized by the company and then constructed on a specific expression plasmid and then produced by Escherichia coli fermentation; it specifically includes the following steps: after the gene corresponding to the above enzyme is sequence optimized, it is ordered to be synthesized by General Biological Company (Chuzhou, Anhui). Then the NdeI / XhoI restriction sites are introduced and subcloned into the pET28a expression vector. The plasmid with confirmed correct sequence is transferred into E. coli (BL21) competent cells for plate culture (Qingke Biological) and monoclonal small-scale liquid culture. The bacteria with correct protein expression are finally amplified in liquid culture step by step. It specifically includes transferring a single colony into 5mL of LB culture medium containing 50μM kanamycin (37℃) for culture. When the cells grow to the logarithmic phase, they are inoculated into 250mL of LB culture medium containing the same antibiotic. When they also grow to the logarithmic phase, they are transferred to a 5L culture fermenter for culture and final protein expression. In a 5L fermentor, when the cell OD value reached ~20, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added at 25°C to induce protein expression for 6 hours. Finally, the cells were harvested by high-speed centrifugation (4000 rpm, 20 minutes) to obtain 25-50 g of wet cells with overexpressed enzymes. A small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0) on ice. The cells were then disrupted by freeze-thaw, centrifuged at high speed to remove cell walls, and the supernatant was run on SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. Bacterial cells expressing the correct protein 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 (~10 g of wet cells: 200 mL of buffer). The cell walls were then disrupted by low-temperature high pressure. After high-speed centrifugation (16,000 rpm, 45 min), the cell walls were removed and the enzyme-containing supernatant was obtained for use (the resulting enzyme activity was 500-1000 U / mL, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature). LB medium was composed of: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate, and 5% glycerol.

[0098] Enzyme immobilization:

[0099] To the crude enzyme supernatant collected above, ammonium sulfate solid was slowly added until protein solids precipitated (20-60% w / v ammonium sulfate: buffer). The protein solids were collected by high-speed centrifugation (10,000 rpm, 10 minutes) and slowly dissolved in 25 mM Tris buffer (pH 8.0) (buffer A). The enzyme solution was then dialyzed against 50 volumes of buffer A (twice, 4 hours apart) to remove ammonium sulfate. Finally, the dialyzate was loaded onto a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column (NaCl gradient elution in buffer: 0-1N NaCl) to obtain pre-purified KTP-LigB and PPK enzyme solutions. The KTP-LigB and PPK enzymes were then immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at an activity unit ratio of 1: (1.5-3) using the following method: 2000 U of the purified enzyme mixture was dissolved in 1 L of 50 mM pH 8.0. 8.0 potassium phosphate solution (buffer B), followed by the addition of 20-60 mM phenoxyacetic acid and 400 g of LX-1000EP epoxy resin. After stirring at room temperature for 8 hours, the immobilized enzyme was filtered out and finally washed twice with water and buffer B, respectively, and stored at low temperature until use. The immobilized enzyme had an initial activity of 60-90%.

[0100] The protein gel used in the present invention is shown in the attached Figure 8 .

[0101] The amino acid ligase provided by the present invention, derived from Bacillus subtilis, can catalyze the condensation of tyrosine and arginine (or arginine amide) to produce kyotomorphin (KTP) and kyotomorphin amide (KTP-NH2). By subjecting the enzyme to site-directed mutagenesis of key amino acid residues in its catalytically active center, its catalytic activity is greatly enhanced, reducing the amount of enzyme used in the production process. This method significantly outperforms the traditional chemical synthesis of kyotomorphin. Therefore, the production process of the present invention exhibits outstanding advantages in terms of production cost, energy consumption, product quality, and green index.

[0102] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0103] The present invention will be further described below in conjunction with the embodiments:

[0104] Example 1: Preparation of Tyr-Arg-OH(KTP) using ligase (KTP-LigA)

[0105]

[0106] 9.05 g of L-tyrosine (50 mM), 9.57 g of L-arginine (55 mM), 27.5 g of adenosine triphosphate monosodium salt (ATPNa, 52 mM) and 50 mL of DMSO were added to 1 L of 100 mM Tris-HCl solution (pH 7.5). The pH of the reaction system was then adjusted to 7.5 with a NaOH aqueous solution. Ligase KTP-LigA1500U was then added to initiate the reaction. The reaction was stirred gently at 30°C while maintaining the pH of the reaction system between 6.5 and 8.5. After 2 hours, the L-arginine raw material was essentially reacted as detected by HPLC (see Figure 1 ). Then, the pH was adjusted to 1.0 with HCl aqueous solution to denature the enzyme in the reaction system and remove the precipitate by centrifugation. The pH of the reaction solution was then adjusted to 7.0 and directly loaded onto a D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 4:1, v:v) to obtain 14.5 grams of L-tyrosine-L-arginine (Tyr-Arg-OH) dipeptide (yield 86%). The sample was sent for mass spectrometry and nuclear magnetic resonance testing, which confirmed that the final product was Tyr-Arg-OH. The chromatographic results are shown in Figure 2 、 Figure 3 ; Among them, the signal attribution of the NMR spectrum is as follows:

[0107] 1 H NMR(400MHz, Deuterium Oxide)δ7.21–7.15(m,2H),6.91–6.85(m,2H),4.19(t,J=6.9Hz,1H),4.10(dd,J=7.7, 5.6Hz,1H),3.21–3.06(m,4H),1.84–1.66(m,2H),1.52(dqd,J=10.2,6.7,2.7Hz,2H).

[0108] Example 2: Preparation of Tyr-Arg-NH2 (KTP-NH2) using ligase (KTP-LigB)

[0109]

[0110] Similar to the above-mentioned preparation method of Tyr-Arg-OH, 9.05 g of L-tyrosine (50 mM), 9.51 g of L-arginine amide (55 mM), 27.5 g of adenosine triphosphate monosodium salt (ATP, 52 mM), and 50 mL of DMSO were added to 1 L of 100 mM Tris-HCl solution (pH 7.5). The pH of the reaction system was then adjusted to 7.5 with aqueous NaOH solution, and 1000 U of ligase KTP-LigB was added to initiate the reaction. The reaction was gently stirred at 30°C while maintaining the pH of the reaction system between 7.0 and 8.5. After 2 hours, the reaction of the L-tyrosine starting material was completely detected by HPLC. The liquid phase spectrum monitoring the reaction process is shown in FIG. Figure 4 . Then, the pH was adjusted to 1.0 with HCl aqueous solution to denature the enzyme in the reaction system and remove the precipitate by centrifugation. After the pH of the reaction solution was adjusted back to 7.0, it was directly loaded onto a D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 3:1, v:v) to obtain 15.5 grams of L-tyrosine-L-arginine amide (Tyr-Arg-NH2) dipeptide (yield 92%). The sample was sent for mass spectrometry and nuclear magnetic resonance, confirming that the obtained product was Tyr-Arg-NH2. The mass spectrum results are attached. Figure 5 ; The signal assignments of the NMR spectrum are as follows:

[0111] 1 H NMR(400MHz,Deuterium Oxide)δ7.19–7.12(d,J=8.0Hz,2H),6.88–6.80(d,J=7.6Hz,2H),4.24-4.20(t,J=6.4,6.8Hz,1H ),3.75-3.73(m,1H),3.63-3.49(m,2H),3.18-3.04(m,2H),1.85-1.65(m,2H),1.52-1.48(m,2H).

[0112] Example 3: Preparation of Tyr-Arg-NH2 (KTP-NH2) using ligase (KTP-LigB) and ATP regeneration system

[0113]

[0114] Similarly, 9.05 g of L-tyrosine (50 mM), 9.51 g of L-arginine amide (55 mM), 2.7 g of adenosine triphosphate monosodium salt (ATP, 5 mM), 10.2 g of sodium metabisphosphate (16.7 mM), and 50 mL of DMSO were added to 1 L of 100 mM Tris-HCl solution (pH 8.0). The pH of the solution was adjusted to 8.0, and then 3000 U of polyphosphate kinase PPK enzyme and 1200 U of ligase KTP-LigB were added. The reaction system was gently stirred at room temperature (25°C) and the pH of the reaction system was maintained between 7.0 and 9.0. After 3 hours, the L-tyrosine reaction was basically complete as detected by HPLC (see Figure 6 The pH was then adjusted to 1.0 with aqueous HCl to denature the enzyme in the reaction system and remove the precipitate by centrifugation. The reaction solution was then adjusted to a pH of 7.0 and directly loaded onto a D201 anion exchange resin to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 3:1, v:v) to obtain 15.0 g of L-tyrosine-L-arginine amide (Tyr-Arg-NH2) dipeptide (yield 89%).

[0115] Example 4: Preparation of Tyr-Arg-NH2 (KTP-NH2) using immobilized ligase (KTP-LigB) and PPK enzyme

[0116]

[0117] Similar to Example 3 above, 9.05 g of L-tyrosine (50 mM), 9.51 g of arginine amide (55 mM), 2.7 g of adenosine triphosphate monosodium salt (ATP, 5 mM), 10.2 g of sodium metabisphosphate (16.7 mM), and 50 mL of DMSO were added to 1 L of 100 mM Tris-HCl solution (pH 8.0); after adjusting the pH of the solution to 8.0, 2000 U of the immobilized KTP-LigB / PPK enzyme was added, and the reaction system was gently stirred at 30° C. while maintaining the pH of the reaction system between 7.5 and 9.0. After 3 hours, the reaction of the raw materials was basically complete as detected by HPLC (see Figure 7The immobilized KTP-LigB / PPK enzyme was then directly recovered by filtration and rinsed three times with 25 mM Tris pH 8.0 buffer for later use. The reaction solution was adjusted to pH 1.0 with aqueous HCl to precipitate protein, which was then removed by centrifugation. The pH of the reaction solution was then adjusted to pH 7.0 and directly loaded onto a D201 anion exchange resin column to remove adenosine diphosphate and free phosphate impurities. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 3:1, v:v) to yield 15.8 g of L-tyrosine-L-arginine amide (Tyr-Arg-NH2) dipeptide (94% yield).

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A mutant of an amino acid ligase, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that Encoding the mutant according to claim 1; The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.5 or SEQ ID NO.

6.

3. An expression vector, characterized in that Comprising the nucleic acid molecule according to claim 2, and an acceptable genetic element; The backbone of the expression vector includes pET-28a.

4. A host cell, characterized in that comprising the nucleic acid molecule according to claim 2 or the expression vector according to claim 3; The host cells include Escherichia coli BL21; The host cells are non-animal and plant species.

5. A composition, characterized in that The method comprises the mutant according to claim 1 and adenosine triphosphate.

6. The composition according to claim 5, wherein Also included are polyphosphate kinase and polyphosphate; The polyphosphoric acid includes hexaphosphate.

7. The composition according to claim 6, wherein The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO.

1.

8. Use of any of the following in the synthesis of kyotomorphin and / or kyotomorphin derivative Tyr-Arg-NH2: (I) The mutant according to claim 1; (II), the nucleic acid molecule according to claim 2; (III) the expression vector according to claim 3; (IV) The host cell according to claim 4; (V) The composition according to any one of claims 5 to 7.

9. A method for preparing kyotomorphin and / or a kyotomorphin derivative Tyr-Arg-NH2, characterized in that: include: (a) mixing the mutant according to claim 1 with amino acids to obtain the kyotomorphin and / or kyotomorphin derivative Tyr-Arg-NH2; or (b) expressing the nucleic acid molecule according to claim 2, and mixing the obtained protein product with amino acids to obtain the kyotomorphin and / or the kyotomorphin derivative Tyr-Arg-NH2; or (c) expressing the expression vector according to claim 3, and mixing the obtained protein product with amino acids to obtain the kyotomorphin and / or the kyotomorphin derivative Tyr-Arg-NH2; or (d) culturing the host cell according to claim 4, and mixing the obtained protein product with amino acids to obtain the kyotomorphin and / or the kyotomorphin derivative Tyr-Arg-NH2; or (e) mixing the composition according to any one of claims 5 to 7 with amino acids to obtain the kyotomorphin and / or the kyotomorphin derivative Tyr-Arg-NH2; When synthesizing the kyotomorphin, the amino acids are L-tyrosine and L-arginine; When synthesizing the kyotomorphin derivative, the amino acids are L-tyrosine and L-arginine amide.

Images