CN115976129A - Method for preparing ergothioneine - Google Patents

Abstract

The invention provides a method for preparing ergothioneine, which comprises the steps of adding Egt1 enzyme into histidine betaine and L-cysteine under the action of ferrous ions and a reducing agent to react to prepare an intermediate CHER, and adding Egt2 enzyme into the intermediate CHER under the action of coenzyme and the reducing agent to prepare the ergothioneine. The invention selects a eukaryotic organism synthesis way with a relatively simple path, screens Egt1 enzyme and Egt2 enzyme with high conversion rate, realizes the in vitro enzyme catalysis preparation of ergothioneine under the conditions of proper specific temperature and pH, can complete the conversion in a short time, can reach higher substrate concentration and conversion rate, has simple process operation, and is suitable for the large-scale production of the ergothioneine.

Description

Method for preparing ergothioneine

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for preparing ergothioneine by using biological enzyme catalysis.

Background

Ergothioneine was a specific amino acid containing thiol found in ergot in 1909, and subsequently found in Neurospora crassa, cyanobacteria, mushrooms, etc. Human beings and other invertebrates cannot synthesize them directly, but they can absorb and accumulate food to various parts of the human body, and they have superior antioxidant ability, which is known to be higher than vitamin E, vitamin C, cysteine, glutathione, etc., which makes them widely used in the industries of food, cosmetics, medicine, etc.

Ergothioneine has the following structural formula:

the synthesis of ergothioneine in aerobic organisms occurs in two pathways derived from bacterial biosynthesis and from fungal biosynthesis. In bacteria, a gene cluster having 5 key enzymes, i.e., gamma-glutamylcysteine synthetase (EgtA), mononuclear non-heme iron enzyme (EgtB), amidotransferase (EgtC), S-adenosylmethionine-dependent histidine methyltransferase (EgtD), pyridoxal phosphate (PLP) -bound C-S lyase (EgtE), is involved. Ergothioneine synthesis in eukaryotes mainly involves two key enzymes, namely Egt1 and Egt2, egt1 catalyzes cysteine and histidine betaine (Hacining) to synthesize an intermediate Hacining cysteine sulfoxide (CHER), and then the CHER is cleaved into ergothioneine by Egt2.

At present, methods for synthesizing ergothioneine mainly comprise a chemical method, a microbial fermentation method and an enzymatic preparation method. The starting materials for the chemical process are expensive and the synthesis is difficult, resulting in insufficient yields. The microbial fermentation by adopting metabolic modification is a hotspot of the current research, and the yield of 108h of final fermentation of the engineering bacteria obtained under the condition that the China patent application with the publication number of CN113234652A clones EgtABCDE into escherichia coli and metabolically modifies a path of a required substrate, reaches 1.1g/L. The Chinese patent application with publication number CN114277042A reports that ergothioneine synthesis genes Egt1 and Egt2 cloned from Neurospora crassa are introduced and integrated into Rhodosporidium toruloides, and the final yield of 8.5g/L can be reached by fermenting for 120 h. For example, chinese patent application with publication No. CN104854245A reports a method for preparing ergothioneine in vitro using histidine or histidine betaine as a substrate, one is prepared by using EgtABCDE, and the other is prepared by using protein OvoA/NcEgtl and EgtE. Although information on relevant enzymes is disclosed, the conversion cycle and the substrate conversion rate are unknown, and whether the actual effect can be applied in a large scale is not clear.

Therefore, the further development of a novel method for synthesizing the ergothioneine through enzyme catalysis, which has the advantages of short reaction time, high conversion rate and simplified process steps, has important significance for promoting the industrial and large-scale production of the ergothioneine.

Disclosure of Invention

The invention aims to provide an enzymatic synthesis method of ergothioneine.

The invention provides a method for preparing ergothioneine by using biological enzyme catalysis, which comprises the following steps:

(1) Under the action of ferrous ions and a reducing agent, adding Egt1 enzyme into histidine betaine and L-cysteine to react under an aerobic environment with the temperature of 25-40 ℃ and the pH of 6.0-8.0 to prepare an intermediate CHER;

(2) Under the action of coenzyme and reducing agent, adding Egt2 enzyme to react at 25-40 deg.c and pH 6.0-9.0 to prepare ergothioneine;

the reaction formula is as follows:

further, the reaction temperature in the step (1) is 25-30 ℃, and the pH value is 6.0-7.5;

and/or the reaction temperature in the step (2) is 25-30 ℃, and the pH is 7.0-8.5.

Further, the reaction temperature in the step (1) is 30 ℃, and the pH value is 6.5-7.0;

and/or the reaction temperature in the step (2) is 30 ℃, and the pH value is 8.0-8.5.

Further, the amino acid sequence of the Egt1 enzyme is shown as SEQ ID NO.2 or SEQ ID NO. 4;

the amino acid sequence of the Egt2 enzyme is shown as SEQ ID NO.6 or SEQ ID NO. 8.

Furthermore, the Egt1 enzyme added in the step (1) is added with wet thalli for expressing the Egt1 enzyme or added with crude enzyme liquid of the Egt1 enzyme; the step (2) of adding the Egt2 enzyme is to add wet thalli for expressing the Egt2 enzyme or to add crude enzyme liquid of the Egt2 enzyme.

Further, the crude enzyme solution of the Egt1 enzyme or the crude enzyme solution of the Egt2 enzyme is prepared by the following steps:

(a) Preparing wet thalli expressing Egt1 enzyme or wet thalli expressing Egt2 enzyme;

(b) And (b) mixing and rotating the wet bacteria prepared in the step (a) by using a PBS buffer solution, homogenizing and crushing, centrifuging and collecting a supernatant.

Furthermore, the wet cells expressing the Egt1 enzyme are cells obtained by protein expression of recombinant Escherichia coli containing the Egt1 gene; the wet thalli for expressing the Egt2 enzyme is thalli after protein expression is carried out on recombinant escherichia coli containing Egt2 genes;

the Egt1 gene sequence is shown as SEQ ID NO.1 or SEQ ID NO.3, and the Egt2 gene sequence is shown as SEQ ID NO.5 or SEQ ID NO. 7.

Further, the wet cell expressing the Egt1 enzyme or the wet cell expressing the Egt2 enzyme can be prepared by the following method:

(a') cloning Egt1 gene or Egt2 gene into an expression vector to obtain recombinant expression plasmid, and transferring the recombinant expression plasmid into escherichia coli to obtain recombinant bacteria; preferably, the expression vector is a PET-28a plasmid;

(b') inoculating and culturing the recombinant bacteria, adding ferrous ions and an inducer to induce protein expression and culture, and centrifugally collecting bacteria to obtain wet bacteria expressing the Egt1 enzyme or wet bacteria expressing the Egt2 enzyme.

Further, the reducing agent in the step (1) is one or more of tris (2-carboxyethyl) phosphine, dithiothreitol or mercaptoethanol, the coenzyme in the step (2) is pyridoxal phosphate, and the reducing agent is one or more of tris (2-carboxyethyl) phosphine, dithiothreitol or mercaptoethanol.

Further, the mass ratio of the histidine betaine, the L-cysteine, the reducing agent and the Egt1 enzyme in the step (1) is (18-23): 15-20): 0.5-1.5): 20-30); preferably 20;

and/or the mass ratio of the coenzyme, the reducing agent and the Egt2 enzyme in the step (2) to the reducing agent in the step (1) is (0.1-0.3): 0.3-0.8): 1-3): 1, preferably 0.2.

The invention has the beneficial effects that: the invention selects a eukaryotic organism synthesis way with a relatively simple path, screens Egt1 enzyme and Egt2 enzyme with high conversion rate, realizes the in vitro enzyme catalysis preparation of ergothioneine under the conditions of proper specific temperature and pH, can complete the conversion in a short time, can reach higher substrate concentration and conversion rate, has simple process operation, and is suitable for the large-scale production of the ergothioneine.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is an HPLC chromatogram of Egt1 catalyzed intermediate CHER.

FIG. 2 is an HPLC plot of Egt2 catalyzed ergothioneine production.

Detailed Description

The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.

Example 1 construction and expression of recombinant E.coli for the Synthesis of ergothioneine enzyme and crude enzyme solution

Looking up the literature and searching the NCBI database, 16 candidates Egt1 and Egt2 from different eukaryotes were identified that might catalyze the synthesis of ergothioneine from histidine betaine, as shown in table 1. The genes corresponding to these enzymes were artificially synthesized and cloned into NcoI and HindIII of the expression vector PET-28a, and the recombinant expression plasmid was transformed into E.coli BL21 (DE 3).

Inoculating an escherichia coli strain containing recombinant plasmids into 50ml of LB liquid culture medium, carrying out shake culture at 37 ℃ for 16h, transferring 8ml of cultured bacterium liquid into 400ml of TB liquid culture medium after the culture is finished, detecting the OD (optical density) value to be 0.6-0.8 after the culture is carried out for 2-3 h, adding ammonium ferrous citrate (providing ferrous ions as an activator of enzyme) with the final concentration of 0.1mM and isopropyl-beta-D-thiogalactoside (inducer) with the final concentration of 0.1mM for inducing protein expression, and carrying out shake culture at 20 ℃ for 18h. The cells were collected by centrifugation at 8000rpm to obtain wet cells.

The prepared wet bacteria containing the expressed target gene were mixed with 0.1M PBS buffer (pH = 7.0), homogenized, disrupted, and centrifuged to collect the supernatant, thereby obtaining a crude enzyme solution.

Example 2 Activity assay of synthetic ergothioneine enzymes

Analysis of catalytic activity of Egt1 enzyme on substrate histidine betaine: to the Egt1 crude enzyme reaction solution, TCEP was added at a final concentration of 1g/L, L-cysteine was added at a final concentration of 8g/L, and histidine betaine was added at a final concentration of 10g/L, the reaction solution was transferred to a 100mL microreactor at a rotation speed of 200rpm, an aeration rate of 0.15vvm, a pH of 7.0, and a temperature of 30 ℃ for 6 hours, 50. Mu.L of the reaction solution was added to 950. Mu.L of a 0.1M hydrochloric acid solution with stirring, centrifuged at 12000rpm for 5 minutes, and the supernatant was filtered and used for HPLC analysis.

Analysis of catalytic activity of Egt2 enzyme on substrate histidine betaine: taking reaction liquid with complete conversion of the histidine betaine by Egt1, respectively adding pyridoxal phosphate (PLP) with the final concentration of 0.1g/L, dithiothreitol (DTT) with the final concentration of 0.5g/L and Egt2 wet thalli with the final concentration of 5g/L, adjusting the pH of the reaction liquid to 7.5, magnetically stirring the reaction liquid in a water bath kettle at the temperature of 30 ℃ for 1 hour, taking 50 microliter of the reaction liquid to be added into 950 microliter of 0.1M hydrochloric acid solution under the condition of stirring, centrifuging the solution at 12000rpm for 5min, taking supernatant, filtering the supernatant and then using the supernatant for HPLC analysis.

The HPLC analysis conditions were: the analytical column Agilent ZORBAX stableBond AQ with a mobile phase of 20mM K ₂ HPO ₄ The solution flow rate is 1ml/min, the column temperature is 30 ℃, the detection wavelength is 210nm, the analysis time is 15min, the retention time of histidine betaine is 5.063min, the retention time of intermediate CHER is 4.316min, and the retention time of ergothioneine is 4.517min.

Table 1: comparison of Egt1 Activity on intermediate CHER for Histone betaine and Egt2

As shown by the activity of each enzyme in Table 1, the enzyme with better histidine betaine activity by Egt1 is derived from Chaetomium thermophilum and Bipolaris sorokiniana and named CtEgt1 and BsEgt1 respectively.

The Egt2 has better activity on the intermediate CHER and is derived from Rhodotorula mucoginosa and Tremella mesenterica, and the intermediates are named as RmEgt2 and TmEgt2 respectively.

And then CtEgt1, bsEgt1, rmEgt2 and TmEgt2 with relatively best effect are used for synthesizing the ergothioneine.

Wherein, the nucleotide sequence of the code BsEgt1 is shown in SEQ ID NO.1:

ATGGCAGCTAATATAATTGATATAAGGGTAGACAAGGCGGAAAGCGATATTCTGGCGGACATCAAGAAAGGTCTGCGTCCTGTTGCGGACGCCGAGAAGACGCTGCCGACCCTGTTGTTGTACGACCAGGAGGGCTTGCGACTGTTCGAACAGATCACGTACCAAGAAGAATATTACTTAACTAACGCCGAGATCGAGGTTCTGGAAACCTACGCGGACAAAATCGCCCAACGCATCAGCCCGGGTTCTATCGTGGTTGAATTGGGCTCTGGTAACCTGCGGAAGGTGAATATTCTCCTCCAGGCGGTGGACCGTCTGGGTAAGGACATCGAGTACTACGCGGTTGATCTTAGCCTGCCTGAGCTGGAACGCACCTTCAAGCAAATCCCAATTGAGGGTTATAGCCATGTTAAGTGCTTTGGCCTGCACGGCACGTACGACGACGCTCTCGAATGGCTGAAATCCCCGGCGGTCGAGGCGAAGCCGAAAACCATCCTATGGCTGGGCTCATCCCTGGGAAACTTTAAACGCCATGAAGTTCCGCCGTTTCTGGCGGGTTTCGGAAAGGTGTTGCAAACCGGCGACACCATGCTCATTGGTATCGATAGTTGTAAAGACCCGAAGCGCGTCTTTCATGCTTACAATGATCGTGACGGTGTGACCCATCGTTTTATTCTGAATGGTCTGAAACACGCGAATGCACTGATGGGCGAGAACGCCTTCAACCTGGATGACTGGGAAGTGATTGGTGAATATGACACGAAAGCGGGCCGTCACCACGCGTTCGTTGCTCCGCGTAAAGACGTGGTGGTTGACGGCGTCCCGATGAAACAAGGTGAACGTATCCGTATCGAGGAATCTTACAAATATAGCCGTGAAGAAGCTAAGAAGCTGTGGGAATTGGCGAAACTGGCGGAGAACGCGGTATGGGCGAACAGCAAGGGCGATTATGGCCTTCACATGGTGAGCAAACCATCGTTCTTTTTCCCGACGACCCCGGAAGAGTATGCGGAAAAACCGGTTCCGTCCTTGACCGAATGGCAGGAGTTGTGGAAAGCCTGGGATGCTGTTTCGAAACAAATGATTCCAAATAGCGAACTGTTGGCAAAGCCGATTAAACTGCGCAACGAGTGCATTTTTTATCTGGGTCACATTCCGACTTTTCTGGATATTCATATTGCGCGCGCGACCGATGGCAAGCCGACCGAACCGGCTTACTTCTGGAAGATCTTCGAGCGCGGTGTCGACCCAGATGTTGACGACCCGACCTTATGCCACGCGCATAGCGAAGTTCCGGAAGAGTGGCCTCCGCTGGGGACCATCCTGCAGTATCAACAAACCATTCGCAAAAACCTTGAGGCGCTGTACGACAGCGGTGAGGCCGAGAAAAACTGCCGTATCAGCCGTGGTCTGTGGATTGCATTTGAACATGAAGCCATGCATCTGGAAACTTTGCTCTACATGTTAATTCAATCCGATAAAGTGCTGCCGCCACCGGGCATCAAGCAGCCGGACTTCGCCGCATTCGCGGCACAGAGCGAGGTTATGGCAGTTGAAAACGAGTGGTTTACCATCCCGGAATCCGACATCGATATCGGCCTGAACGATCCGGAGAAGGACTTTGGTTCCAAGCGTTATTTCGGCTGGGATAACGAACGTCCGTGTCGTAGCGTCCACGTGAAGAGCTTTCGTGCAAAGGCTCGTCCGATCACCAATGGCGAGTACGCAACCTACTTGTTGCAAACGGGTAAAAAAGAGATTCCGGCGAGCTGGTGTGATAAAGCGTACAGCAACGGTCACGATACCAATACCACAAAGCGCGATAGCGTAGTGAACGGTCAGTCTAACGGTAACGGTGAGTCTAGCCAGGGTATTATCGAAGGCAAGTTCGTGCGTACCGTTTATGGTACCATTCCGCTTAAGCTGGCGATGGGTTGGCCGGTAGTGGCCAGCTACGACGAATTGGTTGGCTGCGCACAGTGGATGGGTGGTCGTATCCCAACGATGGAGGAGGCCAGAAGCATCTATGCTTATGTTGACTCGATCAAACCGGAGTTCGAGCAGTCTCTGGGCAACACGATCCCGGCGGTCAACGGTCACCTGTTAAACGAAGGCGTGTTCGAGACGCCGCCGTCCCACCACCTGAGCAATGGCAATTCCGGTGCGGTTACCGGTCTGAAGCCCCGTGATCTGTTTATCGATCTGGAAGGTACAAACGTGGGTTTTAAACATTGGCATCCGGTATCTGTGGCGGAGAAAGGTGATAAGTTGTGCGGTCAGAGTGACCTGGGTGGCGTTTGGGAGTGGACCAGCACCGTTTTAGAAAAGCACGATGGTTTTGAGCCGATGGAATTGTACCCGGGCTATACCGCAGACTTCTTCGATGGGAAGCACAACATTACCCTGGGCGGATCATGGGCAACCCACCCGCGTATAGCGGGCCGTAAGACCTTCGTGAATTGGTATCAGCGTAATTATCCGTACGTGTGGGCTGGCGCACGCATCGTCACTGACCTGTAA

the amino acid sequence of BsEgt1 enzyme is shown in SEQ ID NO.2:

MAANIIDIRVDKAESDILADIKKGLRPVADAEKTLPTLLLYDQEGLRLFEQITYQEEYYLTNAEIEVLETYADKIAQRISPGSIVVELGSGNLRKVNILLQAVDRLGKDIEYYAVDLSLPELERTFKQIPIEGYSHVKCFGLHGTYDDALEWLKSPAVEAKPKTILWLGSSLGNFKRHEVPPFLAGFGKVLQTGDTMLIGIDSCKDPKRVFHAYNDRDGVTHRFILNGLKHANALMGENAFNLDDWEVIGEYDTKAGRHHAFVAPRKDVVVDGVPMKQGERIRIEESYKYSREEAKKLWELAKLAENAVWANSKGDYGLHMVSKPSFFFPTTPEEYAEKPVPSLTEWQELWKAWDAVSKQMIPNSELLAKPIKLRNECIFYLGHIPTFLDIHIARATDGKPTEPAYFWKIFERGVDPDVDDPTLCHAHSEVPEEWPPLGTILQYQQTIRKNLEALYDSGEAEKNCRISRGLWIAFEHEAMHLETLLYMLIQSDKVLPPPGIKQPDFAAFAAQSEVMAVENEWFTIPESDIDIGLNDPEKDFGSKRYFGWDNERPCRSVHVKSFRAKARPITNGEYATYLLQTGKKEIPASWCDKAYSNGHDTNTTKRDSVVNGQSNGNGESSQGIIEGKFVRTVYGTIPLKLAMGWPVVASYDELVGCAQWMGGRIPTMEEARSIYAYVDSIKPEFEQSLGNTIPAVNGHLLNEGVFETPPSHHLSNGNSGAVTGLKPRDLFIDLEGTNVGFKHWHPVSVAEKGDKLCGQSDLGGVWEWTSTVLEKHDGFEPMELYPGYTADFFDGKHNITLGGSWATHPRIAGRKTFVNWYQRNYPYVWAGARIVTDL

the nucleotide sequence of the code CtEgt1 is shown in SEQ ID NO.3:

ATGCCAGGATTAGAAAATCCCGTACTAGCTAGCCAAACTGGTCAGGGCCGTCTGCTGGCAATTAAAGAGAAGAAGCGCTTACCGGATGTGCGCGTGAAAATCGGCGAAAAGGCGTCGTTCGATATTATCGACATCCGTCAAGGTAGCGTGGAAATGAATCTGAAGGTGGAAATCTTGAGCATGTTTCTGACCAAAAACGGCCCTCGTAAGCTGCCGACTCTGTTGTTGTACGATGAGCGTGGTTTACAATTGTTCGAGAAAATTACCTACTTGGAGGAATACTACCTGACTAACGACGAAATCGAAGTTCTGCAGAAGTACTCTGCGGACATCGCGAAGCTGATTCCGGAAGGTGCAATGCTCATTGAACTGGGTTCCGGTAACCTGCGTAAAGTCAATCTGTTGCTGCGCGCGTTCGAGGACGCAGGCAAGTCTATCGACTATTACGCTTTGGACCTGTCCAAGCAAGAACTGGAACGGACCTTAGCGCAGCTGCCGCATTACCAGTATGTCCGTGCACACGGCCTGCTGGGTACGTACGACGATGGTAGAGCGTGGCTGAAACACCCGAGCCGTGCGTCCCGCCAGAAGTGCATCCTGTCGCTGGGCTCTAGCGTCGGAAACTTCGACCGCGCGGATGCCGCTGCTTTCCTGAAAACCTTTGCCGACATCTTAGGTCCGGGTGACACGATGCTCATCGGCTTGGACGCCTGCAATGATCCGGCGCGCGTGTATCATGCATATAACGATAAAGAGGGCGTTACCCACGAGTTGGCGTCCCAGGCCGGTTCCGGCGACAGCGCAGACGAGAGCATTCATCGTTTCATTTTAAATGGTCTGCGCCACGCTAATAAAATCTTGGGTGAAACCGTGTTTGTGGAGGCGGAGTGGCGTGTTATTGGCGAGTACGTGTACGACGGCCAAGGTGGTAGACACCAGGCGTTCTATGCGCCACTGCATGATACCACCGTCCTGGGCCAGCTGATTCGCCCACACGATCGTATTCAGGTTGAACAGTCCCTGAAGTACTCGCCGGCGGAAGCAGAACTTCTGTGGAAACGTGCGGGCATGGAAGAGATCGGTCACTGGCGTTGTCGTGATGAGTACGGCGTTCACATGCTGTCCAAGCCGAAAATGGCGTTCGGCCTGATTCCGTCTGTTTACGCACGTTCTGCTCTGCCGAGCTTGGAGGAGTGGGAAAGCCTGTGGGCAGCGTGGGATACTCTTACGCAGGAGATGCTGCCGCCAGAAGAACTTTTGTCTAAGCCGATCAAGCTGCGCAACGCGTGCATCTTCTACTTGGGTCACATCCCGAATTTTCTGGATGTTCAGCTGAGTAAGGTCACCACGGATCCTCTGACCGACCCGGCTTGGTACAGGCGTATCTTCGAGCGCGGTATTGACCCGGATGTGGACAACCCGGAAATCTGTCATGATCATAGCGAAATTCCGGATGAGTGGCCACCGGCGGATGAGATTTTGGAGTATCAAACCCGTGTTCGTGCACGTTTGCGTAAGTATTATGAAAACGGAGTTGAGAACATTCCGCGTCACATCGGGAGAGCCATTTGGGTTGGTTTCGAGCACGAGATCATGCATCTGGAAACCTTGCTGTATATGATGTTGCAGAGCGATAAGACCCGTCCGCCTCCGAATGTGCCGGTTCCCGACTGGGAAAAATTGGCGGCCAAGGCACGTAGCGAACGCGTACCGAATGAATGGTTTGATATCCCGGAGCAAGAGATCACGATTGGCCTGGACGACCCGGAAGATGAAACCGACCCGAACGTGCATTATGGTTGGGATAATGAAAAACCGGTGCGTCGTGCTAAAGTTCACGCTTTCCAAGCCAAGGGTCGTCCGATTACGAACGAGGAGTACGCGACCTACCTGTATAACACTCATGGTAGCCAGATTCCGGCTTCATGGGCCTACACCAAAGAGAAAGATCCGCAAAATGGCGTTAGCGGTACGAACGGCCACTCCACCATCGCCAACGGCACCGCTCCGCTGCCGGAGAGCTTCCTAGAGGATAAGGCCGTTAAAACCGTGTTCGGCCTGGTCCCGCTCAAATACGCGTTGGATTGGCCAGTGTTTGCATCCTATAATGAGCTTGCCGCGTGCGCTGCCTGGATGGGTGGTCGTATCCCGACCTTTGAGGAAGTGCGTAGCATTTATGCGCACGTTGAGGCGCGCAAGAAACAGAAGGAGGCTCAAAAACATCTCGCGCAAACCGTGCCGGCGGTTAACGCTCACCTGTGCAACAACGGCGTAGAAATCAGCCCGCCCGCGACGCCTCCGGCGGGGACCGCGGCGGCGACCGCGGAAGGAGACGAGTCCGAAAACAGCCTGTTCATCGACCTGGACGGCGCGAACGTTGGTTTTCAGCATTGGCATCCAGTGCCGGTGACAAACCGTGGTGGCGAGCTGGCTGGCCAGGCAGAATGTGGTGGTGTGTGGGAATGGACCAGCAGCGTTCTGCGTCCCTGGGACGGTTTCAAACCGATGACCCTATACCCAGGTTATACCGCGGACTTTTTTGACGAGAAGCACAATATTGTTCTGGGCGGTTCATGGGCGACTCACCCGCGTATAGCCGGTCGCAAGAGTTTCGTGAATTGGTACCAACGTAACTATCCGTATGCGTGGGTTGGCGCTCGCCTTGTTCGCGATCTGCCGTAA

the amino acid sequence of CtEgt1 enzyme is shown in SEQ ID NO.4:

MPGLENPVLASQTGQGRLLAIKEKKRLPDVRVKIGEKASFDIIDIRQGSVEMNLKVEILSMFLTKNGPRKLPTLLLYDERGLQLFEKITYLEEYYLTNDEIEVLQKYSADIAKLIPEGAMLIELGSGNLRKVNLLLRAFEDAGKSIDYYALDLSKQELERTLAQLPHYQYVRAHGLLGTYDDGRAWLKHPSRASRQKCILSLGSSVGNFDRADAAAFLKTFADILGPGDTMLIGLDACNDPARVYHAYNDKEGVTHELASQAGSGDSADESIHRFILNGLRHANKILGETVFVEAEWRVIGEYVYDGQGGRHQAFYAPLHDTTVLGQLIRPHDRIQVEQSLKYSPAEAELLWKRAGMEEIGHWRCRDEYGVHMLSKPKMAFGLIPSVYARSALPSLEEWESLWAAWDTLTQEMLPPEELLSKPIKLRNACIFYLGHIPNFLDVQLSKVTTDPLTDPAWYRRIFERGIDPDVDNPEICHDHSEIPDEWPPADEILEYQTRVRARLRKYYENGVENIPRHIGRAIWVGFEHEIMHLETLLYMMLQSDKTRPPPNVPVPDWEKLAAKARSERVPNEWFDIPEQEITIGLDDPEDETDPNVHYGWDNEKPVRRAKVHAFQAKGRPITNEEYATYLYNTHGSQIPASWAYTKEKDPQNGVSGTNGHSTIANGTAPLPESFLEDKAVKTVFGLVPLKYALDWPVFASYNELAACAAWMGGRIPTFEEVRSIYAHVEARKKQKEAQKHLAQTVPAVNAHLCNNGVEISPPATPPAGTAAATAEGDESENSLFIDLDGANVGFQHWHPVPVTNRGGELAGQAECGGVWEWTSSVLRPWDGFKPMTLYPGYTADFFDEKHNIVLGGSWATHPRIAGRKSFVNWYQRNYPYAWVGARLVRDLP

the nucleotide sequence for coding RmEgt2 is shown in SEQ ID NO.5:

ATGGTACAAAGTCACTCACTATTCTTTTATGGCACCCTGTGTCATGCAGCGGTTCTGCGTCGCGTTATCGGTAACAACGGTGAGCACTCCACCTGCCGTGATGCACTGCTGGAGGATCATGTGCGTCTCCATGTTGCTGGTGAGGACTATCCGGCTGTTGTCTCCGTGGACAGCGCGGAAAGCGCCCGTCTTTTGAAGCGCAATCTGGCACCGACCGAACGTCGTGTACAAGGTGTGGTGGTGCAGGGCTTGACCGATGATGATGTTGCCATGCTGGACGAATTCGAGGGCGACGCTTCCCAGACCGCGGTTCTCGAGCCGGAAGGCCGTGGCGAAATCGCCGACTCAGTGAGCCGTGTCGTTCGCTTGGATTACAGCAGGCGTACCACCGTTTACCTGTGGACCGCTCCGCTGTCTCGCCTGGAGCCGCGTATTTGGAGCTTTACTGACTTCCTGAGAGATTCCGCACACCGTTGGGTTGGCTCCGGTGCTGACTCCAATCCGGATTACGTTGAGGTCGACCGTCGTCGAAACATGAAAGGTGTCATCACGCCGCGTGGCGTACGCGAGGAATCGGCGAAGGTGCTGGAGGGCGCTACTGCCCAATTGAGCCTGGACCGCAAGAACGGTGGTGGCGGTGGCGAGAAGCACGTGAACGGCACCGCGTTCGAGCCCTTTGGCCGTGAACTCGCAAAAAAATACTGGCGTTTCGAAGAGGGCTGGGTTAACCTGAATCATGGTAGCTATGGTGCGGCGCCGTTGCCGGTTGTAGAGAGCCTGCATGCGATTCAGGCAAGATGTGATTCCGCTCCGGACCGTTTCATGCGTGTGGAGTATGAGCAAGAACTCATCGACGTGCGCACCCGTTTAGCCGACTTCGTAGATTGTGAAACGGATGACCTGGTTCTGGTCCCGAATGCAACCAGCGGCGTCAACGAGGCGCTGCGTGGGCTGACCACGGAGTGGAATAAAGGTGATCGACTATTGTTTTTCTCCTCTTCTATTTACAATGCTTGCAGCTCGACGCTGCAGTACATCGTTGACACCCATCGCCACCTGGACTTGGAGTTGCTGCCTGTTGTGTTTACCTACCCGAAACCACACTCGGAAGTGGTTCGGTTAGCGCGCGACGCGATTGAACGTGCTAACAGCGATGGTACCGGTCGCCGCGTGCGTTTGGCGCTCATCGATGCGATCAGCTCAGCCCCAGGCGTTGTTGTGCCGTGGGAAGATCTGGTGGATTTGTTTCGTGAGAAAGATATCATATCTCTAGTGGACGCTGCCCACCAAATTGGCCAGCTGCCGGTGAGCCTGCGTACCTCGAAGCCGGACTTCTGGATTTCTAACTGCCACAAATGGCTGCACGCTCACCGTGGAGTCGCGGTGCTCTATGTTGACAAGAAGTTCCAGCATCTTGTTCACAGCTCCCCGATTGGTCATTATTACGGTGTTAAAGGCGGTTTTGTGAACGAGCACAGTTGGTCTGGTACGGTTGATTGGAGCCCGTATCTGTCCGTTGCGGCCGCGCTGGATTTCCGCCGCGACATTCTGGGTGGAGAGGAACGCATCTATCAGTACTGCTTTGATCTGGCGGTTCAAGGCGGCGACCTGGTCGCCGCGGAATTGGGCACCAAAGTGATGCGTAATGCGACACCGGAAGAAGGCGAACTGATCGCGACTATGGTTAACGTGGAATTGCCGCTGCCGGCACCAAGCACCTTTAGCACCAGCGACCTGAAGCTGCTGAACCCGTTCTGGTTTCGTGAGCTGGCGAGCAAACACCAAACGGTGGTGCCGATCTTTGCACATGGTGACGTGTTCTGGACCCGCCTGAGCGCTCAGGTTTACAACGATCTTGACGACTTCGCCCACGTGGCGAAGGCACTCAAGACCGTGTGCGAACAGATTACGGCGGGTACTTGGCGTTAA

the amino acid sequence of the RmEgt2 enzyme is shown in SEQ ID NO.6:

MVQSHSLFFYGTLCHAAVLRRVIGNNGEHSTCRDALLEDHVRLHVAGEDYPAVVSVDSAESARLLKRNLAPTERRVQGVVVQGLTDDDVAMLDEFEGDASQTAVLEPEGRGEIADSVSRVVRLDYSRRTTVYLWTAPLSRLEPRIWSFTDFLRDSAHRWVGSGADSNPDYVEVDRRRNMKGVITPRGVREESAKVLEGATAQLSLDRKNGGGGGEKHVNGTAFEPFGRELAKKYWRFEEGWVNLNHGSYGAAPLPVVESLHAIQARCDSAPDRFMRVEYEQELIDVRTRLADFVDCETDDLVLVPNATSGVNEALRGLTTEWNKGDRLLFFSSSIYNACSSTLQYIVDTHRHLDLELLPVVFTYPKPHSEVVRLARDAIERANSDGTGRRVRLALIDAISSAPGVVVPWEDLVDLFREKDIISLVDAAHQIGQLPVSLRTSKPDFWISNCHKWLHAHRGVAVLYVDKKFQHLVHSSPIGHYYGVKGGFVNEHSWSGTVDWSPYLSVAAALDFRRDILGGEERIYQYCFDLAVQGGDLVAAELGTKVMRNATPEEGELIATMVNVELPLPAPSTFSTSDLKLLNPFWFRELASKHQTVVPIFAHGDVFWTRLSAQVYNDLDDFAHVAKALKTVCEQITAGTWR

the nucleotide sequence for coding TmEgt2 is shown in SEQ ID NO.7:

ATGACAGTTAGTACGACTGTACACAATTCAACCTCTTTAAAGTGCGTTGTGTTTTTTTATGGCACTCTGTGCGTGCCGGCCATTTTGGCGCGTGTTCTGGGTCATGGCTGCCAGAATTTGACCTTTCAGGATGCTTTGCTGCCGGATTACACCCGTCATTGTGTTGTCGGACAGGATTACCCGGCGGTTATCGATTCCAAATCTACGCGTATCATCAGCGGTGACGACGTCGAGGACACCAGCACCCGTGGTACGCTGGTTAGCGGTCTGAGCGTTGCTGACGTACTGGCCCTCGACATTTTTGAAGGTGATGAGTACACCCGCAAAATCCTTAAGGTGCACACTTTCAGCAGCGCGGCTGCGATCGACCAATTGCCGCAAGAACTAATGAATCCGAACCTGAGAGAAAGCACCATTAACACCCCGCCTTCAATCTATGCAAAAAAGGAGGCGTGGGTTTACACATGGTCGGCTGGTCTCGACCGCCTGGATCCGAAAGTGTGGTCTTTCGCGGAGTTCGTTAAGGAGCGCGCGGATCAGTGGACCGGCAAAGATACCACTGAATTCGATGAGATCGAACACCACCTGGCGATTCGTGCTGAACCGGGCATTCGTGATGAAAAAAGCCAGTCCGATTTCAAACCGACGGGTGTGAAGGGCAAGACCAAAGAAGGTTTTCCGGATTTTGGTCGCAACATGCTGAAGTATTGGGGTTTCCAACCGGGCTATGTCAACCTGAACCACGGTAGCTATGGCTCTCCGCCGAAGCCAGTGATCGAGCGCATGCGTGCGTTGACTGATGAAATCGAACGCTTCCCGGATCTGTTCATGCGTCGTACGTGCCGTCCGATGTTGGACCGTGTGAAGCAGCGTGTCGCCAAATTGATTGGCGCGGACGAGCACGAATGTGTGATTGTTCCAAATACCACCCACGGTATCAACACCGTCATGTATAACATTGACTGGGAAGAGGGCGACGTCATCGTAATTTATAACACCACGTATGGCGCAGTTGGCCAAACCGAGAAGTTTATCTGCGACAAATACCCGGGTGTAAGCCTCGAGCAGATTTCCATCACCTTCCCCTGCAGCCATGAGGAAGTTGTGAAAAAAACCGAAGACGTGCTGGGCCGTTACAATCAACAAGTTAACCCGACCCAATTAGAAATCAAACCGGTAGGTAGAGGTGAAAAGCGCGTGCGTATGGTTGTTGTGGACCAAATAGCGAGCAATCCGGGTGTTGTGTATCCTTGGGAAGACATTGTGCGTCTGTGTAAAAAGTACGGTGCTCTGTCTGTGGTCGACGCGGCGCACGCCATCGGTCAAGTTAAAACCAATGTGAAGTTGGCGGACTGTGATTTCTGGATTTCCAACTGCCATAAATGGCTGATGGCACACCGTGGCTGCTCCGTTCTGTATGTGCCGTTTCGTAATCAGCATCTGATGCGCACTACGTTCCCGACTTCACACGCGTATGAGAGCAGCCGTTACCCGACTCTGGATATTGATGGTCAGCAGGCAGGTCGCCCACGTAGCTTCGAGATGCAGTACGAGTGGACCGGTACGCAAGATTGGGCACCGCTGTTGTCGCCGTCCTTCGCTCTGGACTTCCGTGAGGAGATTGGCGGTGAAGAGCGAATCATGGAATACTGCCATACCCTGGCAGTGGAAGGCGGCAAGCGCATCGCGGAGCGCTGGGAAACCGAGGTTATGGATATCCCGGGTGACATCCTGACCGCAGCGATGGTGAACGTGGCACTGCCTTACATTCCGAGCCCGATGAGCCTGAAAGAGCAGTATACGCAATTACGTTACATCGAGGATACCCTTTTTTCGTCCAACTGCTTTGCTCCGTGTTACGTTCACGGCGGCAAGTGGTGGACCCGTTTTTCTGCACAGGTTTGGAATGAACTGGAGGACTTCGACTACGTTGCGAACATCTTGGAGAAGGCCTGCCTGGACATCAAGAAGGGGCTCCATCTGCAGCATCACACCGTTGACACAGATCACATTCATCAGCCGGCGGACGTGCCAGCGCACGACGAGTAA

the amino acid sequence of the TmEgt2 enzyme is shown in SEQ ID NO.8:

MTVSTTVHNSTSLKCVVFFYGTLCVPAILARVLGHGCQNLTFQDALLPDYTRHCVVGQDYPAVIDSKSTRIISGDDVEDTSTRGTLVSGLSVADVLALDIFEGDEYTRKILKVHTFSSAAAIDQLPQELMNPNLRESTINTPPSIYAKKEAWVYTWSAGLDRLDPKVWSFAEFVKERADQWTGKDTTEFDEIEHHLAIRAEPGIRDEKSQSDFKPTGVKGKTKEGFPDFGRNMLKYWGFQPGYVNLNHGSYGSPPKPVIERMRALTDEIERFPDLFMRRTCRPMLDRVKQRVAKLIGADEHECVIVPNTTHGINTVMYNIDWEEGDVIVIYNTTYGAVGQTEKFICDKYPGVSLEQISITFPCSHEEVVKKTEDVLGRYNQQVNPTQLEIKPVGRGEKRVRMVVVDQIASNPGVVYPWEDIVRLCKKYGALSVVDAAHAIGQVKTNVKLADCDFWISNCHKWLMAHRGCSVLYVPFRNQHLMRTTFPTSHAYESSRYPTLDIDGQQAGRPRSFEMQYEWTGTQDWAPLLSPSFALDFREEIGGEERIMEYCHTLAVEGGKRIAERWETEVMDIPGDILTAAMVNVALPYIPSPMSLKEQYTQLRYIEDTLFSSNCFAPCYVHGGKWWTRFSAQVWNELEDFDYVANILEKACLDIKKGLHLQHHTVDTDHIHQPADVPAHDE

example 3 Synthesis of CHER by CtEgt1

TCEP with the final concentration of 1g/L, L-cysteine with the final concentration of 16g/L and histidine betaine with the final concentration of 20g/L are respectively added into a reactor, 200mL of deionized water is added, the pH is adjusted to be 6.75, 20g of CtEgt1 wet thalli prepared in example 1 is added, the pH of a reaction solution is controlled to be 6.75 in the whole process, the reaction solution is converted for 8 hours at the rotating speed of 200rpm and the aeration rate of 0.15vvm at the temperature of 30 ℃, and then the reaction solution is subjected to HPLC analysis, and the result is shown in figure 1, wherein the conversion rate is more than or equal to 99%.

Example 4 Synthesis of CHER with BsEgt1

TCEP with the final concentration of 1g/L, L-cysteine with the final concentration of 16g/L and histidine betaine with the final concentration of 20g/L are respectively added into a reactor, 200mL of deionized water is added, the pH is adjusted to be 6.75, 30g of Bsegt1 wet thalli prepared in example 1 is added, the pH of a reaction solution is controlled to be 6.75 in the whole process, the reaction solution is converted for 8 hours at the rotating speed of 200rpm and the aeration rate of 0.15vvm at the temperature of 30 ℃, and then the reaction solution is subjected to HPLC analysis, wherein the conversion rate is more than or equal to 98%.

Example 5 Synthesis of ergothioneine from RmEgt2

Taking the CtEgt1 reaction solution in example 2, adding PLP with final concentration of 0.2g/L and DTT with final concentration of 0.5g/L, respectively, adding 2g of RmEgt2 wet thallus prepared in example 1, controlling pH to be 8.2, magnetically stirring in a 30-degree water bath for 2h, and then carrying out HPLC analysis on the reaction solution, wherein the result is shown in figure 2, and the conversion rate is more than or equal to 98%

Example 6 Synthesis of ergothioneine by TmEgt2

Taking the CtEgt1 reaction solution in example 3, respectively adding PLP with final concentration of 0.2g/L and DTT with final concentration of 0.5g/L, adding TmEgt2 wet thalli prepared in example 1, controlling pH to be 8.2, magnetically stirring in a 30 ℃ water bath for 2h, and carrying out HPLC analysis on the reaction solution, wherein the conversion rate is more than or equal to 99 percent

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 optimization of catalytic conditions for synthesizing CHER by Egt1

4 parts of catalytic liquid are prepared as follows: TCEP with a final concentration of 1g/L, L-cysteine with a final concentration of 8g/L, and histidine betaine with a final concentration of 10g/L were added to the crude CtEgt1 enzyme solution of example 1, and the mixture was divided into 4 portions of catalyst solutions, which were placed in a 100mL microreactor, and after the pH was adjusted to 6.0 to 8.0, the mixture was converted at a rotation speed of 200rpm and an aeration rate of 0.15vvm at a temperature of 30 ℃ for 6 hours, and the reaction solution was subjected to HPLC analysis, the results of which are shown in Table 2.

Table 2: egt1 conversion at different pH

PH	6h conversion
		6.0-6.5	79％
6.5-7.0	99％
		7.0-7.5	85％
7.5-8.0	63％

The results show that the conversion obtained under the reaction conditions of pH 6.5-7.0 is significantly better than other pH ranges, and thus pH 6.5-7.0 is the optimum reaction pH condition.

4 parts of catalytic liquid are prepared as follows: the crude enzyme solution CtEgt1 of example 1 was taken, TCEP at a final concentration of 1g/L, L-cysteine at a final concentration of 8g/L, and histidine betaine at a final concentration of 10g/L were added, the mixture was divided into 4 portions of a catalyst solution, the catalyst solution was placed in a 100mL microreactor, the pH of the reaction solution was adjusted to 6.75, the reaction solution was placed at 25 ℃, 30 ℃, 35 ℃, and 40 ℃ respectively, and HPLC analysis was performed on the reaction solution after conversion at 200rpm and an aeration rate of 0.15vvm for 6 hours, and the results are shown in the following table:

table 3: egt1 conversion under different reaction temperature conditions

Temperature of	6h conversion
		25℃	86％
30℃	99％
		35℃	63％
40℃	25％

The results show that under the reaction conditions at a temperature of 30 ℃, the conversion obtained is significantly better than other temperatures, so 30 ℃ is the optimal reaction temperature.

Experimental example 2 optimization of catalytic conditions for synthesizing CHER by Egt2

5 parts of catalytic liquid are prepared as follows: the CtEgt1 reaction solution obtained in example 3 was diluted with Tris-HCl buffer to a substrate concentration of 10g/L and a buffer concentration of 100mM, and after adding 0.1g/L of PLP and 0.5g/L of DTT, the reaction solution was divided into 5 portions, the pH thereof was adjusted to 6.0 to 9.0, and 5g/L of TmEgt2 was added. After magnetically stirring for 2h in a 30 ℃ water bath, the reaction solution was subjected to HPLC analysis, and the results are shown in the following table:

table 4: egt2 conversion at different pH

The results show that under the reaction conditions of pH 8.0-8.5, the conversion rates obtained are significantly better than other pH ranges, so that pH 8.0-8.5 is the optimal reaction pH condition.

4 parts of catalytic liquid are prepared as follows: the CtEgt1 reaction solution in example 3 was diluted with Tris-HCl buffer to a substrate concentration of 10g/L and a buffer concentration of 100mM, 0.1g/L PLP and 0.5g/L DTT were added, the pH was adjusted to 8.0, 5g/L TmEgt2 was added, the reaction solution was divided into 5 portions of catalyst solution, and the catalyst solution was magnetically stirred in water baths of 25 ℃, 30 ℃, 35 ℃ and 40 ℃ for 2 hours, followed by HPLC analysis, and the results are shown in the following table:

table 5: egt2 conversion under different reaction temperature conditions

Temperature of	2h conversion
		25℃	65％
30℃	99％
		35℃	90％
40℃	32％

The results show that under the reaction conditions at a temperature of 30 ℃, the conversion obtained is significantly better than other temperatures, so 30 ℃ is the optimal reaction temperature.

In conclusion, the invention provides a novel method for preparing ergothioneine, selects a eukaryotic biosynthesis pathway with a relatively simple path, screens Egt1 enzyme and Egt2 enzyme with high conversion rate, realizes in-vitro enzyme catalysis preparation of the ergothioneine under the conditions of proper specific temperature and pH, can complete conversion in a short time, can reach higher substrate concentration and conversion rate, has simple process operation, and is suitable for large-scale production of the ergothioneine.

Claims (10)

1. A method for preparing ergothioneine by using biological enzyme catalysis is characterized by comprising the following steps:

(1) Under the action of ferrous ions and a reducing agent, adding Egt1 enzyme into histidine betaine and L-cysteine to react under an aerobic environment with the temperature of 25-40 ℃ and the pH of 6.0-8.0 to prepare an intermediate CHER;

(2) Under the action of coenzyme and reducing agent, adding Egt2 enzyme to react at 25-40 deg.c and pH 6.0-9.0 to prepare ergothioneine;

the reaction formula is as follows:

2. the method of claim 1, wherein the reaction temperature in step (1) is 25 to 30 ℃ and the pH is 6.0 to 7.5;

and/or the reaction temperature in the step (2) is 25-30 ℃, and the pH value is 7.0-8.5.

3. The method of claim 2, wherein the reaction temperature in step (1) is 30 ℃, the pH is 6.5 to 7.0;

and/or the reaction temperature in the step (2) is 30 ℃, and the pH value is 8.0-8.5.

4. The method according to any one of claims 1 to 3, wherein the Egt1 enzyme has an amino acid sequence as shown in SEQ ID No.2 or SEQ ID No. 4;

the amino acid sequence of the Egt2 enzyme is shown as SEQ ID NO.6 or SEQ ID NO. 8.

5. The method according to claim 4, wherein the Egt1 enzyme is added in step (1) by adding wet cells expressing the Egt1 enzyme or by adding crude enzyme solution of the Egt1 enzyme; the step (2) of adding the Egt2 enzyme is to add wet thalli for expressing the Egt2 enzyme or to add crude enzyme liquid of the Egt2 enzyme.

6. The method according to claim 5, wherein the crude enzyme solution of the Egt1 enzyme or the crude enzyme solution of the Egt2 enzyme is prepared by the following method:

(a) Preparing wet thalli expressing Egt1 enzyme or wet thalli expressing Egt2 enzyme;

(b) And (b) mixing and rotating the wet bacteria prepared in the step (a) by using a PBS buffer solution, homogenizing and crushing, centrifuging and collecting a supernatant.

7. The method according to claim 5 or 6, wherein the wet cells expressing the Egt1 enzyme are cells obtained by protein expression of recombinant Escherichia coli containing the Egt1 gene; the wet thallus for expressing the Egt2 enzyme is thallus which contains the protein expression of recombinant escherichia coli containing the Egt2 gene;

the Egt1 gene sequence is shown as SEQ ID NO.1 or SEQ ID NO.3, and the Egt2 gene sequence is shown as SEQ ID NO.5 or SEQ ID NO. 7.

8. The method according to claim 7, wherein the wet cells expressing Egt1 enzyme or the wet cells expressing Egt2 enzyme are prepared by the following method:

(a') cloning Egt1 gene or Egt2 gene into an expression vector to obtain recombinant expression plasmid, and transferring the recombinant expression plasmid into escherichia coli to obtain recombinant bacteria; preferably, the expression vector is a PET-28a plasmid;

(b') inoculating and culturing the recombinant bacteria, adding ferrous ions and an inducer to induce protein expression and culture, and centrifugally collecting bacteria to obtain wet bacteria expressing the Egt1 enzyme or wet bacteria expressing the Egt2 enzyme.

9. The method of any one of claims 1 to 3, wherein the reducing agent of step (1) is one or more of tris (2-carboxyethyl) phosphine, dithiothreitol, or mercaptoethanol, the coenzyme of step (2) is pyridoxal phosphate, and the reducing agent is one or more of tris (2-carboxyethyl) phosphine, dithiothreitol, or mercaptoethanol.

10. The method according to any one of claims 1 to 3, wherein the mass ratio of the histidine betaine, the L-cysteine, the reducing agent and the Egt1 enzyme in step (1) is (18-23), (15-20), (0.5-1.5) and (20-30); preferably 20;

and/or the mass ratio of the coenzyme, the reducing agent and the Egt2 enzyme in the step (2) to the reducing agent in the step (1) is (0.1-0.3): 0.3-0.8): 1-3): 1, preferably 0.2.

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