Mutant enzyme and fermentation production method thereof and application in preparing buckwheat alkaloids

Abstract

The present invention discloses mutant enzymes and fermentation production methods thereof and applications in the preparation of buckwheat alkali. The amino acid sequence of the aminopropanol oxidase mutant enzyme is shown in SEQ.ID.NO.1, the amino acid sequence of the phosphofructoaldehyde zymolyase mutant enzyme is shown in SEQ.ID.NO.3, the amino acid sequence of the benzyl hydrolase mutant enzyme is shown in SEQ.ID.NO.5, and the amino acid sequence of the buckwheat alkali reductase mutant enzyme is shown in SEQ.ID.NO.7. Based on the natural enzyme, the present invention conducts a site-directed mutation test to improve its substrate catalytic activity; at the same time, an error-prone PCR high-throughput screening experiment is used to locate the remote site to be replaced, thereby guiding the experiment to improve the activity and stability of the enzyme. Finally, through the integration of the above aspects, the mutant enzyme SkADH / EcFSA / AlUR / SspIRED with improved performance in all aspects was successfully obtained.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and in particular relates to a mutant enzyme and a fermentation production method thereof and an application thereof in the preparation of buckwheat alkaloids. Background Art

[0002] Fagomine belongs to the piperidine class of compounds and is a naturally occurring polyhydroxyalkaloid found in plants such as buckwheat, mulberry leaves, and wolfberry. Fagomine is also an iminosugar that was originally isolated from buckwheat seeds and is present in the human diet, and is now available as a pure crystalline product. Fagomine can reduce postprandial blood sugar and regulate bacterial adhesion, so it can be used as a dietary supplement ingredient or functional food ingredient to reduce or excessive intake of rapidly digested carbohydrates or the overabundance of potential pathogens. Fagomine can induce changes in the composition and diversity of the intestinal microbiota, similar to those caused by dietary fiber and its anti-inflammatory and weight loss effects; animal experimental tests have shown that the use of fagomine alone or in combination with ω-3 PUFA can reduce the amount of fat accumulated in animals. In summary, fagomine, a natural iminosugar, can offset the short-term effects of a high-energy-density diet on body weight, fasting blood sugar levels, and the proportion of intestinal enterobacteria. Experiments also show that long-term supplementation with fagomine may delay the occurrence of other factors associated with metabolic syndrome.

[0003] Although buckwheat alkaloids are widely present in nature as an iminosugar, their production methods are still very limited. Common preparation methods today include natural product extraction and chemical synthesis. Since buckwheat alkaloids are not abundant in nature, the natural extraction method is very cumbersome, which makes it expensive and cannot be produced on a large scale. In chemical synthesis, since buckwheat alkaloids have three hydroxyl groups and one amino group, multiple steps of selective protection and deprotection are required during the preparation process, which results in a long preparation route, low final yield and low product quality. Summary of the invention

[0004] The object of the present invention is to provide aminopropanol oxidase mutant enzyme, fructose phosphoaldolase mutant enzyme, benzyl hydrolase mutant enzyme and buckwheat alkaloid reductase mutant enzyme, and fermentation production method and application of the same in preparing buckwheat alkaloid.

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

[0006] The aminopropanol oxidase (SkADH) mutant enzyme, 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 aminopropanol oxidase mutant enzyme of the present invention has a natural enzyme (WTSkADH, Uniprot ID: A0A7Y6NJA4, EC 1.1.1.47) derived from Schlegelella koreensis, and its initial activity is to oxidize alcohol sugar to aldose, and the test shows that it has weak aminopropanol oxidation activity. After a large number of mutations, the present invention has high activity for aminopropanol, and its mutation sites are: F17I, G78T, M112L, K113D, N141Q, P177V.

[0008] The mutant enzyme of fructose phosphoaldolase (EcFSA), whose amino acid sequence is shown in SEQ.ID.NO.3, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.4.

[0009] The fructose phosphoaldolase mutant enzyme, whose natural enzyme (WTEcFSA, Uniprot ID: P78055, EC 4.1.2.-) is derived from Escherichia coli (Escherichia Coli.), whose natural substrate is D-glucose-6-phosphate, and it also has a weak condensation ability of Cbz-3-aminopropanal and 1,3-dihydroxyacetone. After mutation modification, the condensation ability is significantly improved, and its mutation sites are: P21A, T89L, L119T, A129S, A165G, Q192R.

[0010] The benzyl hydrolase (AlUR) mutant enzyme, whose amino acid sequence is shown in SEQ.ID.NO.5, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.6.

[0011] The benzyl hydrolase mutant enzyme, whose natural enzyme (WTAlUR, Uniprot ID: A0A327ZJ48) is derived from Actinoplanes lutulentus. The enzyme naturally has a certain hydrolysis ability for benzyl. Using it as a template, its benzyl hydrolysis activity and stability are greatly improved through systematic modification. The final mutation sites are: K76L, V105G, D226N, S244T, W305M.

[0012] The buckwheat alkali reductase (SspIRED) mutant enzyme, whose amino acid sequence is shown in SEQ.ID.NO.7, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.8.

[0013] The buckwheat alkaloid reductase mutant enzyme, whose natural enzyme (WTSspIRED, Uniprot ID: M4ZS15) is derived from Streptomyces sp., is active on pyridine ring natural products and has weak reducing activity on buckwheat alkaloid. On this basis, through multi-site mutation, its activity and chiral selectivity are greatly improved. Its final mutation sites are: H27E, T30S, S37F, N60A, L149G, V151A, R158H, F176Y, W177L.

[0014] The fermentation production method of the mutant enzyme comprises the following steps:

[0015] Synthesize the nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4, SEQ.ID.NO.6 or SEQ.ID.NO.8, subclone it into pET28a plasmid through NdeI / XhoI restriction site, transfer the plasmid into Escherichia coli cells for plate culture, and finally select a single clone for liquid step-by-step amplification culture.

[0016] The step-by-step amplification culture comprises the following steps:

[0017] (1) First, transfer the single colony on the plate into 5 mL of LB culture medium containing 50 μM kanamycin 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 into a 5 L culture fermenter for culture. When the fermentation liquid OD 600 When the value reached 25, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression at 25°C for at least 10 h, and then the wet cells were collected by centrifugation (4000 rpm, 15 min);

[0018] (2) The wet cells were first mixed with Tris-HCl buffer (50 mM, pH 8.0), and then the cells were disrupted by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell wall, and finally a clear solution containing the mutant enzyme was obtained.

[0019] Use of one or more of the above mutant enzymes in the preparation of buckwheat alkaloids.

[0020] A method for preparing buckwheat alkaloids comprises the following steps:

[0021] Cbz-aminopropanol and nicotinamide adenine dinucleotide disodium salt (NAD +), adjusting the pH value of the reaction system to 7-9, adding aminopropanol oxidase mutant enzyme, phosphofructoyl aldolase mutant enzyme, benzyl hydrolase mutant enzyme and buckwheat alkaloid reductase mutant enzyme to start the reaction, the reaction is completed after at least 5 hours, and buckwheat alkaloid is obtained after separation and purification;

[0022] The activity unit ratio of the aminopropanol oxidase mutant enzyme, the fructose phosphoaldolase mutant enzyme, the benzyl hydrolase mutant enzyme and the buckwheat alkali reductase mutant enzyme is (1.2-1.8):(0.8-1.5):1:(1.0-2.0).

[0023] A method for preparing buckwheat alkaloids comprises the following steps:

[0024] (1) Add Cbz-aminopropanol, sodium pyruvate and nicotinamide adenine dinucleotide disodium salt (NAD + ), adjusting the pH value of the reaction system to 7-9, adding aminopropanol oxidase mutant enzyme and lactate dehydrogenase (LDH) to start the reaction, the reaction is completed after at least 3 hours, and Cbz-aminopropanaldehyde is obtained after separation and purification;

[0025] The molar ratio of Cbz-aminopropanol to sodium pyruvate is 1:(0.8-2.0);

[0026] The activity unit ratio of the aminopropanol oxidase mutant enzyme to lactate dehydrogenase is (1.5-3):1;

[0027] The separation and purification to obtain Cbz-aminopropionaldehyde comprises the following steps:

[0028] The pH value of the reaction solution was adjusted to below 1 to inactivate the enzyme, and then the pH value of the solution was adjusted back to 7.0 and centrifuged to remove solid impurities. The supernatant was separated and concentrated using Seplite AB-8 weak polar exchange resin to obtain Cbz-aminopropionaldehyde;

[0029] (2) Cbz-aminopropionaldehyde, sodium formate and reduced nicotinamide adenine dinucleotide disodium salt (NADH) are added to a buffer, the pH value of the reaction system is adjusted to 7-9, and a phosphofructoyl aldolase mutant, a benzyl hydrolase mutant, a buckwheat alkaloid reductase mutant and a formate dehydrogenase (FDH) are added to start the reaction. The reaction is completed after at least 6 hours, and buckwheat alkaloids are obtained after separation and purification;

[0030] The molar ratio of Cbz-aminopropionaldehyde to sodium formate is 1:(1.0-3.0);

[0031] The activity unit ratio of the fructose phosphoaldolase mutant enzyme, the benzyl hydrolase mutant enzyme, the buckwheat alkali reductase mutant enzyme and the formate dehydrogenase is (2.0-4.0):1:(1.5-3.0):(2.0-4.0).

[0032] In the above two methods for preparing buckwheat alkaloids:

[0033] The buffer solution has a pH value of 7 to 9, preferably a Tris.HCl solution with a pH value of 8.0.

[0034] Preferably, a cosolvent can be added at the same time as Cbz-aminopropanol or Cbz-aminopropanal to promote the dissolution of both; further preferably, the cosolvent is isopropanol; particularly preferably, the amount of isopropanol accounts for 10-20% of the volume of the reaction system.

[0035] The separation and purification to obtain buckwheat alkaloids comprises the following steps:

[0036] The pH value of the reaction solution is adjusted to below 1 to inactivate the enzyme, and then the pH value of the solution is adjusted back to 7.0 and centrifuged to remove solid impurities. The supernatant is analyzed and purified using D201 anion exchange resin to remove NAD and unreacted raw materials in the reaction to obtain crude buckwheat alkaloids. Finally, the crude product is desalted using a reverse osmosis membrane and concentrated and crystallized to obtain buckwheat alkaloids.

[0037] The enzyme can be used after being immobilized;

[0038] The immobilization of the mutant enzyme comprises the following steps:

[0039] (1) adding ammonium sulfate solid to the clear solution containing the mutant enzyme gradually until the enzyme precipitates, then slowly dissolving it in a buffer solution with a pH value of 7 to 9, and finally desalting it through a G25 size exclusion chromatography column, and separating the mutant enzyme using a DEAE Seplite FF anion exchange column;

[0040] (2) Dissolve one or more mutant enzymes in a buffer solution with a pH value of 7 to 9 according to the activity unit ratio of SkADH / EcFSA / AlUR / SspIRED mutant enzymes (1.2 to 1.8):(0.8 to 1.5):1:(1.0 to 2.0), then add phenoxyacetic acid and epoxy resin to the buffer solution, stir at room temperature for at least 10 hours, filter out the immobilized enzyme, and finally wash with clean water and a buffer solution with a pH value of 7 to 9.

[0041] Compared with the prior art, the present invention has the following advantages and effects:

[0042] 1. Based on the natural enzyme, the present invention conducts site-directed mutagenesis tests to improve its substrate catalytic activity; at the same time, error-prone PCR high-throughput screening experiments are used to locate the remote sites to be replaced, thereby guiding the experiment to improve the activity and stability of the enzyme. Finally, through the integration of the above aspects, the mutant enzyme SkADH / EcFSA / AlUR / SspIRED with improved performance in all aspects was successfully obtained.

[0043] 2. In the whole enzyme method for preparing buckwheat alkaloids provided by the present invention, the initial raw material used is cheap benzyloxycarbonyl (Cbz) protected aminopropanol, which can be converted into Cbz-aminopropanal by an alcohol oxidase ADH contained in Schlegel's body; a condensing enzyme EcFSA in Escherichia coli can then condense Cbz-aminopropanal with 1,3-dihydroxyacetone, and a UR enzyme in sludge actinomycetes can hydrolyze Cbz to achieve its deprotection, and finally an imine reductase in Streptomyces is used to reduce the final product buckwheat alkaloids. The buckwheat alkaloids biological method is very direct and efficient. It can be achieved in one step or in steps during the specific implementation process. The coenzyme NAD used in the conversion process + / NADH can be recycled by the regeneration system, thereby further reducing its production price. Therefore, the patented buckwheat alkaloid preparation process has many advantages such as being green, cheap, and easy to scale up. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a SDS-PAGE gel detection diagram of the mutant enzyme of the present invention; wherein, lane 1: SkADH mutant enzyme; lane 2: EcFSA mutant enzyme; lane 3: AlUR mutant enzyme; lane 4: SspIRED mutant enzyme.

[0045] Figure 2 It is buckwheat alkaloids 1 H-NMR spectrum; D2O as solvent, Varian 600MHz NMR. DETAILED DESCRIPTION

[0046] 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.

[0047] Example 1: Transformation and fermentation production of mutant enzymes

[0048] Enzyme modification:

[0049] The basic idea of ​​the modification of this patented enzyme is to achieve enzyme amino acid site replacement by rationally / randomly replacing the gene template bases corresponding to the natural enzyme, thereby ultimately changing the performance of the enzyme.

[0050] In the practice of this patent, we firstly conduct homologous multiple sequence alignment on the WTSkADH / WTEcFSA / WTAlUR / WTSspIRED enzymes, preliminarily determine the overall analysis of the conserved amino acid sites of the family, and then use the commercial software Discovery Studio to perform homologous modeling and ligand docking on the WTSkADH / WTEcFSA / WTAlUR / WTSspIRED enzymes, and finally determine the substrate binding site; under the guidance of the above two aspects, site-directed mutagenesis tests are conducted to improve the catalytic activity of the substrate; at the same time, error-prone PCR high-throughput screening experiments can locate the remote sites to be replaced, thereby guiding the experiment to improve the activity and stability of the enzyme. Finally, through the integration of the above aspects, the mutant enzyme SkADH / EcFSA / AlUR / SspIRED with improved performance in all aspects was successfully obtained.

[0051] The aminopropanol oxidase (SkADH) mutant enzyme, whose amino acid sequence is shown in SEQ.ID.NO.1, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.2.

[0052] The aminopropanol oxidase mutant enzyme of the present invention has a natural enzyme (WTSkADH, Uniprot ID: A0A7Y6NJA4, EC 1.1.1.47) derived from Schlegelella koreensis, and its initial activity is to oxidize alcohol sugar to aldose, and the test shows that it has weak aminopropanol oxidation activity. After a large number of mutations, the present invention has high activity for aminopropanol, and its mutation sites are: F17I, G78T, M112L, K113D, N141Q, P177V.

[0053] The mutant enzyme of fructose phosphoaldolase (EcFSA), whose amino acid sequence is shown in SEQ.ID.NO.3, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.4.

[0054] The fructose phosphoaldolase mutant enzyme, whose natural enzyme (WTEcFSA, Uniprot ID: P78055, EC 4.1.2.-) is derived from Escherichia coli (Escherichia Coli.), whose natural substrate is D-glucose-6-phosphate, and it also has a weak condensation ability of Cbz-3-aminopropanal and 1,3-dihydroxyacetone. After mutation modification, the condensation ability is significantly improved, and its mutation sites are: P21A, T89L, L119T, A129S, A165G, Q192R.

[0055] The benzyl hydrolase (AlUR) mutant enzyme, whose amino acid sequence is shown in SEQ.ID.NO.5, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.6.

[0056] The benzyl hydrolase mutant enzyme, whose natural enzyme (WTAlUR, Uniprot ID: A0A327ZJ48) is derived from Actinoplanes lutulentus. The enzyme naturally has a certain hydrolysis ability for benzyl. Using it as a template, its benzyl hydrolysis activity and stability are greatly improved through systematic modification. The final mutation sites are: K76L, V105G, D226N, S244T, W305M.

[0057] The buckwheat alkali reductase (SspIRED) mutant enzyme, whose amino acid sequence is shown in SEQ.ID.NO.7, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.8.

[0058] The buckwheat alkaloid reductase mutant enzyme, whose natural enzyme (WTSspIRED, Uniprot ID: M4ZS15) is derived from Streptomyces sp., is active on pyridine ring natural products and has weak reducing activity on buckwheat alkaloid. On this basis, through multi-site mutation, its activity and chiral selectivity are greatly improved. Its final mutation sites are: H27E, T30S, S37F, N60A, L149G, V151A, R158H, F176Y, W177L.

[0059] The fermentation production of the mutant enzyme comprises the following steps:

[0060] The nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4, SEQ.ID.NO.6 or SEQ.ID.NO.8 was synthesized, subcloned into the pET28a plasmid through the NdeI / XhoI restriction site, and the plasmid was transferred into E.coli (BL21) cells for plate culture. Finally, a single clone was selected for liquid step-by-step amplification culture.

[0061] The following is the basic process of cell amplification culture. First, transfer the single colony on the plate into 5mL LB culture medium containing 50μM kanamycin (37℃) for culture. When the cells grow to the logarithmic phase, inoculate them into 250mL LB culture medium containing the same antibiotics, and finally transfer them to a 5L culture fermenter for culture. When the cell OD is ~25, add 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25℃ to induce protein expression for 12 hours, and then centrifuge (4000rpm, 15min) to collect 40-60 grams of wet cells. To verify the expression of the enzyme, first take a small amount of cells and mix them evenly with tris-hydroxymethylaminomethane hydrochloric acid (Tris.HCl) buffer (50mM, pH 8.0), then use the freeze-thaw method to break the cells, and take the supernatant after high-speed centrifugation to run SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) to determine the soluble expression of the protein ( Figure 1 ); 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 the above buffer), and then the cells are crushed by high pressure and centrifuged at high speed (16000rpm, 45min) to remove the cell wall. The enzyme-containing clear liquid obtained at last can be used directly in subsequent use (the liquid enzyme activity is 450-600U / mL, U is the amount of enzyme required to convert 1μmol of substrate in one minute at room temperature) or further purified and immobilized for use (in case of solid enzyme reaction).

[0062] LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0063] Example 2

[0064] Step 1: Using Cbz-aminopropanol as raw material, liquid enzyme (SkADH, LDH) two-step conversion to prepare Cbz-aminopropanal

[0065]

[0066] 20.9 g of Cbz-aminopropanol (100 mM), 13.2 g of sodium pyruvate (120 mM), 150 mL of isopropanol and 1.42 g of oxidized nicotinamide adenine dinucleotide disodium NAD+ (2 mM) were added to 1 L of 50 mM Tris.HCl solution at pH 8.0. The pH value of the reaction solution was adjusted to 8.0, and then 2000U SkADH crude enzyme solution and 1000U LDH were added to start the reaction. The reaction solution was slowly stirred at room temperature for 3 hours, and then a dilute HCl aqueous solution was added to adjust the pH value of the reaction solution to 1.0 to denature the SkADH and LDH enzymes and terminate the reaction. The solution pH was then adjusted back to 7.0 and centrifuged at 12000 rpm for 15 minutes to remove solid impurities. The supernatant was separated and concentrated using Seplite AB-8 (Xi'an Lanxiao Technology New Materials Co., Ltd.) weak polar exchange resin to obtain 17.3 g of Cbz-aminopropionaldehyde off-white solid (yield 83%).

[0067] Step 2: Using Cbz-aminopropionaldehyde as raw material, liquid enzyme (EcFSA, AlUR, SspIRED, FDH) four-step transformation to prepare buckwheat alkaloids

[0068]

[0069] Similar to the conditions in the above example, 20.9 g of Cbz-aminopropanal (100 mM) prepared in the first step, 7.48 g of sodium formate (110 mM), 150 mL of isopropanol and 1.42 g of reduced nicotinamide adenine dinucleotide disodium salt NADH (2 mM) were added to 1 L of 50 mM pH 8.0 tris-hydroxymethylaminomethane hydrochloric acid (Tris.HCl) solution, and then the pH value of the solution was adjusted back to 8.0; before the formal reaction, 3000 U of EcFSA, 1000 U of AlUR, 2000 U of SspIRED crude enzyme solution and 3000 U of The reaction was started by FDH enzyme, and the reaction solution was slowly stirred at room temperature for 6 hours, and then a dilute HCl aqueous solution was added to adjust the pH value of the reaction solution to 1.0 to denature EcFSA, AlUR, SspIRED, and FDH enzymes and terminate the reaction. The solution pH was then adjusted back to 7.0 and centrifuged to remove solid impurities. The supernatant was analyzed and purified using D201 anion exchange resin to remove NAD and unreacted raw materials in the reaction to obtain a crude product of buckwheat alkaloids. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=2:1, V:V) to obtain 11.2 g of buckwheat alkaloids as a white solid ( Figure 1 , 76% yield).

[0070] Example 3

[0071] Using Cbz-aminopropanol as raw material, liquid enzyme (SkADH, EcFSA, AlUR, SspIRED) four-step enzyme conversion of buckwheat alkaloids

[0072]

[0073] The reaction conditions are similar to those in the above example, but Cbz-aminopropanol is used as the raw material, and no LDH / FDH enzyme is added to regenerate NAD + / NADH.

[0074] Add 20.9 g of Cbz-aminopropanol (100 mM), 150 mL of isopropanol and 1.42 g of nicotinamide adenine dinucleotide disodium salt NAD to 1 L of 50 mM Tris-HCl solution at pH 8.0. + (2mM). The pH value of the reaction solution was adjusted to 8.0, and then 3000U SkADH, 2000UEcFSA, 2000U AlUR and 3000U SspIRED crude enzyme solution were added to start the reaction. The reaction solution was slowly stirred at room temperature for 5 hours, and a dilute HCl aqueous solution was added to adjust the pH value of the reaction solution to 1.0 to denature and precipitate the enzyme used in the reaction solution and terminate the reaction. The solution pH was then adjusted back to 7.0 and centrifuged to remove solid impurities. The supernatant was analyzed and purified using D201 anion exchange resin to remove NAD and unreacted raw materials in the reaction to obtain a crude product of buckwheat alkaloids. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 2:1, V:V) to obtain 13.4 grams of a white solid (91% yield).

[0075] Example 4

[0076] Using Cbz-aminopropanol as raw material, the immobilized mixed enzyme was used to convert buckwheat alkaloids into buckwheat alkaloids in one step.

[0077]

[0078] The reaction is similar to that of Example 3, but an immobilized mixed enzyme is used for the conversion, and the enzyme can be recycled after the reaction.

[0079] Add 20.9 g of Cbz-aminopropanol (100 mM), 150 mL of isopropanol and 1.42 g of nicotinamide adenine dinucleotide disodium salt NAD to 1 L of 50 mM Tris-HCl solution at pH 8.0. +(2mM). The pH value of the reaction solution was adjusted to 8.0 and 10000U of immobilized mixed enzyme was added to start the reaction. The reaction solution was slowly stirred at room temperature for 10 hours, and then the immobilized enzyme was filtered to recover and terminate the reaction (the immobilized mixed enzyme still retained 78% of the initial activity after being used 10 times). The filtered clear liquid was analyzed and purified using D201 anion exchange resin to remove NAD and unreacted raw materials in the reaction to obtain a crude product of buckwheat alkaloids. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 2:1, V:V) to obtain 12.8 grams of white solid (87% yield).

[0080] The mixed immobilization of enzymes includes the following steps:

[0081] Solid ammonium sulfate was gradually added to the clear solution containing aminopropanol oxidase (SkADH) mutant enzyme, phosphofructoacetic acid aldolase (EcFSA) mutant enzyme, benzyl hydrolase (AlUR) mutant enzyme, and buckwheat alkali reductase (SspIRED) mutant enzyme collected in Example 1 until the enzyme precipitated (40%-60%, w / v ammonium sulfate / buffer). The enzyme solid was then collected by centrifugation (10000rpm, 12min), and slowly dissolved in 25mM Tris buffer at pH 8.0, and finally desalted by G25 size exclusion chromatography column (purchased from Sigma) and separated by DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain pre-purified liquid enzymes SkADH, EcFSA, AlUR, and SspIRED, and the enzyme solution can be directly used for subsequent enzyme immobilization.

[0082] In the mixed immobilization of the SkADH / EcFSA / AlUR / SspIRED mutant enzyme, the pre-purified enzyme was mixed and immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) according to the activity unit ratio of 3:2:2:3. The basic method of fixation is: 10000U mixed enzyme mixed according to the above activity unit ratio is dissolved in 4L 50mM pH8.0 potassium phosphate solution, then 50mM phenoxyacetic acid and 1000 grams of LX-1000EP epoxy resin are added to the buffer solution, and the immobilized enzyme is filtered out after stirring at room temperature for 12 hours, and finally washed three times with clean water and 25mM pH8.0 phosphate buffer, and then dried at low temperature for use; the SkADH / EcFSA / AlUR / SspIRED immobilized mixed enzyme has 81-87% of the activity of the corresponding liquid enzyme.

[0083] Comparative Example

[0084] Using Cbz-aminopropanol as raw material, natural liquid enzymes (WTSkADH, WTEcFSA, WTAlUR, WTSspIRED) were used to convert buckwheat alkaloids in one step.

[0085] Similar to the above Example 3, the high-activity mutant enzyme was replaced by the low-activity natural enzyme throughout the reaction, and 1.6 g of gray solid was finally obtained (11% yield).

[0086] The performance comparison of the mutant enzyme involved in the present invention and its corresponding natural enzyme is as follows:

[0087] Table 1: Performance comparison of the mutant enzyme of the present invention and its natural enzyme

[0088]

[0089] It can be seen from Table 1 that, compared with the natural enzyme, the mutant enzyme obtained after modification has obvious improvements in activity, stability and expression level.

[0090] From the comparison between Example 3 and the comparative example, it can be seen that the yield of buckwheat alkaloids prepared by using the mutant enzyme is significantly improved.

[0091] 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 mutant enzyme of aminopropanol oxidase, characterized in that The amino acid sequence is shown in SEQ.ID.NO.

1.

2. A phosphofructoacetic acid aldolase mutant enzyme, characterized in that The amino acid sequence is shown in SEQ.ID.NO.

3.

3. A benzyl hydrolase mutant enzyme characterized in that The amino acid sequence is shown in SEQ.ID.NO.

5.

4. A buckwheat alkaloid reductase mutant enzyme, characterized in that The amino acid sequence is shown in SEQ.ID.NO.

7.

5. The fermentation production method of the mutant enzyme according to any one of claims 1 to 4, characterized in that The following steps are involved: Synthesize the nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4, SEQ.ID.NO.6 or SEQ.ID.NO.8, subclone it into pET28a plasmid through NdeI / XhoI restriction site, transfer the plasmid into Escherichia coli cells for plate culture, and finally select a single clone for liquid step-by-step amplification culture.

6. Use of the mutant enzyme according to any one of claims 1 to 4 in the preparation of buckwheat alkaloids.

7. A method for preparing buckwheat alkaloids, characterized in that The following steps are involved: Cbz-aminopropanol and nicotinamide adenine dinucleotide disodium salt are added to the buffer, the pH value of the reaction system is adjusted to 7-9, the aminopropanol oxidase mutant enzyme according to claim 1, the phosphofructoyl aldolase mutant enzyme according to claim 2, the benzyl hydrolase mutant enzyme according to claim 3 and the buckwheat alkaloid reductase mutant enzyme according to claim 4 are added to start the reaction, the reaction is completed after at least 5 hours, and buckwheat alkaloid is obtained after separation and purification.

8. A method for preparing buckwheat alkaloids, characterized in that The following steps are involved: (1) adding Cbz-aminopropanol, sodium pyruvate and nicotinamide adenine dinucleotide disodium salt to a buffer, adjusting the pH value of the reaction system to 7-9, adding the aminopropanol oxidase mutant enzyme and lactate dehydrogenase described in claim 1 to start the reaction, and completing the reaction after at least 3 hours, and obtaining Cbz-aminopropanaldehyde after separation and purification; (2) Cbz-aminopropionaldehyde, sodium formate and reduced nicotinamide adenine dinucleotide disodium salt are added to a buffer solution, the pH value of the reaction system is adjusted to 7-9, the phosphofructoyl aldolase mutant enzyme described in claim 2, the benzyl hydrolase mutant enzyme described in claim 3, the buckwheat alkaloid reductase mutant enzyme described in claim 4 and formate dehydrogenase are added to start the reaction, the reaction is completed after at least 6 hours, and buckwheat alkaloid is obtained after separation and purification.

9. The method according to claim 7 or 8, characterized in that: While adding the Cbz-aminopropanol or Cbz-aminopropionaldehyde, add the co-solvent.

10. The method according to claim 7 or 8, characterized in that: The enzyme is used after being immobilized.

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