A method for synthesizing ergothioneine using enzyme

Abstract

The present invention provides a method for synthesizing thioneine using an enzyme, comprising the following steps: step 1 is to use histidine betaine as a substrate, L-cysteine, L-ascorbic acid, ferrous sulfate heptahydrate, tris (2-carboxyethyl) phosphine hydrochloride as a cofactor, adjust the pH value of the solution, add the co-expression enzyme to carry out a catalytic reaction, and obtain a crude thioneine product after the reaction is completed; step 2 is to add a thiol reducing agent and PLP to the crude thioneine product, react under heating conditions, and obtain the thioneine product after the reaction is completed. The method for synthesizing thioneine using an enzyme of the present invention constructs the EGTB enzyme sequence into an expression vector, and transfers it into Escherichia coli for heterologous expression, and expresses it by fusion with the EGTB enzyme through a specific fusion tag, thereby improving the enzyme activity of the EGTB enzyme, thereby improving the conversion rate of the substrate.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a method for synthesizing ergothioneine by utilizing an enzyme. Background Art

[0002] Ergothioneine is a natural amino acid with antioxidant properties, widely used in food, cosmetics and medicine.

[0003] At present, there are three methods for synthesizing ergothioneine, namely extraction method, chemical synthesis method and biological method. The extraction method uses the method of natural mushroom fermentation to synthesize ergothioneine, but the method has low yield and high extraction cost, which is difficult to meet market demand. The chemical method for synthesizing ergothioneine faces the problems of lengthy synthesis route, complex process and difficult operation. The biological method mainly uses engineered bacteria to ferment ergothioneine, which has low production cost, but has the problems of long production cycle, low yield and difficulty in large-scale production. Summary of the invention

[0004] In view of this, the present invention aims to overcome the defects of the prior art and proposes a method for synthesizing ergothioneine utilizing enzyme.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A co-expressed enzyme is obtained by fusion expression of a fusion tag and an EGTB enzyme. The amino acid sequence of the EGTB enzyme is shown in SEQ ID NO.1.

[0007] Furthermore, the fusion tag is Flag.

[0008] The present invention also provides an application of the co-expressed enzyme in synthesizing ergothioneine.

[0009] The co-expressed enzyme is prepared by a method comprising the following steps:

[0010] Step 1 is to amplify the recombinant vector that fused the tag and expressed the EGTB enzyme;

[0011] Step 2 is to assemble the fusion tag into a recombinant vector expressing the EGTB enzyme, and transform it into a host cell, and then obtain the co-expressed enzyme after screening and shake flask expression.

[0012] Furthermore, the assembly step in step 2 is performed using a homologous recombinase.

[0013] Furthermore, the host cell in step 2 is Escherichia coli.

[0014] The present invention also provides a method for synthesizing ergothioneine using an enzyme, comprising the steps of:

[0015] Step 1 is to use histidine betaine as a substrate, L-cysteine, L-ascorbic acid, ferrous sulfate heptahydrate, and tris(2-carboxyethyl)phosphine hydrochloride as cofactors, adjust the pH value of the solution, add the co-expressed enzyme to carry out a catalytic reaction, and obtain a crude ergothioneine product after the reaction is completed;

[0016] Step 2 is to add a thiol reducing agent and PLP to the crude thioneine product, react under heating conditions, and obtain the thioneine product after the reaction is completed.

[0017] Furthermore, the molar ratio of histidine betaine, L-cysteine, L-ascorbic acid, ferrous sulfate heptahydrate and tris(2-carboxyethyl)phosphine hydrochloride in step 1 is 1:1-2:0.1-0.5:0.01-0.2:0.1-0.5.

[0018] Preferably, the molar ratio of histidine betaine, L-cysteine, L-ascorbic acid, ferrous sulfate heptahydrate and tris(2-carboxyethyl)phosphine hydrochloride in step 1 is 1:1-1.5:0.1-0.25:0.01-0.1:0.1-0.25.

[0019] More preferably, the molar ratio of histidine betaine, L-cysteine, L-ascorbic acid, ferrous sulfate heptahydrate and tris(2-carboxyethyl)phosphine hydrochloride in step 1 is 1:1.25:0.15:0.05:0.15.

[0020] Furthermore, the mass ratio of histidine betaine to the co-expressed enzyme in step 1 is 1:1-4.

[0021] Preferably, the mass ratio of histidine betaine to the co-expressed enzyme in step 1 is 1:1-3.

[0022] More preferably, the mass ratio of histidine betaine to the co-expressed enzyme in step 1 is 1:2.

[0023] Furthermore, the pH value of the solution in the step of adjusting the pH value of the solution in step 1 is 4.5-5.5; the temperature of the catalytic reaction in step 1 is 20-30° C., and the time is 2-5 hours.

[0024] Preferably, the pH value of the solution in the step of adjusting the pH value of the solution in step 1 is 5-5.5; the temperature of the catalytic reaction in step 1 is 25° C., and the time is 3.5-4.5 hours.

[0025] More preferably, the pH value of the solution in the step of adjusting the pH value of the solution in step 1 is 5.0.

[0026] Furthermore, the molar ratio of the histidine betaine in step 1 to the thiol reducing agent and PLP in step 2 is 1:1-5:0.1-0.5.

[0027] Furthermore, the thiol reducing agent in step 2 is at least one of β-mercaptoethanol, dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride.

[0028] Preferably, the molar ratio of the histidine betaine in step 1 to the thiol reducing agent and PLP in step 2 is 1:1-3:0.1-0.25.

[0029] More preferably, the molar ratio of the histidine betaine in step 1 to the thiol reducing agent and PLP in step 2 is 1:1-2:0.15.

[0030] Furthermore, the temperature of the heating step in step 2 is 30-50° C., and the time is 2-10 hours.

[0031] Preferably, the temperature of the heating step in step 2 is 37° C., and the time is 5-10 hours.

[0032] More preferably, the temperature of the heating step in step 2 is 37° C. and the time is 6 hours.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] The method for synthesizing ergothioneine by using an enzyme of the present invention constructs an EGTB enzyme sequence onto an expression vector, transfers the vector into Escherichia coli for heterologous expression, and fuses the vector with the EGTB enzyme through a specific fusion tag, thereby significantly improving the enzymatic activity of the EGTB enzyme and thus improving the conversion rate of the substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The nuclear magnetic spectrum of ergothioneine described in the embodiment of the present invention 1;

[0036] Figure 2 It is the conversion rate of thioneine described in Examples 1-3 of the present invention and Comparative Example 1;

[0037] Figure 3 Be the transformation efficiency of the ergothioneine described in Example 1 of the present invention and Comparative Example 1-3. DETAILED DESCRIPTION

[0038] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0039] The present invention will be described in detail below with reference to the embodiments.

[0040] Example 1

[0041] 1. Construction of co-expression enzyme:

[0042] Design a 15-20 bp homology arm, amplify the fusion tag and the recombinant vector expressing EGTB by PCR amplification technology, assemble the fusion tag Flag into the recombinant vector expressing EGTB using homologous recombinase and transform it into E. coli The DH5α strain was inoculated onto LB solid medium containing 30 mg / L kanamycin and cultured at 37°C for 10 h. Finally, transformants were selected for DNA sequencing to detect whether the sequence was correct.

[0043] The plasmid with the correct sequence was transformed into E. coli (BL21) competent cells were plate cultured, and single colonies on the plates were picked and transferred to 5 ml LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells grew to the logarithmic phase, they were inoculated into 250 ml TB culture medium containing the same antibiotics. When they also grew to the logarithmic phase, they were transferred to a 5 L culture fermenter for culture and final fermentation. In the 5L fermenter culture, when the cell OD = 20, 0.25 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added at 16°C to induce protein expression for 18 hours, and finally the bacterial sludge was collected by high-speed centrifugation (6500 rpm, 5 min). The collected bacterial sludge was used as a co-expression enzyme to participate in subsequent reactions.

[0044] 2. Synthesis of ergothioneine using enzymes:

[0045] 2 kg of histidine betaine (10 M), 1.52 kg of L-cysteine ​​(12.5 M), 267 g of L-ascorbic acid (1.5 M), 433 g of TCEP (1.5 M), and 140 g of ferrous sulfate heptahydrate (500 mM) were weighed into a reactor, and 60 L of purified water was added to the reactor and stirred; 4% hydrochloric acid was added to adjust the pH to 5.0, and the temperature was raised and maintained at 25° C. After the temperature was raised to 25° C., 4 kg of FLag-EgtB cells were weighed and added to 40 L of purified water, stirred and mixed, and poured into the reactor; the temperature was kept constant at 25° C., and the pH was maintained at 5.0 with sodium hydroxide and stirred for 4 hours; the conversion rate of ergothioneine was 70-80%; 5 kg of TCEP (20 M) and 370 g of PLP (1.5 M) were weighed and added to the reactor within half an hour after the first step of the reaction was completed, and the temperature was raised to 37° C. and stirred for 6 hours at a constant temperature. The conversion rate of ergothioneine is 99%. The NMR spectrum of ergothioneine is as follows Figure 1 shown.

[0046] Example 2

[0047] The only difference from Example 1 is that the thiol reducing agent is β-mercaptoethanol.

[0048] Example 3

[0049] The only difference from Example 1 is that the thiol reducing agent is dithiothreitol.

[0050] Comparative Example 1

[0051] The only difference from Example 1 is that the fusion tag is GFP.

[0052] Comparative Example 2

[0053] 2 kg of histidine betaine (10 M), 1.52 kg of L-cysteine ​​(12.5 M), 267 g of L-ascorbic acid (1.5 M), 433 g of TCEP (1.5 M), and 140 g of ferrous sulfate heptahydrate (500 mM) were weighed into a reactor, and 60 L of purified water was added to the reactor and stirred; 4% hydrochloric acid was added to adjust the pH to 5.0, and the temperature was raised and maintained at 25° C. After the temperature was raised to 25° C., 4 kg of EgtB cells were weighed and added to 40 L of purified water, stirred and mixed, and poured into the reactor; the temperature was kept constant at 25° C., and the pH was maintained at 5.0 with sodium hydroxide and stirred for 4 hours; the conversion rate of ergothioneine was 70-80%; 5 kg of TCEP (20 M) and 370 g of PLP (1.5 M) were weighed and added to the reactor within half an hour after the first step of the reaction was completed, the temperature was raised to 37° C., and the reaction was stirred at a constant temperature for 6 hours; the conversion rate of ergothioneine was 91%.

[0054] Comparative Example 3

[0055] 1. Construction of co-expression enzyme:

[0056] Design a 15-20 bp homology arm, amplify the fusion tag and the recombinant vector expressing EGTB by PCR amplification technology, assemble the fusion tag Flag into the recombinant vector expressing EGTB using homologous recombinase and transform it into E. coli The strain (DH5α) was inoculated on LB solid medium containing 30 mg / L kanamycin and cultured at 37°C for 10 h. Finally, transformants were selected for DNA sequencing to detect whether the sequence was correct. The plasmid with the correct sequence was transferred into E. coli (BL21) Competent cells were cultured on plates and in small amounts in liquid culture for single clones, and finally in liquid culture for step-by-step expansion;

[0057] 2. Synthesis of ergothioneine using enzymes:

[0058] 2 kg of histidine betaine (10 M), 1.52 kg of L-cysteine ​​(12.5 M), 267 g of L-ascorbic acid (1.5 M), 433 g of TCEP (1.5 M), and 140 g of ferrous sulfate heptahydrate (500 mM) were weighed into a reactor, and 60 L of purified water was added to the reactor and stirred to mix; 4% hydrochloric acid was added to adjust the pH to 5.0, and the temperature was raised and maintained at 25° C. After the temperature was raised to 25° C., 4 kg of Flag-EGTB cells were weighed and added to 40 L of purified water, stirred and mixed, and poured into the reactor; the temperature was kept constant at 25° C., and the pH was maintained at 5.0 with sodium hydroxide, and the reaction was stirred for 4 hours, and the conversion rate of ergothioneine was 74%.

[0059] like Figure 2 As shown, the thiol reducing agents β-mercaptoethanol and dithiothreitol used in Examples 2 and 3 are less effective than the thiol reducing agent TCEP used in Example 1; Figure 3 Shown, Example 1 is compared with Comparative Example 1, and the ability of catalysis and generation of thioneine after EGTB and Flag fusion expression is significantly higher than the combination of EGTB and GFP, and Example 1 is compared with Comparative Example 2, and the ability of catalysis and generation of thioneine after EGTB and fusion tag fusion expression is significantly improved, and Example 1 is compared with Comparative Example 3, and increasing one-step chemical reaction improves the yield of thioneine.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for synthesizing ergothioneine using an enzyme, characterized in that: The steps include: Step 1 is to use histidine betaine as a substrate, L-cysteine, L-ascorbic acid, ferrous sulfate heptahydrate, and tris(2-carboxyethyl)phosphine hydrochloride as cofactors, adjust the pH value of the solution, add a co-expressed enzyme to carry out a catalytic reaction, and obtain a crude ergothioneine product after the reaction is completed; Step 2 is to add a thiol reducing agent and PLP to the crude thioneine product, react under heating conditions, and obtain the thioneine product after the reaction is completed; The co-expressed enzyme is obtained by fusion expression of a fusion tag and EGTB enzyme; the fusion tag is Flag; The molar ratio of histidine betaine, L-cysteine, L-ascorbic acid, ferrous sulfate heptahydrate and tris(2-carboxyethyl)phosphine hydrochloride co-expressed enzyme in step 1 is 1:1-2:0.1-0.5:0.01-0.2:0.1-0.5; the mass ratio of histidine betaine to co-expressed enzyme in step 1 is 1:1-4; the molar ratio of histidine betaine in step 1 to the thiol reducing agent and PLP in step 2 is 1:1-5:0.1-0.5; the thiol reducing agent in step 2 is tris(2-carboxyethyl)phosphine hydrochloride; The amino acid sequence of the EGTB enzyme is shown in SEQ ID NO.

1.

2. the method utilizing enzyme to synthesize ergothioneine according to claim 1, is characterized in that: The pH value of the solution in the step of adjusting the pH value of the solution in step 1 is 4.5-5.5; the temperature of the catalytic reaction in step 1 is 20-30° C. and the time is 2-5 hours.

3. the method utilizing enzyme to synthesize ergothioneine according to claim 1, is characterized in that: The temperature of the heating step in step 2 is 30-50° C., and the time is 2-10 hours.

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