CN118272325A - Amino substitution enzyme mutant, complex enzyme, immobilized enzyme and preparation method of ergothioneine - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a preparation method of amino-substituted enzyme mutants, complex enzymes, immobilized enzymes and ergothioneine. The invention can conveniently and efficiently convert low-cost histidine into high-added-value ergothioneine by utilizing the continuous catalytic capability of amino substitution enzyme (HisMuTa), trimethyl oxidase (HerOxy) and lyase (HERCYLYASE). The invention also makes certain attempts on the catalytic process, wherein the use of polypeptide pairing (RAID-RIDD) further improves the conversion speed, and the use of immobilized enzyme can well recycle the enzyme. The method has the obvious advantages of simple production process, high substrate conversion rate, high product concentration, less impurities and environmental protection compared with the traditional chemical method and fermentation production method.

Description

Amino substitution enzyme mutant, complex enzyme, immobilized enzyme and preparation method of ergothioneine

Technical Field

The invention relates to the field of biotechnology, in particular to a preparation method of amino-substituted enzyme mutants, complex enzymes, immobilized enzymes and ergothioneine.

Background

Ergothioneine is a naturally occurring, rare amino acid, structurally it is a thiourea derivative of histidine, which contains a sulfur atom in the imidazole ring. Relatively few organisms in nature, such as actinomycetes, "cyanobacteria" and certain fungi, are able to biosynthesize ergothioneine. Ergothioneine was discovered in 1909 and named after its first purified ergot, and its chemical structure was determined in 1911. Although the role of ergothioneine in humans is still under investigation, and its specific physiological role is not clear, numerous studies have demonstrated that humans accumulate ergothioneine in erythrocytes, bone marrow, liver, kidneys, semen and eyes by diet. Part of researches also show that the ergothioneine has unique physiological functions of scavenging free radicals, maintaining normal growth of cells, cellular immunity, whitening, resisting aging and the like. Ergothioneine was listed in the new resource food list of the european union in 2018 and is now mainly sold as a dietary supplement. The European food safety agency dietary product group reports a daily safety margin of 2.82 mg/kg body weight for infants, 3.39 mg/kg, and 1.31 mg/kg for adults (including pregnant and lactating women). Due to the effectiveness and safety, the ergothioneine has wide application prospect in industries such as foods, cosmetics, other functional foods, biological medicines and the like. The popularization and application of the ergothioneine are greatly limited by the high price so far, so that the development of a cheap, efficient and easily-scaled production process of the ergothioneine is particularly urgent.

The methods for preparing ergothioneine are relatively many in the market today, but can be basically divided into chemical synthesis methods and biological fermentation production methods. The L-histidine esterified in the chemical synthesis process is used as a starting material, amino groups can be subjected to trimethyl and then to aza-cycle sulfuration after selective protection by functional groups (reference documents: patent U.S. Pat. No. 3, 7767826, 20090093642 A1), or 2-thio-histidine is prepared first and then amino groups are subjected to trimethyl (reference document ：Ashley,J.N.,andHarington,C.R.,J.Chem.Soc.,1930,2586;Heath,H.,Lauson,A.,Rimington,C.,Nature,1950,166,106).), however, all chemical preparation processes have the disadvantages of complicated process, low yield, poor product quality (racemate can be produced) and high production environment cost, so far, the industrialized production of the ergothioneine is mainly fermentation production, and different subject groups can be used for realizing the fermentation production of the ergothioneine by implanting and modifying ergothioneine biosynthesis genes on industrial common strains such as escherichia coli (escherichia coli), saccharomyces cerevisiae) and corynebacterium glutamicum (Corynebacteriumglutamicum) (reference document ：WO2014/100752Al;StevenA.vanderHoeketal,Front.Bioeng.Biotech.2019,7,262;Kim,M.,Jeong,D.W.,J.Agric.Food.Chem.2022,70,1516);, however, the ideal fermentation efficiency of the modified strain still can still reach the maximum market price per gram of the product directly because the biological synthesis path of the compound is clear in organisms.

Disclosure of Invention

In view of this, the present invention provides methods for preparing amino-substituted enzyme mutants, complex enzymes, immobilized enzymes, and ergothioneine. The invention provides amino-substituted enzyme mutants capable of replacing the amino group of histidine with trimethylamine to produce trimethylhistidine (HERCYNINE); and then binding to the enzyme trimethylhistidine oxidase (HerOxy, uniprotID: A7IP 38) of xanthobacter autotrophicum (Xanthobacterautotrophicus), which is capable of transferring cysteine-SH to trimethylhistidine azacyclic rings; and finally, the lyase (HERCYLYASE, UNIPROTID: A0A1G7NE 74) in the klebsiella (Klenkiabrasiliensis) body lyses the oxidase product of the last step to obtain ergothioneine.

The invention uses the ergothioneine in vitro enzymatic preparation method to jump out the known synthetic path in organisms, creatively combines a novel amino acid substitution enzyme (HisMuTa), trimethylhistidine oxidase (HerOxy) and lyase (HERCYLYASE) for use, and directly converts histidine into ergothioneine with high yield at one time. The method has the characteristics of high concentration of catalytic substrate, simple operation, high purity of the product and the like, so that the method is suitable for large-scale production of ergothioneine.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides amino-displacing enzyme mutants comprising any one or more of the following site mutations:

L8I; and/or

Q70V; and/or

D71K; and/or

A186L; and/or

S192T; and/or

L201V; and/or

V205C; and/or

S207M; and/or

N304I; and/or

M308S; and/or

D443H; and/or

E470M。

In some embodiments of the invention, the amino-displacing enzyme mutant has:

(I) An amino acid sequence shown as SEQ ID NO. 1; or (b)

(II) a sequence of 1 or more amino acids substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (I); or (b)

(III) a sequence having 90% or more homology with the amino acid sequence represented by (I) or (II).

The invention also provides a complex enzyme comprising the amino-substituted enzyme mutant, trimethylhistidine oxidase and lyase.

In some embodiments of the invention, trimethylhistidine oxidase (HerOxy) is derived from xanthobacter autotrophicum (Xanthobacterautotrophicus, uniprotID: A7IP 38).

In some embodiments of the invention, the lyase (HERCYLYASE) is derived from Klebsiella (Klenkiabrasiliensis) in vivo (UniprotID: A0A1G7NE 74).

In some embodiments of the invention, the amino-substituted enzyme mutant further comprises a protein pairing unit RIAD at the N-terminus; and/or

The N-terminal of the trimethylhistidine oxidase also comprises a protein pairing unit RIDD.

The invention also provides an immobilized enzyme comprising any of the following:

(I) The amino-substituted enzyme mutant; or (b)

(II) the complex enzyme.

The invention also provides a nucleic acid molecule encoding the amino-substituted enzyme mutant, the complex enzyme or the immobilized enzyme, the nucleic acid molecule encoding the amino-substituted enzyme mutant having:

(I) A nucleotide sequence shown as SEQ ID NO. 6; or (b)

(II) a nucleotide sequence which encodes the same protein as the nucleotide sequence shown in (I) but which differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (b)

(III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (I) or (II), and functionally identical or similar to the nucleotide sequence shown in (I) or (II); or (b)

(IV) a nucleotide sequence having a sequence homology of 90% or more with the nucleotide sequence of (I), (II) or (III).

The invention also provides a recombinant vector comprising the nucleic acid and a backbone vector.

The invention also provides a host comprising the recombinant vector.

The invention also provides the application of any of the following in the preparation of ergothioneine:

(I) The amino-substituted enzyme mutant; and/or

(II) the complex enzyme; and/or

(III) the immobilized enzyme; and/or

(IV) the nucleic acid molecule; and/or

(V) the recombinant vector; and/or

(VI), the host.

The invention also provides a method for preparing ergothioneine, comprising preparing ergothioneine using any of the following:

(I) The amino-substituted enzyme mutant; and/or

(II) the complex enzyme; and/or

(III) the immobilized enzyme; and/or

(IV) the nucleic acid molecule; and/or

(V) the recombinant vector; and/or

(VI), the host.

In some embodiments of the invention, the method of making comprises:

taking L-histidine, L-cysteine, dithiothreitol DTT and trimethylamine hydrochloride, regulating the pH value in the presence of a buffer solution, mixing with the amino substitution enzyme mutant, trimethylhistidine oxidase and lyase, connecting, removing enzymes, desalting, concentrating and crystallizing to obtain the ergothioneine;

The pH comprises 8.0;

The buffer solution comprises 100mmol/L of tris (hydroxymethyl) aminomethane hydrochloride solution with pH of 8.0.

In some embodiments of the invention, the amino-substituted enzyme comprises an amino-substituted enzyme, RIAD-amino-substituted enzyme, or an immobilized amino-substituted enzyme.

In some embodiments of the invention, the trimethylhistidine oxidase comprises trimethylhistidine oxidase, RIDD-trimethylhistidine oxidase, or immobilized trimethylhistidine oxidase.

In some embodiments of the invention, the ratio of L-histidine to amino-substituted enzyme, trimethylhistidine oxidase and lyase is comprised of (100-200 mmol): (1000-1500U): (2000-4000U): 1000U.

In some embodiments of the invention, the pH at the time of the attachment comprises 7 to 9.

In some embodiments of the invention, the time of connection comprises 3 to 8 hours.

The invention provides the following beneficial effects:

The invention uses cheap L-histidine, trimethylamine and cysteine as raw materials, and obtains ergothioneine through one-time conversion of amino substitution enzyme, trimethylhistidine oxidase and lyase, so that the enzyme can be crude enzyme liquid or immobilized enzyme. Compared with other traditional chemical and fermentation preparation processes, the preparation method has the advantages of simple preparation route, high substrate conversion rate and high product concentration.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows HisMuta, herOxy, herCyLyase crude enzyme liquid one pot reaction data; wherein, (a) is the content detection of ergothioneine based on an enzymatic method: adding ergothioneine lyase into the diluted reaction solution, reacting for 30min at 30 ℃, and monitoring the change of absorbance at 311 nm; (b) is a liquid-phase spectrogram of the crude enzyme liquid one-pot method reaction.

FIG. 2 shows a spectrum of ergothioneine characterization; wherein, (a) is nuclear magnetic hydrogen spectrum, 1H NMR (400 mhz, d2 o) delta 6.80 (s, 1H), 3.93 (dd, j=11.6, 4.0hz, 1H), 3.29 (s, 11H); (b) Is a nuclear magnetic carbon spectrum, 13CNMR (101 mhz, d2 o) δ 170.06, 155.97, 123.81, 115.25, 77.06, 52.16, 22.83;

FIG. 3 shows RIAD-HisMuta, RIDD-HerOxy, herCyLyase crude enzyme liquid one pot reaction data; wherein, (a) is histidine consumption detection based on enzymatic method: adding histidine lyase into the diluted reaction solution, reacting for 30min at 30 ℃, and monitoring the change of the absorbance at 277 nm; (b) A liquid phase spectrogram of the reaction of the crude enzyme liquid one-pot method (RIAD/RIDD combination);

FIG. 4 shows immobilized HisMuta, herOxy, herCyLyase one-pot reaction data; wherein, (a) is the detection of the ergothioneine production amount based on an enzymatic method: adding ergothioneine lyase into the diluted reaction solution, reacting for 30min at 30 ℃, and monitoring the change of absorbance at 311 nm; (b) a liquid phase spectrogram of the immobilized enzyme one-pot method reaction;

FIG. 5 shows a protein expression profile; wherein 1 is HisMuTa;2 is RIAD-HisMuTa;3 is HerOxy;4 is RIDD-HerOxy;5 is HERCYLYASE;

FIG. 6 shows the route of the ergothioneine enzymatic process of the present invention;

FIG. 7 shows a one-pot process route to ergothioneine using HisMuta, herOxy and HERCYLYASE crude enzyme solutions;

FIG. 8 shows a one-pot process route for ergothioneine using RIAD-HisMuta, RIDD-HerOxy, and HERCYLYASE crude enzyme solutions;

FIG. 9 shows the route to ergothioneine using immobilized HisMuta, herOxy and HERCYLYASE enzyme one-pot methods.

Detailed Description

The invention discloses a preparation method of amino substitution enzyme mutant, complex enzyme, immobilized enzyme and ergothioneine, and a person skilled in the art can properly improve process parameters by referring to the content of the amino substitution enzyme mutant, complex enzyme, immobilized enzyme and ergothioneine. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

The invention aims at providing an amino-substituted enzyme (HisMuTa), experimental tests show that an ammonia lyase (UniprotID: I0W5P 8) in rhodococcus (Rhodococcusimtechensis) body can reversibly catalyze the amino/trimethylamine cleavage capacity of histidine/trimethylhistidine, and by utilizing the characteristic, the amino-substituted enzyme can be finally obtained by directionally evolving the enzyme to obtain a novel and high-activity amino-substituted enzyme (HisMuTa), and the amino-substituted enzyme can replace the amino group of histidine by trimethylamine to generate trimethylhistidine (HERCYNINE); and then binding to the enzyme trimethylhistidine oxidase (HerOxy, uniprotID: A7IP 38) of xanthobacter autotrophicum (Xanthobacterautotrophicus), which is capable of transferring cysteine-SH to trimethylhistidine azacyclic rings; and a lyase (HERCYLYASE, UNIPROTID: A0A1G7NE 74) in the body of Klebsiella (Klenkiabrasiliensis) finally cleaves the product of the above oxidase to obtain ergothioneine (FIG. 6).

The invention also aims to provide a novel preparation method of the ergothioneine, which has the advantages of complicated chemical preparation process route, low yield and low product quality of the ergothioneine at present, and the concentration of the product obtained by the strain fermentation production process is still low, so that the purification cost of the final product is high and the whole production price is high. The preparation method of ergothioneine provided by the invention can ensure the production quality of the product and effectively reduce the production price. Through the excavation, test and modification of the new enzyme functions of the enzyme library, the invention successfully constructs an amino-substituted enzyme to prepare trimethylhistidine, and then the trimethylhistidine oxidase and the lyase can be effectively converted into ergothioneine.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention converts low-cost histidine into high-added-value ergothioneine conveniently and efficiently by utilizing the continuous catalytic capability of an amino substitution enzyme (HisMuTa), trimethyl oxidase (HerOxy) and lyase (HERCYLYASE). The invention also makes certain attempts on the catalytic process, wherein the use of polypeptide pairing (RAID-RIDD) further improves the conversion speed, and the use of immobilized enzyme can well recycle the enzyme. The method has the obvious advantages of simple production process, high conversion rate, less impurities and environmental protection compared with the traditional chemical method and fermentation production method.

The amino acid sequence of the amino substitution enzyme (HisMuTa) used in the invention is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 8; the enzyme is an ammonia lyase (UniprotID: I0W5P 8) derived from Rhodococcus (Rhodococcusimtechensis) at the micro-research institute, which has obtained high activity on histidine and trimethylhistidine by laboratory evolution, and the specific mutation sites are (L8I, Q70V, D71K, A186L, S192T, L201V, V205C, S207M, N304I, M308S, D443H, E470M); RIAD-HisMuTa (amino acid sequence shown as SEQ ID NO:2, nucleotide sequence shown as SEQ ID NO: 9) is obtained by fusion construction of protein pairing unit RIAD (19 AA) at the N-terminus of HisMuTa enzyme.

The amino acid sequence of the trimethylhistidine oxidase (HerOxy) used in the invention is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 10; it is derived from xanthobacter autotrophicum (Xanthobacterautotrophicus, uniprotID: A7IP 38), which is capable of transferring cysteine-SH to trimethylhistidine azacyclic; RIDD-HerOxy (the amino acid sequence is shown as SEQ ID NO:4, the nucleotide sequence is shown as SEQ ID NO: 11) is formed by fusion of a protein pairing unit RIDD (50 AA) at the N-terminal of HerOxy enzyme. And (3) injection: the protein fragment RIAD and RIDD can be paired and combined in a ratio of 1:2, and the combined enzyme has higher catalytic activity and catalytic stability.

The amino acid sequence of the lyase (HERCYLYASE) used in the invention is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 12; it is derived from Klebsiella (Klenkiabrasiliensis) in vivo (UniprotID: A0A1G7NE 74), and can efficiently cleave the product of trimethylhistidine oxidase (HerOxy) to obtain ergothioneine.

The tool enzyme for testing the reaction progress used in the invention is ergothioneine lyase (Uniprot)

ID: M2BPW 8), the amino acid sequence is shown as SEQ ID NO. 6, and the nucleotide sequence is shown as SEQ ID NO. 13. Also histidine lyase (UniprotID: A0A429B4Z 1), the amino acid sequence is shown in SEQ ID NO. 7, and the nucleotide sequence is shown in SEQ ID NO. 14.

The amino acid sequence and DNA sequence of the enzyme adopted by the invention are shown in tables 1 and 2:

table 1 amino acid sequence of enzyme

TABLE 2 DNA sequence of enzymes

The amino-substituted enzyme mutant, the complex enzyme, the immobilized enzyme and the raw materials and reagents used in the preparation method of ergothioneine provided by the invention can be purchased from the market unless otherwise specified.

The invention is further illustrated by the following examples:

example 1 preparation of enzyme

Fermentation production of enzyme:

the enzyme required by the invention is prepared by constructing a specific expression plasmid after the company synthesizes corresponding genes and then fermenting and producing the specific expression plasmid by escherichia coli; the method specifically comprises the following steps:

The genes corresponding to the enzymes are synthesized by general biological company (Chuzhou Anhui) after sequence optimization, and then subcloned into a pET 28a expression vector. Plasmid with correct sequence was confirmed to be transferred into E.coli (BL 21) competent cells plate culture (of the species Prinsepia) and monoclonal miniculture, the bacteria with correct protein expression are finally amplified and cultured step by step. The method specifically comprises the steps of transferring a single colony into 5mL of LB culture solution (37 ℃) containing 50 mu mol/L kanamycin for culture, inoculating the cell into 250mL of LB culture solution containing the same antibiotics after growing to a logarithmic phase, transferring the cell into a 5L culture fermentation tank for culture when growing to the same logarithmic phase, and carrying out final protein expression. In the culture of a 5L fermentation tank, 0.5mmol/L isopropyl-beta-D-thiopyran galactoside (IPTG) is added at 25 ℃ to induce the protein to express for 6 hours when the cell OD-20 is cultured, and finally the cell is collected by high-speed centrifugation (4000 rpm,20 min) to obtain 30-60 g of wet cell with over-expressed enzyme. A small amount of cells are firstly mixed with a Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) buffer solution (50 mmol/L, pH 8.0) on an ice basin uniformly, then the cells are broken by a freeze thawing method, and the clear liquid after cell wall removal by high-speed centrifugation is subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. The results of the protein are shown in FIG. 5. Cells with correct protein expression were used for the next catalytic experiment, specifically, the remaining cells were mixed with Tris-HCl buffer (50 mmol/L, pH 8.0) at low temperature (200 mL buffer mixing with 10 g wet cells), then the cell walls were crushed at low temperature Gao Yapo and centrifuged at high speed (16000 rpm,45 min) to remove the cell walls and obtain enzyme-containing supernatant (the enzyme activities obtained for the three enzymes were varied from 100 to 1000U/mL, U was the amount of enzyme required for converting 1. Mu. Mol of substrate at 37℃for one minute). LB medium consisted of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate and 5% glycerol.

Immobilization of enzymes:

The three enzymes HisMuta, herOxy and HERCYLYASE are mixed and immobilized at a certain ratio.

The above collected crude enzyme supernatant was slowly added with ammonium sulfate solids to precipitate out protein solids (30-50% w/v ammonium sulfate: buffer), which were collected by high-speed centrifugation (10000 rpm,10 minutes) and slowly dissolved in 25mmol/L Tris buffer (buffer A) at pH8.0, then dialyzed (twice, 4 hours apart) in 50 volumes of buffer A to remove ammonium sulfate from the enzyme solution, finally the dialysate was loaded onto DEAE SEPLITE FF (Siemens blue) anion exchange column (NaCl gradient elution: 0-1 NNaCl) to obtain initially purified HisMuta, herOxy and HERCYLYASE enzyme solutions, and these enzymes were then purified using LX-1000EP epoxy (Siemens blue) in accordance with activity unit 1:2:1 is mixed and fixed at one time by the following method:

4000U of purified mixed enzyme is dissolved in 1L of 50mmol/L potassium phosphate (buffer B) solution with pH of 8.0, then 20-60 mmol/L phenoxyacetic acid and 800 g LX-1000EP epoxy resin are added until the mixture is stirred at room temperature for 10 hours, the mixture is filtered to obtain immobilized enzyme, and finally the immobilized enzyme is washed twice by clean water and buffer B and then is preserved at low temperature for standby, wherein the immobilized enzyme has 50-80% of initial activity.

Example 2 comparison of amino-substituted enzyme Effect

To 1L 100mmol/L of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) solution at pH 8.0 were added 31.0 g of L-histidine (200 mmol/L), 36.3 g of L-cysteine (300 mmol/L), 3.1 g of dithiothreitol DTT (20 mmol/L) and 38.2 g of trimethylamine hydrochloride (400 mmol/L); the pH value of the reaction liquid is adjusted to 8.0, 1500U of amino substitution enzyme is added one by one, 4000U He rOxy and 1000U HerCyLyase enzyme are used for starting the reaction, air is continuously introduced into the reaction system for stirring, the pH value of the reaction system is controlled to be maintained between 7 and 9 by utilizing acid-base regulation, and the reaction system is slightly stirred for 3 hours at 37 ℃. Mutants 1,2,3, which have fewer wild-type and mutant sites, all showed very low yields. The reaction is greatly enhanced when the mutation points of the key catalytic residues, such as mutants 4 and 5, are added. Mutant 6 was optimal, under this condition, the conversion was 42%. The results of ergothioneine yield are shown in Table 3.

TABLE 3 yields of ergothioneine by different enzymes

Example 3 one pot method of ergothioneine Using HisMuta, herOxy and HERCYLYASE crude enzyme solution

FIG. 7 shows a route for ergothioneine prepared in one pot using HisMuta, herOxy and HERCYLYASE crude enzyme solution prepared in example 1, 31.0 g L-histidine (200 mmol/L), 36.3 g L-cysteine (300 mmol/L), 3.1 g dithiothreitol DTT (20 mmol/L) and 38.2 g trimethylamine hydrochloride (400 mmol/L) were added to 1L of 100mmol/L Tris/L pH8.0 solution; the pH value of the reaction liquid is adjusted to 8.0, 1500U HisMuta,4000U HerOxy and 1000U HerCyLyase enzymes are added one by one to start the reaction, air is continuously introduced into the reaction system for stirring, the pH value of the reaction system is controlled to be between 7 and 9 by utilizing acid-base regulation, and the reaction system is slightly stirred for 8 hours at 37 ℃. The maximum yield of ergothioneine was found by HPLC detection using ergothioneine lyase (311 nm absorbance) (see FIG. 1). Finally, the pH was adjusted to 1.0 with aqueous HCl. Enzyme in the reaction system is denatured, precipitated and centrifugally removed, the pH of the reaction solution is adjusted to 7.0, the reaction solution is sampled and purified by D201 anion exchange resin, then the crude product is desalted by a reverse osmosis membrane, concentrated and crystallized (ethanol: water, 1:1, v: v) to obtain 29.5 g of pale yellow ergothioneine solid, the crystallization purity is 98.2% (the final yield is 64%), and the structural characterization is carried out on the pale yellow ergothioneine solid, as shown in figure 2.

Example 4 preparation of ergothioneine in one pot Using RIAD-HisMuta, RIDD-HerOxy and HERCYLYASE crude enzyme solution

A scheme of the method for preparing ergothioneine by using RIAD-HisMuta, RIDD-HerOxy and HERCYLYASE crude enzyme liquid one-pot method is shown in FIG. 8, wherein RIAD-HisMuTa (the amino acid sequence is shown as SEQ ID NO:2, the nucleotide sequence is shown as SEQ ID NO: 9) is formed by fusing a protein pairing unit RIAD (19 AA) at the N-end of HisMuTa enzyme, and RIDD-HerOxy (the amino acid sequence is shown as SEQ ID NO:4, the nucleotide sequence is shown as SEQ ID NO: 11) is formed by fusing a protein pairing unit RIDD (50 AA) at the N-end of HerOxy enzyme. Similar to example 2, but with a different amount of enzyme and reaction time. To 1L 100mmol/L of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) solution at pH 8.0 were added 31.0 g of L-histidine (200 mmol/L), 36.3 g of L-cysteine (300 mmol/L), 3.1 g of dithiothreitol DTT (20 mmol/L) and 28.6 g of trimethylamine hydrochloride (300 mmol/L); the pH value of the reaction liquid is adjusted to 8.0, 1500U RIAD-HisMuta,2000U RIDD-HerOxy and 1000U HerCyLyase enzyme are added one by one to start the reaction, air is continuously introduced into the reaction system to stir, the pH value of the reaction system is controlled to be maintained between 7and 9 by utilizing acid-base adjustment, and the reaction system is slightly stirred for 4 hours at 37 ℃; the consumption of the starting material L-histidine was found to be complete by histidine cleavage (277 nm absorbance) and HPLC detection (see FIG. 3). Finally, regulating the pH value to 1.0 by using a hydrochloric acid aqueous solution, carrying out denaturation precipitation and centrifugal removal on enzyme in a reaction system, regulating the pH value of a reaction solution to 7.0, then carrying out crude purification by using a D201 anion exchange resin, and then desalting, concentrating and crystallizing the crude product by using a reverse osmosis membrane (ethanol: water, 1:1, v: v) to obtain 41 g of pale yellow ergothioneine solid, wherein the crystallization purity is more than 98% (the final yield is 89%).

Example 5 one pot method of ergothioneine Using immobilized HisMuta, herOxy and HERCYLYASE enzyme

The route patterns of the immobilized HisMuta, herOxy and HERCYLYASE enzyme one-pot method prepared in example 1 are shown in FIG. 9, which are similar to those of examples 2 and 3, but the immobilized enzyme reaction is a heterogeneous reaction (the enzyme is solid and the raw material is solution), so that the immobilized enzyme can be well post-treated and recycled.

15.5 G L-histidine (100 mmol/L), 18.2 g L-cysteine (150 mmol/L), 3.1 g dithiothreitol DTT (20 mmol/L) and 19.1 g trimethylamine hydrochloride (200 mmol/L) were added to 1L 100mmol/L of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) solution at pH 8.0; the pH value of the reaction solution is adjusted to 8.0, immobilized amino-substituted enzyme HisMuta U, immobilized oxidase HerOxy U, immobilized lyase HERCYLYASE U and immobilized enzyme are added one by one to start the reaction, air is continuously introduced into the reaction system in the reaction process, the pH value of the reaction system is controlled between 7 and 9, the reaction system is slightly stirred for 6 hours at 30 ℃, the maximum ergothioneine yield of the reaction system is monitored by utilizing ergothioneine lyase (311 nm absorption value) and HPLC (see figure 4), and finally the immobilized enzyme is directly filtered to terminate the reaction and recycle the enzyme. The immobilized enzyme mixture is washed three times with 25mmol/L cold Tris-HCl buffer (pH 8.0) for standby (the immobilized enzyme has 85% initial activity after 8 continuous use); the reaction solution is directly loaded on D201 anion exchange resin for crude purification, and finally, the crude product is desalted by a reverse osmosis membrane, concentrated and crystallized (ethanol: water, 1:1, v: v) to obtain 16.3 g of ergothioneine solid, and the purity of the crystallized product is more than 98 percent (yield 71 percent).

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Amino acid substitution enzyme mutant, characterized by comprising any one or more of the following site mutations:

L8I; and/or

Q70V; and/or

D71K; and/or

A186L; and/or

S192T; and/or

L201V; and/or

V205C; and/or

S207M; and/or

N304I; and/or

M308S; and/or

D443H; and/or

E470M。

2. A complex enzyme comprising the amino acid substitution enzyme mutant, trimethylhistidine oxidase and lyase according to claim 1.

3. The complex enzyme of claim 2, wherein the amino-substituted enzyme mutant further comprises a protein pairing unit RIAD at the N-terminus; and/or

The N-terminal of the trimethylhistidine oxidase also comprises a protein pairing unit RIDD.

4. An immobilized enzyme, comprising any of the following:

(I) An amino acid substitution enzyme mutant according to claim 1; or (b)

(II) the complex enzyme according to claim 2 or 3.

5. A nucleic acid molecule encoding the amino acid substitution enzyme mutant according to claim 1, the complex enzyme according to claim 2 or 3 or the immobilized enzyme according to claim 4, wherein the nucleic acid molecule encoding the amino acid substitution enzyme mutant has:

(I) A nucleotide sequence shown as SEQ ID NO. 6; or (b)

(II) a nucleotide sequence which encodes the same protein as the nucleotide sequence shown in (I) but which differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (b)

(III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (I) or (II), and functionally identical or similar to the nucleotide sequence shown in (I) or (II); or (b)

(IV) a nucleotide sequence having a sequence homology of 90% or more with the nucleotide sequence of (I), (II) or (III).

6. A recombinant vector comprising the nucleic acid according to claim 5 and a backbone vector.

7. A host comprising the recombinant vector of claim 6.

8. Use of any of the following in the preparation of ergothioneine:

(I) An amino acid substitution enzyme mutant according to claim 1; and/or

(II) the complex enzyme of claim 2 or 3; and/or

(III) the immobilized enzyme of claim 4; and/or

(IV) the nucleic acid molecule of claim 5; and/or

(V) the recombinant vector of claim 6; and/or

(VI) the host of claim 7.

9. A process for the preparation of ergothioneine, comprising preparing ergothioneine using any of the following:

(I) An amino acid substitution enzyme mutant according to claim 1; and/or

(II) the complex enzyme of claim 2 or 3; and/or

(III) the immobilized enzyme of claim 4; and/or

(IV) the nucleic acid molecule of claim 5; and/or

(V) the recombinant vector of claim 6; and/or

(VI) the host of claim 7.

10. The method of manufacturing according to claim 9, wherein the method of manufacturing comprises:

taking L-histidine, L-cysteine, dithiothreitol DTT and trimethylamine hydrochloride, regulating the pH value in the presence of a buffer solution, mixing with the amino substitution enzyme mutant, trimethylhistidine oxidase and lyase, connecting, removing enzymes, desalting, concentrating and crystallizing to obtain the ergothioneine;

The pH comprises 8.0;

the buffer solution comprises 100mmol/LpH8.0 of tris hydrochloride solution.