Enzyme composition and its application in preparing allylcysteine

Abstract

The present invention relates to the field of enzyme engineering technology, and in particular to an enzyme composition and its application in the preparation of allylcysteine. The present invention provides an efficient allylcysteine holoenzymatic synthesis process, wherein the overall process uses allyl benzoate and glutathione as raw materials, and directly obtains the target product through three consecutive reactions of thioester ligase catalytic coupling, metal-dependent hydrolase directional cleavage, and amidohydrolase demodification. The process can use a crude enzyme liquid system to complete the conversion in one step, or can use an immobilized enzyme to achieve the recycling of the enzyme catalyst. The coenzyme involved in the reaction can be regenerated in situ by a cascade regeneration system, further reducing material costs. Therefore, the allylcysteine production process developed by the present invention has the comprehensive advantages of a simple reaction path, mild conditions, high product purity, and easy industrial scale-up.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, in particular to an enzyme composition and application thereof in the preparation of allylcysteine. Background Art

[0002] Allylcysteine is a sulfur-containing amino acid derivative with a chemical structure similar to cysteine but with an allyl side chain. It was first discovered in garlic and other lily plants, and subsequent studies have shown its presence in onions, leeks, and other plants. This substance possesses a unique thioether bond, forming stable complexes with heavy metal ions (such as mercury and lead). It can inhibit the activity of enzymes containing metal cofactors, such as cytochrome oxidase, affecting mitochondrial energy metabolism. It also regulates the glutathione peroxidase system, enhancing cellular antioxidant capacity. Physiologically, allylcysteine exhibits antihypertensive, antithrombotic, and lipid metabolism-regulating properties. It is used as a natural antioxidant in the food industry and as a feed additive in the livestock industry to improve cardiovascular health. Its metabolite, allylthiol, can be further converted into organosulfur compounds with antimicrobial activity in vivo.

[0003] The preparation methods of allylcysteine mainly include natural extraction and chemical synthesis. The natural extraction method was initially based on ion exchange chromatography separation of aqueous extracts from lily plants such as garlic and onion. However, it was limited by the low content of active ingredients in the raw materials (only 0.2%-0.5%), the cumbersome separation steps, and the easy oxidation of the product, resulting in a yield of less than 40%. The chemical synthesis method uses 3-bromopropene and L-cysteine as raw materials and synthesizes it in a one-step nucleophilic substitution reaction under alkaline conditions. This method has a yield of 70.8% and a purity exceeding 98%, but it has problems such as the high toxicity of bromopropene and the strict control of reaction conditions. In recent years, an electrolytic reduction method using cystine as a precursor has been developed. The intermediate is obtained through metal membrane concentration and FeCl3 oxidation, and then the target product is obtained through electrolytic reduction. The yield has been increased to 92.5%, but it requires multiple purification steps and has high equipment costs. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an enzyme composition and its application in the preparation of allyl cysteine. The present invention provides a method for synthesizing the target product using allyl benzoate and glutathione as raw materials through a three-step enzymatic reaction of allyl glutathione synthetase-catalyzed coupling, short peptide hydrolase-directed cleavage, and deglutaminase modification. The reaction conditions are mild, no by-products are generated, the enzyme system can be recycled, and the product purity is excellent, which is suitable for industrial production.

[0005] The present invention provides a mutant of an enzyme comprising at least one of allyl glutathione synthetase, short peptide hydrolase and deglutaminase, wherein:

[0006] The allyl glutathione synthetase is derived from Gossypium anomalum, and the mutation sites of the allyl glutathione synthetase include at least one of L11G, K12N, P25D, P64V, Q74E, M89I, W100N, F101L, D122Q, E176F, N206I, E238N and K245D;

[0007] The short peptide hydrolase is derived from the fungus Xylariales sp., and the mutation sites of the short peptide hydrolase include at least one of D35K, E113I, P114S, R128D, V259I, T330D and R413W;

[0008] The deglutaminase is derived from Thermoplasmatales archaeon, and the mutation sites of the deglutaminase include at least one of S21L, S22T, R65V, L84Y, E102D, S201I, D226V, K318N, H390Q and Q426C.

[0009] In some embodiments, the enzymes include allylglutathione synthetase, short peptide hydrolase, and deglutaminase.

[0010] In some embodiments, the enzyme mutant comprises at least one of a mutant of allyl glutathione synthetase, a mutant of short peptide hydrolase, and a mutant of deglutaminase, wherein:

[0011] The mutant of the allyl glutathione synthetase has an amino acid sequence as shown in SEQ ID NO: 1, the mutant of the short peptide hydrolase has an amino acid sequence as shown in SEQ ID NO: 2, and the mutant of the deglutaminase has an amino acid sequence as shown in SEQ ID NO: 3.

[0012] The present invention provides an enzyme composition or an immobilized enzyme thereof, including mutants of the enzyme.

[0013] The present invention provides a nucleic acid molecule encoding at least one of the following (1) to (3):

[0014] (1) A mutant of the enzyme;

[0015] (2) The enzyme composition or the immobilized enzyme thereof.

[0016] In some embodiments, the present invention comprises a nucleic acid molecule encoding a mutant of allyl glutathione synthetase, a nucleic acid molecule encoding a mutant of short peptide hydrolase, and a nucleic acid molecule encoding a mutant of deglutaminase, wherein:

[0017] The nucleic acid molecule encoding the mutant of allyl glutathione synthetase has a nucleotide sequence as shown in SEQ ID NO: 4, the nucleic acid molecule encoding the mutant of short peptide hydrolase has a nucleotide sequence as shown in SEQ ID NO: 5, and the nucleic acid molecule encoding the mutant of deglutaminase has a nucleotide sequence as shown in SEQ ID NO: 6.

[0018] The present invention provides an expression vector or a host cell, wherein the expression vector comprises the nucleic acid molecule;

[0019] The host cell is transfected or transformed with the expression vector.

[0020] The present invention provides the use of at least one of the following ① to ④ in the preparation of allyl cysteine:

[0021] 1. A mutant of the enzyme;

[0022] ②. The enzyme composition or immobilized enzyme thereof;

[0023] ③. the nucleic acid molecule;

[0024] ④. The expression vector or host cell.

[0025] The present invention provides a method for preparing allyl cysteine, which uses allyl benzoate and glutathione as raw materials and undergoes at least one of the following conversions I to IV to obtain allyl cysteine;

[0026] Ⅰ. mutants of the enzyme;

[0027] II. the enzyme composition or the immobilized enzyme thereof;

[0028] III. the nucleic acid molecule;

[0029] IV. The expression vector or host cell.

[0030] In some embodiments, the preparation method includes step-by-step preparation or one-time preparation:

[0031] The step-by-step preparation comprises:

[0032] Allyl benzoate and glutathione are used as raw materials, and allyl glutathione is produced by catalysis of the mutant allyl glutathione synthetase;

[0033] The allyl glutathione is catalyzed by the mutant of the short peptide hydrolase to generate allyl glutamine cysteine;

[0034] The allyl glutamine cysteine is catalyzed by the mutant of the deglutaminase to generate allyl cysteine.

[0035] The one-time preparation comprises taking allyl benzoate, glutathione, a mutant of allyl glutathione synthetase, a mutant of short peptide hydrolase and a mutant of deglutaminase, and mixing them to obtain allyl cysteine.

[0036] In some embodiments, the ratio of allyl benzoate, glutathione, mutant of allyl glutathione synthetase, mutant of short peptide hydrolase and mutant of deglutaminase is (80~110mM): (90~120mM): (3000~5000U): (1500~2500U): (1500~2500U).

[0037] In some specific embodiments, the ratio of allyl benzoate, glutathione, the mutant of allyl glutathione synthetase, the mutant of short peptide hydrolase and the mutant of deglutaminase is 100mM:110mM:4000U:2000U:2000U.

[0038] The present invention provides an efficient holoenzymatic synthesis process for allylcysteine. The overall process uses allyl benzoate and glutathione as raw materials, and directly obtains the target product through three consecutive reactions: thioester ligase-catalyzed coupling, metal-dependent hydrolase-directed cleavage, and amidohydrolase demodification. This process can use a crude enzyme solution system to complete the conversion in one step, and can also use immobilized enzymes to achieve the recycling of enzyme catalysts. The coenzymes involved in the reaction can be regenerated in situ through a cascade regeneration system, further reducing material costs. Therefore, the allylcysteine production process developed by the present invention has comprehensive advantages such as a simple reaction path, mild conditions, high product purity, and ease of industrial scale-up. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 1 is a graph showing an SDS-PAGE gel assay of the enzyme prepared by the present invention, wherein M is a protein standard marker, 1 is TaGlyHydro, 2 is GaAGSyn, and 3 is Xspetidase;

[0040] Figure 2 The synthetic reaction formula of Example 2 is shown;

[0041] Figure 3 The synthetic reaction formula of Example 3 is shown;

[0042] Figure 4 The synthetic reaction formula of Example 4 is shown;

[0043] Figure 5 The synthetic reaction formula of Example 5 is shown;

[0044] Figure 6 The allyl cysteine prepared in Example 5 is shown in FIG. 1H-NMR, D2O as solvent, Varian 600 MHz NMR;

[0045] Figure 7 The mass spectrometry results of allylcysteine prepared in Example 5 are shown, Agilent Ultivo LC / TQ;

[0046] Figure 8 The HPLC detection spectrum of allylcysteine is shown, wherein the column is Water XBridge-C18 (4.6mmX 250mm), 5uM, 215nm, mobile phase is acetonitrile / water (0.1% phosphoric acid), flow rate is 1.0ml / min;

[0047] Figure 9 The synthetic reaction formula of Example 6 is shown;

[0048] Figure 10 The synthetic reaction formula of the comparative example is shown. DETAILED DESCRIPTION

[0049] The present invention provides an enzyme composition and its application in the preparation of allylcysteine. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0050] This invention uses allyl benzoate and glutathione as raw materials. Allyl glutathione synthetase (a thioester ligase) catalyzes the formation of an allyl glutathione intermediate. Subsequently, a short peptide hydrolase (a member of the metal-dependent hydrolase family pfam01443) specifically cleaves the γ-glutamyl bond to form an allylcysteine-glutamate complex. Finally, deglutaminase (an amidohydrolase) catalyzes the removal of the glutamate group to yield allylcysteine. Through directed evolution, each of these enzymes has significantly improved their catalytic efficiency and reaction specificity for the target substrate, making them suitable for large-scale catalytic production.

[0051] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.

[0052] Example 1 Preparation of three liquid enzymes

[0053] This example provides three liquid enzymes, including allylglutathione synthetase (GaAGSyn), deglycinase (Xspetidase), and deglutaminase (TaGluhydro).

[0054] 1. Specific information of enzyme

[0055] Allyl glutathione synthetase (GaAGSyn): Derived from Gossypium anomalum (Uniprot ID: A0A8J5YM71), the native enzyme (WTGaAGSyn) has a relatively broad substrate catalysis range, with weak activity towards the substrate allyl-benzoic acid, and its expression is also suboptimal. Through comprehensive modification, the activity and expression level of the mutant enzyme (GaAGSyn) have been improved. The specific mutation sites are: L11G, K12N, P25D, P64V, Q74E, M89I, W100N, F101L, D122Q, E176F, N206I, E238N, K245D.

[0056] Amino acid sequence of GaAGSyn:

[0057] MSTAVALSSSSGNHNLALGIRFPQNDSFYRRNGPKVSTFRTFTVAMAAINTTPLEICVKASVTTVNKLGDCPFCERVLLTMEEKHLPYEIKLVDLSNKPENLLRISPEGKVPVVKFDEKWVPQSDVIAQSLEEKYPDPPLVTPQEKA LVGSKIFSTFIGFLKSKDPSDGTEQALLDFLSSFNDYIKENVRPLSGSLNVLRFVYSLHIRFILFSLKGPFINGEKISAADLSLAPKLYHLNIALGHYDKWSIPDTLPYTKSYMKTIFSMDSFTKTRASPDDVIAGWRPKVMG (SEQ IDNO:1)

[0058] The nucleotide sequence of GaAGSyn is:

[0059] atgagcaccgcggtggcgctgagcagcagcggcaaccataacctggcgctgggcattcgctttccgcagaacgatagcttttatcgccgcaacggcccgaaagtgagcacctttcgcacctttaccgtggcgatggcggcgattaacaccaccccgctggaaatttgcgtgaaagcgagcgtgaccaccgtgaacaaactgggcgattgcccgttttgcgaacgcgtgctgctgaccatggaagaaaaacatctgccgtatgaaattaaactggtggatctgagcaacaaaccggaaaacctgctgcgcattagcccggaaggcaaagtgccggtggtgaaatttgatgaaaaatgggtgccgcagagcgatgtgattgcgcagagcctggaagaaaaatatccggatccgccgctggtgaccccgcaggaaaaagcgctggtgggcagcaaaatttttagcacctttattggctttctgaaaagcaaagatccgagcgatggcaccgaacaggcgctgctggattttctgagcagctttaacgattatattaaagaaaacgtgcgcccgctgagcggcagcctgaacgtgctgcgctttgtgtatagcctgcatattcgctttattctgtttagcctgaaaggcccgtttattaacggcgaaaaaattagcgcggcggatctgagcctggcgccgaaactgtatcatctgaacattgcgctgggccattatgataaatggagcattccggataccctgccgtataccaaaagctatatgaaaaccatttttagcatggatagctttaccaaaacccgcgcgagcccggatgatgtgattgcgggctggcgcccgaaagtgatgggctaa(SEQ ID NO:4)

[0060] Short peptide hydrolase (Xspetidase): Derived from the fungus (Xylariales sp, Uniprot ID: A0A1V1TJW2), this natural enzyme (WTXspetidase) has high substrate hydrolysis activity, but there are some problems with expression. Through sequence modification, a mutant (Xspetidase) with relatively good expression and catalytic activity was finally obtained. The specific mutation sites are: D35K, E113I, P114S, R128D, V259I, T330D, R413W.

[0061] Amino acid sequence of Xspetidase:

[0062] MAPQLDGYFAEVDKLSNHFIDRLAKAVAIPSISSKASRRPDVVRMGEFLAAELKNLGASVEMRPLGKQPDKPDLDLPPVVLARYGSDKNKRTILVYGHYDVQPAEKSDGWSTISFTLTVG EDGRMFGDGSTDDKGPVLGWLNAIEAHQKAGVDFPVNLLMCFEGMEEYGSEGLDELIEKEGKGYFADADAVCISDNYWLGTEKPCLTYGLRGCNYYSVEISGPGADLHSGVFGGTAQEPM TDLVRVLGSLVDTNGKIQIPGIYEQVAPVTSDEEGLYDGIAFTMETLHESLGSKTTVFEDKKSTLMARWRYPSLSVHGVEGAFSAPGAKDVIPAKVIGKFSIRTVPDMDIDKTNEAVYSY VKQVFAKLNSKNSLKVYAQHTGKWWVASPNHWNFRAAGKATERVWGVKPDFTWEGGSIPVTLTFEQATGKNVLLLPMGSSTDGAHSINEKLDKRNYIEGIKLLGAYLHYVAEEPQN (SEQ ID NO:2)

[0063] Nucleotide sequence of Xspetidase:

[0064]

[0065] Deglutaminase (TaGluhydro): Derived from the Thermoplasmatales archaeon (Uniprot ID: A0AA90ZEK2), this native enzyme (WT TaGluhydro) exhibits good substrate hydrolysis activity, but its expression and stability were suboptimal. Through directed evolution, both performance characteristics were improved. The mutant TaGluhydro contains the following mutations: S21L, S22T, R65V, L84Y, E102D, S201I, D226V, K318N, H390Q, and Q426C.

[0066] The amino acid sequence of TaGluhydro:

[0067] (SEQ ID NO: 3)

[0068] The nucleotide sequence of TaGluhydro:

[0069]

[0070] 2. Enzyme fermentation production

[0071] The enzymes used in the present invention are all produced by laboratory fermentation. The following is the basic operation process for preparing the enzyme. First, the gene sequence corresponding to the enzyme is synthesized by a gene company (Anhui General Biotechnology), and then subcloned into the pET28a plasmid through the NdeI / XhoI restriction site, and the plasmid is transferred into E. coli (BL21) (Qingke Biotechnology) cells for plate culture. Finally, a single clone is selected for liquid step-by-step amplification culture. The following is the basic process for step-by-step cell amplification culture. First, a single colony on the plate is transferred into 5 ml of LB culture medium containing 50 μM kanamycin (37 o C) culture, and when the cells grow to the logarithmic phase, inoculate them into 250 ml of LB culture medium containing the same antibiotics, and finally transfer them into a 5 L culture fermenter for culture; when the cell OD reaches 25, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 25 o Protein expression was induced by C for 8 hours, followed by centrifugation (4000 rpm, 15 minutes) to collect 25-30g of wet cells. To verify enzyme expression, a small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0). The cells were then disrupted by freeze-thaw and centrifuged at high speed, and the supernatant was run on an SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel) to confirm soluble protein expression. The remaining cells, confirmed to be correct, were then mixed with buffer (10g of wet cells with approximately 200ml of commercially available buffer), followed by high-pressure cell disruption and high-speed centrifugation (16000 rpm, 25 minutes) to remove the cell walls. The resulting enzyme-containing supernatant was used directly in subsequent reactions (the liquid enzyme activity ranged from 400 to 1500 U / mL, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature) or further purified and immobilized for use (for solid enzyme reactions). The LB medium consists of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol. The crude enzyme solution containing allyl glutathione synthetase (GaAGSyn), short peptide hydrolase (Xspetidase), and deglutamic acidase (TaGluhydro) was subjected to SDS-PAGE gel detection. The results are as follows: Figure 1 shown.

[0072] 3. Mixed immobilization of enzymes

[0073] To the crude enzyme solutions of allyl glutathione synthetase (GaAGSyn), short peptide hydrolase (Xspetidase), and deglutamic acidase (TaGluhydro) collected above, ammonium sulfate solid was gradually added until the enzyme precipitated (40%-60%, w / v ammonium sulfate / buffer). The enzyme solid was then collected by centrifugation (10,000 rpm, 12 min) and slowly dissolved in 25 mM Tris buffer, pH 8.0. Finally, the solution was desalted on a G25 size exclusion chromatography column (purchased from Sigma) and separated using a DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain pre-purified liquid enzymes GaAGSyn, Xspetidase, and TaGluhydro. The enzyme solution can be directly used for subsequent enzyme immobilization. In the mixed immobilization of GaAGSyn / Xspetidase / TaGluhydro enzymes, the pre-purified enzymes were mixed and immobilized using LX-1000EP epoxy resin (Xi'an Lanxiao Company) at an activity unit ratio of 2:1:1. The basic immobilization method is as follows: 8000U of the enzyme mixture, mixed according to the above activity unit ratio, is dissolved in 2L of 50 mM potassium phosphate solution, pH 8.0. Subsequently, 40 mM phenoxyacetic acid and 600g of LX-1000 EP epoxy resin are added to the buffer. After stirring at room temperature for 6 hours, the immobilized enzyme is filtered out and finally washed three times with clean water and 25 mM pH 8.0 phosphate buffer, and then dried at low temperature for use. The immobilized GaAGSyn / Xspetidase / TaGluhydro enzyme mixture has 80-95% of the activity of the corresponding liquid enzyme.

[0074] 4. Comparison of enzyme activity

[0075] Using the same method as in step 2 above, crude enzyme solutions of wild-type allyl glutathione synthetase (WTGaAGSyn), short peptide hydrolase (WTXspetidase), and deglutaminase (WTTaGluhydro) were prepared, and their enzyme activities, thermal stability, and expression levels were compared. The results are shown in Table 1 below.

[0076] Table 1 Comparison of properties of different enzymes

[0077]

[0078] The results showed that the enzyme activity, thermal stability and expression level of the three mutant enzymes provided by the present invention were much higher than those of the wild type.

[0079] Example 2 Preparation of allyl glutathione using allyl benzoate and glutathione as raw materials and liquid enzyme (GaAGSyn)

[0080] The synthetic reaction formula is as follows Figure 2Specifically, 8.1 g of allyl benzoate (50 mM), 16.9 g of glutathione (55 mM) and 2.0 g of magnesium chloride hexahydrate (10 mM) were added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris-HCl) solution, and then the pH value of the solution was adjusted back to 8.0. Finally, 3000 U of GaAGSyn crude enzyme solution was added at once to start the reaction; the reaction was continued for 30 min. o The mixture was stirred gently at C. The pH was maintained at approximately 7.0-9.0 throughout the reaction. After 4 hours, the reaction was complete and the reaction solution was acidified with dilute aqueous hydrochloric acid to inactivate the reaction. The pH was then adjusted to 7.0 and the crude product was purified using D101 non-polar resin. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=2:1, V:V) to obtain 14.9 g of a white solid (final yield 89%).

[0081] Example 3 Preparation of allyl glutamine cysteine using liquid enzyme (Xspetidase) using allyl glutathione as raw material

[0082] The synthetic reaction formula is as follows Figure 3 Specifically, 66.4 g of allyl glutathione (200 mM) and 2.0 g of magnesium chloride hexahydrate (10 mM) were added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris-HCl) solution, and then the pH value of the solution was adjusted back to 8.0. Finally, 2000 U of Xspetidase crude enzyme solution was added at once to start the reaction; the reaction was continued for 30 min. o The mixture was stirred gently at C. The pH was maintained at approximately 7.0-8.0 throughout the reaction. After 4 hours, the reaction was complete and the reaction solution was acidified with dilute aqueous hydrochloric acid to inactivate the reaction. The pH was then adjusted to 7.0 and the crude product was purified using D101 non-polar resin. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=4:1, V:V) to obtain 51.2 g of a white solid (final yield 92%).

[0083] Example 4 Preparation of allyl cysteine using allyl glutamine cysteine as raw material and liquid enzyme (TaGluHydro)

[0084] The synthetic reaction formula is as follows Figure 4 Specifically, 55.2 g of allyl glutamine cysteine (200 mM) and 2.0 g of magnesium chloride hexahydrate (10 mM) were added to 1 L of 100 mM pH 7.5 tris-hydroxymethylaminomethane hydrochloric acid (Tris-HCl) solution, and then the pH value of the solution was adjusted back to 7.5. Finally, 2000 U of TaGluHydro crude enzyme solution was added at once to start the reaction; the reaction was continued for 30 min. oThe mixture was stirred gently at C. The pH was maintained at approximately 7.0-8.0 throughout the reaction. After 6 hours, the reaction was complete and the reaction solution was acidified with dilute aqueous hydrochloric acid to inactivate the reaction. The pH was then adjusted to 7.0 and the crude product was purified using D101 non-polar resin. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=3:1, V:V) to obtain 30.4 g of a white solid (final yield 94%).

[0085] Example 5 Using allyl benzoate and glutathione as raw materials, liquid enzymes (GaAGSyn, Xspetidase, TaGluHydro) were used to prepare allyl cysteine in one step.

[0086] The synthetic reaction formula is as follows Figure 5 Specifically, 16.2 g of allyl benzoate (100 mM), 34 g of glutathione (110 mM), and 4.1 g of magnesium chloride hexahydrate (20 mM) were added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris-HCl) solution, and then the pH value of the solution was adjusted back to 8.0. Finally, 4000 U of GaAGSyn crude enzyme solution, 2000 U of spetidase crude enzyme solution, and 2000 U of TaGluHydro crude enzyme solution were added at once to start the reaction; the reaction was continued for 30 min. o The mixture was stirred gently at C. The pH was maintained at approximately 7.0-8.0 throughout the reaction. After 6 hours, the reaction was complete and the reaction solution was acidified with dilute aqueous hydrochloric acid to inactivate the reaction. The pH was then adjusted to 7.0 and the crude product was purified using D101 non-polar resin. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=3:1, V:V) to obtain 14.2 g of a white solid (final yield 88%).

[0087] The product was verified by nuclear magnetic resonance spectroscopy, mass spectrometry and HPLC. Figures 6-8 The results showed that the purity of allylcysteine in the final product was 99.2%.

[0088] Example 6: Allyl benzoate and glutathione as raw materials, immobilized mixed enzyme, one-time preparation of allyl cysteine

[0089] The reaction is similar to that of Example 5 above, but the immobilized enzyme prepared in Example 1 is used to achieve enzyme recycling.

[0090] The synthetic reaction formula is as follows Figure 9Specifically, 16.2 g of allyl benzoate (100 mM), 34 g of glutathione (110 mM), and 4.1 g of magnesium chloride hexahydrate (20 mM) were added to 1 L of 100 mM Tris-HCl (pH 8.0). The pH was then adjusted back to 8.0, and 8,000 U of the immobilized enzyme mix was added in one go to initiate the reaction. The reaction was gently stirred at 30°C, and the pH was maintained between 7.0 and 9.0 throughout. After 10 hours, the reaction was complete, and the immobilized enzyme mix was collected by filtration (the enzyme mix retained 90% of its initial activity after six uses). The supernatant was then purified using D101 non-polar resin to collect the crude product. Finally, the product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H₂O = 3:1, V:V) to yield 14.8 g of a white solid (92% final yield).

[0091] Comparative Example: Allyl benzoate and glutathione were used as raw materials and liquid enzymes (WTGaAGSyn, WTXspetidase, WTTaGluHydro) were used to prepare allyl cysteine in one step.

[0092] The reaction is similar to that of Example 5 above, but the reaction is carried out using natural enzymes instead of modified enzymes.

[0093] The synthetic reaction formula is as follows Figure 10 Specifically, 8.1 g of allyl benzoate (50 mM), 17 g of glutathione (55 mM), and 4.1 g of magnesium chloride hexahydrate (20 mM) were added to 1 L of 100 mM pH 8.0 tris(hydroxymethyl)aminomethane hydrochloric acid (Tris-HCl) solution, and then the pH value of the solution was adjusted back to 8.0. Finally, 6000 U WT GaAGSyn crude enzyme solution, 4000 U WTXspetidase crude enzyme solution, and 4000 U WT TaGluHydro crude enzyme solution were added at once to start the reaction; the reaction was continued for 30 min. o The mixture was stirred gently at C. The pH was maintained at approximately 7.0-8.0 throughout the reaction. After 12 hours, the reaction was complete and the reaction solution was acidified with dilute aqueous hydrochloric acid to inactivate the reaction. The pH was then adjusted to 7.0 and the crude product was purified using D101 non-polar resin. Finally, the crude product was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:H2O=3:1, V:V) to obtain 0.8 g of a gray solid (final yield 11%).

[0094] The above are only preferred embodiments 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 the scope of protection of the present invention.

Claims

1. A composition of enzyme mutants, characterized in that The composition comprises a mutant of allyl glutathione synthetase, a mutant of short peptide hydrolase and a mutant of deglutaminase, wherein: The amino acid sequence of the mutant of allyl glutathione synthetase is shown in SEQ ID NO: 1, the amino acid sequence of the mutant of short peptide hydrolase is shown in SEQ ID NO: 2, and the amino acid sequence of the mutant of deglutaminase is shown in SEQ ID NO:

3.

2. An immobilized enzyme, characterized in that A composition comprising the enzyme mutant according to claim 1.

3. A nucleic acid molecule encoding (1) and / or (2): (1) The composition of the enzyme mutant according to claim 1; (2) The immobilized enzyme according to claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that The invention comprises a nucleic acid molecule encoding a mutant of allyl glutathione synthetase, a nucleic acid molecule encoding a mutant of short peptide hydrolase, and a nucleic acid molecule encoding a mutant of deglutaminase, wherein: The nucleotide sequence of the nucleic acid molecule encoding the mutant of allyl glutathione synthetase is shown in SEQ ID NO: 4, the nucleotide sequence of the nucleic acid molecule encoding the mutant of short peptide hydrolase is shown in SEQ ID NO: 5, and the nucleotide sequence of the nucleic acid molecule encoding the mutant of deglutaminase is shown in SEQ ID NO:

6.

5. An expression vector or host cell, characterized in that The expression vector comprises the nucleic acid molecule according to claim 3 or 4; The host cell is transfected or transformed with the expression vector.

6. Use of at least one of the following ① to ④ in the preparation of allylcysteine:

1. The composition of the enzyme mutant according to claim 1; ②. The immobilized enzyme according to claim 2; ③. The nucleic acid molecule according to claim 3 or 4; 4. The expression vector or host cell according to claim 5.

7. A method for preparing allylcysteine, characterized in that: Allyl benzoate and glutathione are used as raw materials and allyl cysteine is prepared by at least one of the following transformations Ⅰ to Ⅳ; Ⅰ. The composition of the enzyme mutant according to claim 1; II. The immobilized enzyme according to claim 2; III. The nucleic acid molecule according to claim 3 or 4; IV. The expression vector or host cell according to claim 5.

8. The preparation method according to claim 7, characterized in that The preparation method comprises: Allyl benzoate and glutathione are used as raw materials, and allyl glutathione is produced by catalysis of the mutant allyl glutathione synthetase; The allyl glutathione is catalyzed by the mutant of the short peptide hydrolase to generate allyl glutamine cysteine; The allyl glutamine cysteine is catalyzed by the mutant of the deglutaminase to generate allyl cysteine.

9. The preparation method according to claim 8, characterized in that The ratio of allyl benzoate, glutathione, mutant of allyl glutathione synthetase, mutant of short peptide hydrolase and mutant of deglutaminase is (80-110 mM): (90-120 mM): (3000-5000 U): (1500-2500 U): (1500-2500 U).

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