Mutant enzymes of serine kinase and sulfhydryl lyase and fermentation production method thereof and application in production of mimosaside

Abstract

The present application discloses mutant enzymes of serine kinase and sulfolyase and fermentation production methods thereof and applications in the production of mimosa, the amino acid sequence of serine kinase mutant enzyme (AhSerK) is shown in SEQ.ID.NO.1, and the amino acid sequence of sulfolyase mutant enzyme (Aphydry) is shown in SEQ.ID.NO.3. A method for producing mimosa, comprising the following steps: adding L-serine, 3,4-dihydroxypyridine, adenosine triphosphate disodium salt ATP and magnesium chloride hexahydrate to a buffer, and finally adding serine kinase mutant enzyme and sulfolyase mutant enzyme to start the reaction; the reaction is complete after at least 4 hours, and the reaction solution is adjusted to acidity to precipitate enzyme inactivation. The present invention, on the basis of the corresponding natural enzyme, obtains serine kinase mutant enzyme and sulfolyase mutant enzyme through directional transformation, improves activity and stability, so as to achieve higher conversion rate and large-scale application performance.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and in particular relates to mutant enzymes of serine kinase and sulfhydryl lyase and a fermentation production method thereof, and application of the mutant enzymes in the production of mimosaside. Background Art

[0002] Mimosa is a non-protein amino acid with chemical properties similar to tryptophan. It was first isolated from Mimosa pudica and was later found to be present in certain other plants and fungi. Mimosa alkaloids can chelate iron and copper and have been shown to reduce iron overload in animal models. Mimosa alkaloids can also inhibit certain iron- or copper-containing enzymes, including arginase, polyphenol oxidase, and dopamine hydroxylase. At the same time, mimosa alkaloids also inhibit the iron-containing enzyme deoxyhippurine hydroxylase, thereby preventing the synthesis of eukaryotic initiation factor 5A and blocking the cell cycle. Mimosa alkaloids have many physiological functions. In animal husbandry, mimosa alkaloids are used as industrial depilatories. In ruminants, mimosic acid is degraded into 3,4- and 2,3-dihydroxypyridones.

[0003] The more common methods for preparing mimosain today are natural plant separation and chemical synthesis. Leucaena leucophylla seeds contain 3% to 5% mimosain. In the early days, mimosain was obtained by separating and purifying extracts from these plants, but its preparation process was cumbersome and the yield was very low. Later, a chemical synthesis method was gradually formed to prepare mimosain in the laboratory. As early as 1962, Ian D. Spenser reported a method for chemically preparing mimosain, using 3-methoxy-4-pyrone as the starting material, and preparing mimosain through multi-step functional group protection, deprotection and condensation reactions. However, this preparation method has a long route, a low overall yield, and is not suitable for large-scale production. Summary of the invention

[0004] The purpose of the present invention is to provide mutant enzymes of serine kinase and sulfhydryl lyase and fermentation production methods thereof and application of the mutant enzymes in the production of mimosaic acid.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A serine kinase mutant enzyme (AhSerK), whose amino acid sequence is shown in SEQ.ID.NO.1 and whose encoding nucleotide sequence is shown in SEQ.ID.NO.2;

[0007] The serine kinase mutant enzyme is a wild-type serine kinase (Uniprot ID: H5V5G1) derived from Atlantibacter hermannii, with the following sites mutated: K71L, F118T, E202V, E204A, N205Q, S281M, D285L, Q461N, R465I. After mutation, the enzyme obtains relatively high activity and stability.

[0008] Sulfur lyase mutant enzyme (Aphydry), the amino acid sequence of which is shown in SEQ.ID.NO.3, and the encoding nucleotide sequence of which is shown in SEQ.ID.NO.4;

[0009] The sulfhydrylase mutant enzyme is a wild-type sulfhydrylase (Uniprot ID: Q9YBL2) from Aeropyrum pernix with mutations at the following sites: D62T, S125Q, R129N, V210I, T238S, A252V. After the mutation, the expression level, catalytic stability and catalytic activity of the enzyme are significantly improved.

[0010] The fermentation production method of serine kinase mutant enzyme and sulfhydrylase mutant enzyme comprises the following steps:

[0011] (1) Synthesize the nucleotide sequence shown in SEQ.ID.NO.2 or SEQ.ID.NO.4, then subclone into pET28a plasmid through NdeI / XhoI restriction sites, and transfer the plasmid into Escherichia coli cells for plate culture, and finally select a single clone for liquid step-by-step amplification culture;

[0012] (2) Transfer the single colony on the plate into LB culture medium containing at least 50 μM kanamycin for culture. When the cells grow to the logarithmic phase, inoculate them into LB culture medium containing the same antibiotic, and finally transfer them to a fermenter for culture. When the OD value of the fermentation liquid reaches 25, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to induce protein expression for more than 8 hours, and then collect the wet cells by centrifugation.

[0013] (3) The wet cells were mixed with the buffer solution, and then the cells were broken by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell wall, and finally the enzyme-containing clear solution was obtained;

[0014] The pH value of the buffer in step (3) is 7.0-9.0, preferably 50 mM Tris-HCl buffer.

[0015] The serine kinase mutant enzyme and / or the sulfhydryl lyase mutant enzyme can be used to produce mimosin.

[0016] A method for producing mimosa pudica comprises the following steps:

[0017] L-serine, 3,4-dihydroxypyridine, adenosine triphosphate disodium salt ATP and magnesium chloride hexahydrate are added to the buffer, the pH value of the reaction system is adjusted to 7.0-9.0, and finally the serine kinase mutant enzyme and the sulfur lyase mutant enzyme are added to start the reaction; the reaction is stirred slightly at 25-37° C., and the pH value of the reaction system is maintained at 7.0-9.0 during the entire reaction process. After at least 4 hours, the reaction is complete, and the reaction solution is adjusted to acidity to inactivate the enzyme and precipitate;

[0018] After the reaction is completed, the mimosaside solid is obtained by separation and purification.

[0019] The molar ratio of 3,4-dihydroxypyridine to L-serine is (1.2-1.8):1;

[0020] In the method, polyphosphate kinase can be added to achieve ATP cycle regeneration in the reaction system;

[0021] In the method, the enzyme can be immobilized and the multiple enzyme system can be recycled for multiple times, thereby further reducing the production price and improving the convenience of the production process.

[0022] The pH value of the buffer solution is 7.0 to 9.0; the buffer solution is preferably a Tris-HCl solution with a pH of 8.0;

[0023] The enzyme immobilization comprises the following steps:

[0024] Dissolve the serine kinase mutant enzyme, the sulfur lyase mutant enzyme, and the polyphosphate kinase in a buffer solution at an activity unit ratio of 1:(1.8-2.5):(2.5-4.0), then add phenoxyacetic acid and epoxy resin, stir at room temperature for at least 5 hours, filter out the immobilized enzyme, and finally wash with water and buffer solution three times each, and then dry for use;

[0025] In the method, the ratio of enzyme to epoxy resin is (5-20) U:1 g;

[0026] The pH value of the buffer solution is 8.0, preferably 50 mM potassium phosphate solution or 25 mM phosphate buffer;

[0027] The epoxy resin is preferably LX-1000EP epoxy resin.

[0028] The separation and purification comprises the following steps:

[0029] After the reaction, the precipitate is removed by centrifugation, the pH value of the solution is adjusted to neutral, and then the phosphoric acid impurities are removed by anion exchange resin. Then, the crude product is purified and collected by non-polar resin. Finally, the mimosa solid is obtained after desalination by reverse osmosis membrane, concentration and crystallization.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] 1. Based on the corresponding natural enzymes (wild enzymes), the present invention obtains serine kinase mutant enzymes and sulfhydrylase mutant enzymes through directional transformation, improves activity and stability, thereby achieving higher conversion rate and large-scale application performance.

[0032] 2. The present invention provides a kind of mimosa holoenzyme preparation process, and its overall process is to use L-serine and 3,4-dihydroxypyridine as raw materials, and directly obtain mimosa by two-step continuous conversion of serine kinase mutant enzyme and sulfide lyase mutant enzyme. This route can be completed in one step using crude enzyme solution, or recyclable immobilized enzyme can be used, and adenosine triphosphate (ATP) used in the first step of the reaction can also be recycled using corresponding enzymes to further reduce production costs. Therefore, this patent preparation process of mimosa has many advantages such as full green, simple process, cheap production and easy large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The SDS-PAGE gel detection images of the prepared enzymes are as follows: 1) AhSerK, 2) Aphydry, 3) SiPPK.

[0034] Figure 2 Mimosa 1 H-NMR spectrum. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0036] The information of the enzymes used in this patent is as follows:

[0037] Polyphosphate kinase (SiPPK) (Uniprot ID: K1KSJ6): Derived from Solibacillus isronensis, this enzyme can naturally use polyphosphate to convert ADP into ATP.

[0038] Example 1: Enzyme modification

[0039] Serine kinase mutant enzyme (AhSerK): The wild-type serine kinase (WTAhSerK) (Uniprot ID: H5V5G1) is derived from Atlantibacter hermannii. The enzyme tends to phosphorylate small molecular hydroxyls and has a certain initial activity for L-serine. Through systematic modification, the enzyme obtains relatively high activity and stability; its specific mutation sites are: K71L, F118T, E202V, E204A, N205Q, S281M, D285L, Q461N, R465I.

[0040] Sulfur lyase mutant enzyme (Aphydry): Wild-type sulfur lyase (WTAphydry) (Uniprot ID: Q9YBL2) is derived from the thermophilic archaeon (Aeropyrum pernix). The enzyme has a high ability to synthesize mimosacin, but there are some problems in expression. By modifying its sequence, we finally obtained a mutant with better expression level, catalytic stability and catalytic activity. The specific mutation sites are: D62T, S125Q, R129N, V210I, T238S, A252V.

[0041] The basic idea of ​​transformation is to achieve enzyme amino acid site replacement by rationally / randomly replacing the gene template base corresponding to the modified enzyme, and finally change the performance of the enzyme. In this patent, firstly, multiple sequence alignment of homologous proteins with the same function of H5V5G1 and Q9YBL2 enzymes is performed, and specifically no less than 100 homologous proteins are selected, and the overall sequence similarity is between 40% and 60%, and the conservative amino acid sites are determined based on this for subsequent mutation activity testing; at the same time, the structures of H5V5G1 (AF-H5V5G1-F1) and Q9YBL2 (PDB: 3VSD) are analyzed, and the Discovery Studio commercial software flexibly docks the ligands L-serine and O-phospho-L-serine into their structural cavities respectively, and finally determines the enzyme-substrate binding site through the association between the sites, so that fixed-point or combined site replacement activity tests can be performed; on this basis, the error-prone PCR technology is further used to randomly mutate the natural WTAhSerK / WTAphydry enzyme. Since this mutation method has a wide coverage, it can further locate the remote related sites to be modified. The entire experimental process needs to be combined with the enzyme's thermal stability test and quantitative characterization of activity, and finally successfully realizes the mutant enzyme AhSerK / Aphydry with significantly improved catalytic performance in all aspects.

[0042] Example 2: Fermentation production method of mutant enzyme

[0043] (1) The nucleotide sequence shown in SEQ.ID.NO.2 or SEQ.ID.NO.4 was synthesized, and then subcloned into the pET28a plasmid through the NdeI / XhoI restriction site, and the plasmid was transferred into E.coli (BL21) (Qingke Biotechnology) cells for plate culture, and finally a single clone was selected for liquid step-by-step amplification culture.

[0044] (2) First, transfer the single colony on the plate into 5 mL of LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells grow to the logarithmic phase, inoculate them into 250 mL of LB culture medium containing the same antibiotic, and finally transfer them to a 5 L culture fermenter for culture. When the OD value of the fermentation liquid is 25, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25°C to induce protein expression for 8 hours, and then centrifuge (4000 rpm, 15 min) to collect 20-35 g of wet cells.

[0045] (3) To verify the expression of the enzyme, a small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0), and then the cells were broken by freeze-thaw method. After high-speed centrifugation, the supernatant was run on SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) to determine the soluble expression of the protein; the results are as follows Figure 1 shown.

[0046] The remaining cells confirmed to be correct are first mixed with the buffer (10 grams of wet cells are mixed with about 200 mL of buffer), and then the cells are crushed by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell wall. The enzyme-containing clear liquid obtained can be used directly in subsequent use (the liquid enzyme activity is 200-450 U / ml, U is the amount of enzyme required to convert 1 μmol of substrate in one minute at room temperature) or further purified and immobilized for use (in solid enzyme reaction).

[0047] The LB medium consists of 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0048] Mixed immobilization of enzymes:

[0049] Ammonium sulfate solid was gradually added to the crude enzyme solutions of serine kinase (AhSerk), sulfhydrylase (Aphydry), and polyphosphate kinase (SiPPK) collected above until the enzyme precipitated (40%-60%, w / v ammonium sulfate / buffer). The enzyme solid was then collected by centrifugation (10000 rpm, 12 min), and slowly dissolved in 25 mM Tris buffer at pH 8.0, and finally desalted by G25 size exclusion chromatography column (purchased from Sigma) and purified by DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column was used to separate the pre-purified liquid enzymes AhSerK, Aphydry, and SiPPK, and the enzyme solution can be directly used for subsequent enzyme fixation. In the mixed fixation of AhSerk / Aphydry / SiPPK enzymes, the pre-purified enzymes were mixed and fixed using LX-1000EP epoxy resin (Xi'an Lanxiao Company) according to the activity unit of 1:2:3. The basic method of fixation is: 6000U mixed enzymes mixed according to the above activity unit ratio are dissolved in 2L 50mM pH 8.0 potassium phosphate solution, followed by adding 40mM phenoxyacetic acid and 600 grams of LX-1000EP epoxy resin to the buffer solution, and the immobilized enzymes are filtered out after stirring at room temperature for 6 hours, and finally washed three times with clean water and 25mM pH8.0 phosphate buffer, and then dried at low temperature for use; AhSerK / Aphydry / SiPPK / immobilized mixed enzymes have 75-90% activity of the corresponding liquid enzymes.

[0050] Example 3: Using L-serine and 3,4-dihydroxypyridine as raw materials, two liquid enzymes (AhSerK and Aphydry) were used to prepare mimosin at one time

[0051]

[0052] 10.4 g of L-serine (100 mM) was added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 16.6 g of 3,4-dihydroxypyridine (150 mM), 60.8 g of adenosine triphosphate disodium salt ATP (110 mM), and 4.2 g of magnesium chloride hexahydrate (20 mM), and then the pH value of the solution was adjusted back to 8.0, and finally 1000 U AhSerK crude enzyme solution and 2000 U Aphydry crude enzyme solution were added at once to start the reaction; the reaction was stirred lightly at 30°C, and the reaction pH was maintained at about 7.0-9.0 during the entire reaction process. After 4 hours, the reaction was completely adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the enzyme in the reaction and precipitate, and then the solution was adjusted to pH after the precipitate was removed by centrifugation. 7.0, D201 anion exchange resin was used to remove phosphoric acid impurities, and then D101 non-polar resin was used to purify and collect the crude product, and finally a reverse osmosis membrane was used to desalinate, concentrate, and crystallize (ethanol: H2O = 3:1, V:V) to obtain 13.3 g of white solid (final yield 67%).

[0053] Example 4: Using L-serine and 3,4-dihydroxypyridine as raw materials, three liquid enzymes (AhSerK, Aphydry, SiPPK) were used to prepare mimosa in one go

[0054]

[0055] Similar to Example 1, but SiPPK enzyme is added to the reaction solution to recycle ATP.

[0056] 20.8 g of L-serine (200 mM) was added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 27.8 g of 3,4-dihydroxypyridine (250 mM), 5.5 g of adenosine triphosphate disodium salt ATP (10 mM), 81.4 g of sodium hexaphosphate (133.4 mM), and 4.2 g of magnesium chloride hexahydrate (20 mM). The pH value of the solution was then adjusted back to 8.0. Finally, 1500 U AhSerk crude enzyme solution, 3000 U Aphydry crude enzyme solution, and 4000 U SiPPK crude enzyme solution were added at once to start the reaction. The reaction was stirred lightly at 30°C, and the pH of the reaction was maintained at about 7.0-9.0 during the entire reaction. After 5 hours, the reaction was completely adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the enzyme in the reaction, and then adjusted to pH 7.0, D201 anion exchange resin was used to remove phosphoric acid impurities, and then D101 non-polar resin was used to purify and collect the crude product. Finally, a reverse osmosis membrane was used to desalinate, concentrate, and crystallize (ethanol: H2O = 3:1, V:V) to obtain 32.8 g of white solid (final yield 83%).

[0057] Example 5: Using L-serine and 3,4-dihydroxypyridine as raw materials, immobilized mixed enzymes were used to prepare mimosin at one time

[0058]

[0059] The reaction is similar to the above embodiment, but the immobilized enzyme can be recovered and reused later.

[0060] 10.4 g of L-serine (100 mM) was added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 20.0 g of 3,4-dihydroxypyridine (180 mM), 5.5 g of adenosine triphosphate disodium salt ATP (10 mM), 40.7 g of sodium metahexaphosphate (66.7 mM), and 4.2 g of magnesium chloride hexahydrate (20 mM). The pH value of the solution was then adjusted back to 8.0, and finally 6000 U of immobilized mixed enzyme (prepared in Example 2) was added at once to start the reaction; the reaction was stirred lightly at 30° C., and the reaction pH was maintained at about 7.0-9.0 during the entire reaction process. After 10 hours, the reaction was complete and the immobilized mixed enzyme was collected by filtration (the mixed enzyme retained 90% of its initial activity after being used 5 times). The clear solution was then subjected to D201 anion exchange resin to remove phosphoric acid-containing impurities, and then purified using D101 non-polar resin to collect the crude product, which was finally desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 3:1, V:V) to obtain 17.6 g of a white solid (final yield 89%).

[0061] Comparative Example: Using L-serine and 3,4-dihydroxypyridine as raw materials, two natural liquid enzymes (WTAhSerK and WTAphydry) were used to prepare mimosadin

[0062]

[0063] The conditions were similar to those in Example 1 above, but the native enzyme (WT) was used.

[0064] 10.4 g of L-serine (100 mM) was added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris.HCl) solution, followed by 16.6 g of 3,4-dihydroxypyridine (150 mM), 60.8 g of adenosine triphosphate disodium salt ATP (110 mM), and 4.2 g of magnesium chloride hexahydrate (20 mM), and then the pH value of the solution was adjusted back to 8.0, and finally 5000 U of WTAhSerK crude enzyme solution and 3000 U of WTAphydry crude enzyme solution were added at once to start the reaction; the reaction was stirred lightly at 30°C, and the reaction pH was maintained at about 7.0-9.0 during the entire reaction process. After 12 hours, the reaction was completely adjusted to acidic with dilute hydrochloric acid aqueous solution to inactivate the enzyme in the reaction and precipitate, and the protein precipitate was removed by centrifugation, and then the solution was adjusted to pH 7.0, and then use D201 anion exchange resin to remove phosphoric acid impurities, and then use D101 non-polar resin to purify and collect the crude product, and finally use reverse osmosis membrane to desalinate, concentrate and crystallize (ethanol: H2O = 3:1, V:V) to obtain 1.8 grams of gray solid (final yield 9%).

[0065] The solid mimosapin obtained in each embodiment and comparative example was subjected to hydrogen nuclear magnetic resonance spectroscopy, with D2O as solvent and Varian 600 MHz nuclear magnetic resonance. The results are as follows: Figure 2 As shown, it is proved that the product is mimosaside.

[0066] Table 1: Performance comparison of the mutant enzyme of the present invention and its wild-type enzyme

[0067]

[0068] Note: The expression levels of the enzymes were determined after fermentation according to the method of Example 2.

[0069] It can be seen from the results of Table 1, the examples and the comparative examples that, compared with the corresponding wild-type enzymes, the activity, thermal stability and expression level of the mutant enzymes of the present invention are significantly improved; and when used for synthesizing mimosapin, the yield is greatly increased.

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A serine kinase mutant enzyme, characterized in that: The amino acid sequence is shown in SEQ.ID.NO.1, and the encoding nucleotide sequence is shown in SEQ.ID.NO.

2.

2. A sulfhydrylase mutant enzyme, characterized in that: The amino acid sequence is shown in SEQ.ID.NO.3, and the encoding nucleotide sequence is shown in SEQ.ID.NO.

4.

3. A fermentation method for producing a serine kinase mutant enzyme and a sulfhydrylase mutant enzyme, characterized in that The following steps are involved: (1) Synthesize the nucleotide sequence shown in SEQ.ID.NO.2 or SEQ.ID.NO.4, then subclone into pET28a plasmid through NdeI / XhoI restriction sites, and transfer the plasmid into Escherichia coli cells for plate culture, and finally select a single clone for liquid step-by-step amplification culture; (2) Transfer the single colony on the plate into LB culture medium containing at least 50 μM kanamycin for culture. When the cells grow to the logarithmic phase, inoculate them into LB culture medium containing the same antibiotic, and finally transfer them to a fermenter for culture. When the OD value of the fermentation liquid reaches 25, add 0.5 mM isopropyl-β-D-thiogalactopyranoside to induce protein expression for more than 8 hours, and then collect the wet cells by centrifugation. (3) The wet cells are mixed with the buffer solution, and then the cells are broken by high pressure and centrifuged at high speed to remove the cell walls, and finally the enzyme-containing clear solution is obtained.

4. Use of the serine kinase mutant enzyme according to claim 1 and / or the sulfhydrylase mutant enzyme according to claim 2 in the production of mimosapin.

5. A method for producing mimosaside, characterized in that The following steps are involved: L-serine, 3,4-dihydroxypyridine, adenosine triphosphate disodium salt ATP and magnesium chloride hexahydrate are added to the buffer, the pH value of the reaction system is adjusted to 7.0-9.0, and finally the serine kinase mutant enzyme according to claim 1 and the sulfur lyase mutant enzyme according to claim 2 are added to start the reaction; the reaction is slightly stirred at 25-37° C., and the pH value of the reaction system is maintained at 7.0-9.0 during the entire reaction process. The reaction is complete after at least 4 hours, and the reaction solution is adjusted to acidity to inactivate the enzyme and precipitate.

6. The method according to claim 5, characterized in that: The molar ratio of the 3,4-dihydroxypyridine to L-serine is (1.2-1.8):

1.

7. The method according to claim 5, characterized in that: Polyphosphate kinase was added at the same time as the serine kinase mutant enzyme and the sulfhydrylase mutant enzyme.

8. The method according to any one of claims 5 to 7, characterized in that: The enzyme is immobilized before use.

Images