Method for detecting histidine betaine through enzyme coupling

Abstract

The invention discloses an application of an ergothioneine trimethylamine lyase in detection of an ergothioneine precursor compound, namely L-histidine betaine. L-histidine betaine is catalyzed by ergothioneine trimethylamine lyase to be hydrolyzed into urocanic acid, the light absorption value of the urocanic acid at 290 nm is detected through a colorimetric method, and the content of L-histidine betaine can be determined. The method is high in sensitivity and capable of detecting 2-39 mg / L of L-histidine betaine, the lower limit of detection is 50 times of the lower limit of HPLC detection, the method can be carried out in a common laboratory, operation is easy and convenient, and a new strategy for measuring the content of L-histidine betaine is developed. The invention also provides the ergothioneine trimethylamine lyase with high enzyme activity.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to an enzyme-coupled method for detecting the ergothioneine precursor compound L-histidine betaine, specifically involving the application of an ergothioneine trimethylamine lyase in the detection of L-histidine betaine. Background Technology

[0002] L-Ergothioneine is an important non-natural amino acid. This compound possesses physiological protective functions such as antioxidation, free radical scavenging, immune enhancement, anti-radiation, and anti-aging. It has broad application prospects in the food, cosmetics, functional food, and biopharmaceutical industries.

[0003] L-histidine betaine is a key precursor compound for the synthesis of ergothioneine. As reported in patent CN 117083377B, this compound can be synthesized from L-histidine and formaldehyde to N,N-dimethyl-L-histidine. Subsequently, using iodomethane as a methylating agent, N,N-dimethyl-L-histidine is converted to L-histidine betaine via a methyl substitution reaction, followed by separation and purification to obtain the final product.

[0004]

[0005] The purity of L-histidine betaine determines the quality of ergothioneine, thus requiring strict quality control of this compound. However, existing technologies only disclose a method for detecting L-histidine betaine using HPLC (CN118330050A). This method has low sensitivity and struggles to distinguish common impurities in the synthesis of L-histidine betaine, such as N,N-dimethyl-L-histidine and L-histidine. Furthermore, HPLC detection of L-histidine betaine requires a high-performance chromatograph, a dedicated HPLC workroom, and specialized operators, which are prohibitively expensive for ordinary laboratories. Summary of the Invention

[0006] To achieve rapid, accurate, and simple detection of L-histidine betaine, we attempted to use its light absorption characteristics for quantitative detection via spectrophotometry (colorimetry). Spectrophotometers are readily available in any chemical laboratory, offering inexpensive, space-saving, and easy-to-operate qualitative and quantitative analysis. However, experiments revealed that due to the presence of the imidazole group, the absorption peak of L-histidine betaine is very close to that of N,N-dimethyl-L-histidine and L-histidine, making spectrophotometric detection impossible.

[0007] Therefore, we devised a method to detect the content of L-histidine betaine using urocanic acid, a decomposition product of L-histidine betaine. Urocanic acid has unique light absorption characteristics different from amino acids, with an absorption peak at 290 nm.

[0008] Uroic acid, chemically known as β-imidazolium acrylic acid (CAS number 104-98-3), is an acrylic acid compound containing an imidazolium group and is soluble in water.

[0009]

[0010] Our research found that certain ergothionases (EGTs) can catalyze the breakdown of L-histidine betaine into uric acid and trimethylamine.

[0011]

[0012] We screened ergothioneine trimethylamine lyases from various microbial sources, and experiments confirmed that the ergothioneine trimethylamine lyase from *Blautia producta* (Uniport ID A0A6P1Z204) possesses this function. Furthermore, to improve enzyme activity, based on the constructed enzyme model pocket structure, mutations were performed at certain amino acid sites, resulting in the screening of a K386L mutant with significantly enhanced enzyme activity, capable of efficiently converting L-histidine betaine into uric acid and trimethylamine. Based on the above research results, this invention includes the following technical solution:

[0013] The first aspect of this invention provides the use of ergothionase (EGT) in the detection of the ergothion precursor compound -L-histidine betaine.

[0014] Preferably, the ergothionase described above is an ergothionase derived from Blautiaproducta with an amino acid sequence as shown in SEQ ID NO: 1 (Uniport ID: A0A6P1Z204) or a conserved variant polypeptide thereof. The conserved variant polypeptide is a mutant with an amino acid sequence that has more than 90% homology with SEQ ID NO: 1, preferably more than 92% homology, preferably more than 95% homology, preferably more than 96% homology, preferably more than 97% homology, preferably more than 98% homology, more preferably more than 99% homology, and with increased enzyme activity.

[0015] MQEKVIRLTGSELTISQIKEIAFENAKVEVDAEAMTRVQKARELIFELDKRGVAVYGLNTGVGWNKDKVVYADRYDAYNKNLLRSHMIGVGPECSIPETRTIMAVRLNGFLCGHTGVAPEIVQYYVE FLNRGVHPVIKSRGSVGEADIATLPAIGLTAIGEGEAYYQNQCMESAKALELSGLEPLKLGPKDGLGIVSSNAQGAAFAAMGVIEAEQFIERYRRVFCLALEGLNGVLDPMDESVNRERGYQGQMES ARKCRELLKGSYLEKPWEGRALQDPLSFRCQSAITGSVMDALAYLKQQLGVELNATDDNPCLLPEEDRMCGSPNFEPLTWVLAVEMASTGLAHMSKMISQQILRIGDPGFTKLNRFLTPAEGAVIAY GTIQKTVSYLDTENRMYANPCSLDFLSMAGHIEDTASNSTMAASNMRKIIDNLYYMAAIELMHAAQAVDLREIPQLGEGTKPLFEAYRKKVPYLDNDRNMSVDIQKTYEFLKSYKALEHHHHHH (SEQ ID NO: 1).

[0016] Furthermore, the above-mentioned conserved variant polypeptide is an ergothionein trimethylamine lyase mutant with the amino acid sequence shown in SEQ ID NO: 3 (which is the K386L mutant of the wild-type enzyme EGT, referred to as EGT-386 in this article).

[0017] MQEKVIRLTGSELTISQIKEIAFENAKVEVDAEAMTRVQKARELIFELDKRGVAVYGLNTGVGWNKDKVVYADRYDAYNKNLLRSHMIGVGPECSIPETRTIMAVRLNGFLCGHTGVAPEIVQYYVE FLNRGVHPVIKSRGSVGEADIATLPAIGLTAIGEGEAYYQNQCMESAKALELSGLEPLKLGPKDGLGIVSSNAQGAAFAAMGVIEAEQFIERYRRVFCLALEGLNGVLDPMDESVNRERGYQGQMES ARKCRELLKGSYLEKPWEGRALQDPLSFRCQSAITGSVMDALAYLKQQLGVELNATDDNPCLLPEEDRMCGSPNFEPLTWVLAVEMASTGLAHMSKMISQQILRIGDPGFTKLNRFLTPAEGAVIAY GTIQLTVSYLDTENRMYANPCSLDFLSMAGHIEDTASNSTMAASNMRKIIDNLYYMAAIELMHAAQAVDLREIPQLGEGTKPLFEAYRKKVPYLDNDRNMSVDIQKTYEFLKSYKALEHHHHHH (SEQ ID NO: 3).

[0018] A second aspect of the present invention provides a method for detecting L-histidine betaine, comprising the following steps:

[0019] (1) Weigh the analyte (which may contain L-histidine betaine) and dissolve it in water or aqueous solution A, or dilute it with water or aqueous solution A to prepare a transparent aqueous solution B;

[0020] (2) Adjust the pH of aqueous solution B to 8-9, preferably around pH 8.5, and add 0.02-0.2 U / mL, preferably around 0.5 U / mL (corresponding to an EGT-386 concentration of around 8.3 μg / mL). React at 20-35℃ for 15-45 minutes, for example, at room temperature for 30 minutes, to ensure that the L-histidine betaine reaction is complete, and obtain reaction solution C;

[0021] (3) The absorbance of reaction solution C at 290 nm was detected by spectrophotometry (colorimetric method). The content of L-histidine betaine in the analyte was determined by using the standard curve of uric acid aqueous solution and the equimolar relationship between uric acid and L-histidine betaine.

[0022] Optionally, in step (1) above, if the analyte is added to water to form a turbid liquid, a transparent aqueous solution B is obtained by centrifugation / filtration and / or decolorization with activated carbon.

[0023] Furthermore, in step (1) above, the aqueous solution A is a buffer solution with pH 8-9, preferably around pH 8.5, such as phosphate buffer solution (PBS).

[0024] Unless otherwise defined, in describing numerical characteristics herein, the terms “about,” “approximately,” or “around” refer to a range that reasonably fluctuates around the stated value. The range of numerals used in this invention includes both the numeral itself and any number within that range.

[0025] In one embodiment, in step (3) above, the spectrophotometry (colorimetric method) is performed using a spectrophotometer or an enzyme-linked immunosorbent assay (ELISA) reader.

[0026] Preferably, the analyte is a synthetic product of L-histidine betaine.

[0027] A third aspect of the present invention provides an ergothionein trimethylamine lyase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0028] A fourth aspect of the present invention provides a gene encoding the ergothioneine trimethylamine lyase mutant SEQ ID NO: 3 as described above.

[0029] Preferably, the nucleotide sequence of the encoding gene of the above-mentioned ergothionein trimethylamine lyase mutant SEQ ID NO: 3 is shown in SEQ ID NO: 4.

[0030] A fifth aspect of the present invention provides a DNA molecule comprising the genes described above.

[0031] A sixth aspect of the present invention provides a kit for detecting L-histidine betaine, which determines the content of L-histidine betaine in the analyte by colorimetric method. The kit includes: ergothionein trimethylamine lyase as described above; a buffer or buffer solution such as phosphate buffered solution (PBS); and uric acid standard for preparing a standard curve.

[0032] Furthermore, the kit also includes centrifuge tubes, cups, stir bar, colorimetric tubes or multi-well plates, activated carbon, etc.

[0033] This invention, through gene mining, obtained an ergothionase (EGT) derived from *Blautia producta* capable of catalyzing the breakdown of L-histidine betaine to produce uric acid. Based on this, directed evolution of EGT yielded a mutant, EGT-386, with significantly enhanced enzyme activity. This mutant can be used for the precise detection of L-histidine betaine, thus utilizing the absorbance of uric acid at 290 nm to develop a novel method for detecting L-histidine betaine. The enzyme-coupled detection method for L-histidine betaine developed in this invention exhibits high sensitivity, capable of detecting 2-39 mg / L L-histidine betaine, with a detection limit 50 times higher than that of HPLC. This method also demonstrates good specificity, detecting only L-histidine betaine and is unaffected by impurities involved in the chemical synthesis route of L-histidine betaine, such as histidine or N,N-dimethyl-L-histidine. Attached Figure Description

[0034] Figure 1 The plasmid map of the expression vector pET24b-EGT for wild-type ergothionein trimethylamine lyase is shown.

[0035] Figure 2 The standard curve of uric acid established in the examples is shown.

[0036] Figure 3 The test parameters and chromatograms for detecting enzyme activity using the HPLC method are shown. The top figure shows the HPLC test parameters; the bottom figure shows the HPLC chromatogram.

[0037] Figure 4 The L-histidine betaine standard curve for detecting L-histidine betaine content by enzyme coupling method in the examples is shown.

[0038] Figure 5 The standard curve for the detection of mixtures containing L-histidine betaine using the EGT-386 enzyme-coupled method is shown. Detailed Implementation

[0039] We have, for the first time, proposed a spectrophotometric (colorimetric) method to replace the costly and technically demanding HPLC method for the detection of L-histidine betaine. Utilizing the unique 290 nm absorption peak of uric acid, a degradation product of L-histidine betaine, which differs from histidine and N,N-dimethyl-L-histidine, we developed an enzyme-coupled detection method. Compared to HPLC, the detection method developed in this invention exhibits high sensitivity, a linear detection range of 2-39 mg / L for L-histidine betaine, and good specificity, unaffected by interference from histidine and N,N-dimethyl-L-histidine.

[0040] The key to achieving enzyme-coupled detection of L-histidine betaine is discovering an enzyme that can break down L-histidine betaine to produce uric acid. Fortunately, based on bioinformatics big data, we experimentally discovered that an ergothionase (EGT) possesses this function.

[0041] Ergothioneine trimethylamine lyase is a lyase that can break down ergothioneine into trimethylamine and thiouric acid in an anaerobic environment. The intermediate products generated may be further metabolized by other microorganisms to obtain energy.

[0042] High enzyme activity is a prerequisite for the industrial application of enzyme catalysis. Therefore, we also conducted mutation screening on the wild-type enzyme (UniportID A0A6P1Z204) in hopes of obtaining mutants with the highest possible enzyme activity. Site-directed saturation mutagenesis screening yielded several mutants with increased enzyme activity, among which the K386L mutant showed the greatest increase.

[0043] In this article, the terms “(enzyme activity) increase,” “enhancement,” or “increase” used above mean an increase of at least 50% or more compared to the reference level, such as at least 80% or more, at least about 1, at least about 2, at least about 3, at least about 4, or at least about 5 times the reference level.

[0044] In this article, the terms “wild-type ergothioneine trimethylamine lyase” and “wild-type enzyme” have the same meaning, both referring to the ergothioneine trimethylamine lyase with the amino acid sequence SEQ ID NO: 1 (Uniport ID A0A6P1Z204).

[0045] Correspondingly, the terms "ergothioneine trimethylamine lyase mutant," "mutant ergothioneine trimethylamine lyase," "mutant," and "mutant enzyme" have the same meaning, all referring to mutants of ergothioneine trimethylamine lyase with increased enzyme activity, such as SEQ ID NO: 3. For the sake of brevity and convenience, wild-type ergothioneine trimethylamine lyase and its mutants may be collectively referred to as "ergothioneine trimethylamine lyase" in this invention, as long as it is not confused with the wild-type enzyme SEQ ID NO: 1.

[0046] The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated into another aliphatic residue or another nonpolar residue.

[0047] The ergothioneine trimethylamine lyase mutant of the present invention has 505 amino acids and a well-defined sequence. Therefore, those skilled in the art can easily obtain its encoding gene, expression cassettes (DNA molecules) containing these genes, plasmids, and transformants containing the plasmids.

[0048] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0049] In order to optimally express ergothionein trimethylamine lyase SEQ ID NO: 1 and its mutant SEQ ID NO: 3 in microbial hosts such as Escherichia coli, which is most commonly used in genetic engineering, the present invention has optimized the codons of its expression gene.

[0050] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Consequently, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0051] After codon optimization, the gene encoding wild-type ergothionein trimethylamine lyase SEQ ID NO: 1 can be SEQ ID NO: 2, while the gene encoding the mutant ergothionein trimethylamine lyase SEQ ID NO: 3 can be SEQ ID NO: 4.

[0052] Ergothionein trimethylamine lyase, obtained by fermentation with engineered microorganisms, can be used for the detection of L-histidine betaine.

[0053] Those skilled in the art will readily understand that, in order to promote and popularize this new use of ergothioneine trimethylamine lyase, it can be offered to the market in the form of a kit containing ergothioneine trimethylamine lyase and urocanic acid standards, as well as colorimetric tubes / multiwell plates.

[0054] Since ergothionein trimethylamine lyase does not catalyze the reaction between L-histidine and N,N-dimethyl-L-histidine, it improves the accuracy and specificity of the purity / content of the target product in the synthesis of L-histidine betaine.

[0055] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the invention.

[0056] Example

[0057] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0058] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0059] Materials and methods:

[0060] The primer synthesis and gene sequencing in the examples were performed by Nanjing Genscript Biotech Co., Ltd.

[0061] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017. Specific experimental conditions can be determined through simple experiments if necessary.

[0062] PCR amplification experiments should be performed according to the reaction conditions provided by the reagent supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0063] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium with an additional 20 g / L agar powder.)

[0064] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5.

[0065] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0066] It should be noted that, for the sake of convenience, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share the same number. This is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0067] Example 1: Construction of EGT-overexpressing engineered bacteria

[0068] We obtained a potential ergothionase (EGT, Uniport ID A0A6P1Z204) from *Blautia producta*, with its amino acid sequence shown in SEQ ID NO: 1. After obtaining the corresponding FASTA sequence from the Uniprot library, we commissioned Nanjing Genscript Biotech Co., Ltd. to perform codon optimization based on *E. coli*, obtaining the gene sequence SEQ ID NO: 2 suitable for expression in *E. coli*. The nucleotide sequence SEQ ID NO: 2 was inserted into plasmid pET24b, with NdeI and XhoI restriction sites at the 5' and 3' ends, respectively, to remove the stop codon inherent in the target sequence, thus enabling the target protein to carry a C-terminal his tag. The constructed plasmid was named pET24b-EGT, and its map is shown below. Figure 1 As shown.

[0069] The plasmid pET24b-EGT was transformed into BL21(DE3) competent cells to obtain engineered bacteria EGT. The bacteria were cultured overnight at 37°C on kanamycin-resistant plates. One single colony was selected and inoculated into a test tube containing LB medium and a final concentration of 50 mg / L kanamycin. The bacteria were cultured overnight and then preserved.

[0070] Example 2: Fermentation of engineered bacteria and purification of EGT protein

[0071] The engineered strain EGT was inoculated at a ratio of 1% into a 1000 mL shake flask containing 400 mL LB medium and a final concentration of 50 mg / L kanamycin, and incubated at 37°C and 220 rpm for approximately 6 hours. OD 600 The culture temperature was increased to 0.8-1.0. IPTG was then added to a final concentration of 0.5 mM for induction, the temperature was lowered to 18°C, and the culture speed was adjusted to 120 rpm for 16 hours. The cells were then obtained by centrifugation. After obtaining wet cells, the collected cells were disrupted using an ultrasonic homogenizer. The protein was purified according to the Thermo Fisher HisPur™ Ni-NTA Resin standard protocol. The purified protein was quantified using the Bradford assay.

[0072] Example 3: Enzyme Activity Assay

[0073] 1. Establishment of the standard curve for uric acid

[0074] Since uric acid exhibits a maximum absorption peak at 290 nm, its concentration can be quantitatively analyzed based on this. A 1 g / L stock solution of uric acid was prepared using 0.1 M NaOH solution as the solvent. Standard solutions of uric acid with concentrations of 4, 8, 12, 16, and 20 mg / L were obtained through serial dilution with 0.2 M phosphate buffer (pH 8.5). 300 μL of each solution was added to an ELISA plate, and the absorbance was measured at 290 nm. A standard curve for uric acid was established based on these readings. (See [link to relevant documentation]). Figure 2 .

[0075] 2. Enzyme activity assay reaction system

[0076] The enzyme activity assay reaction system consisted of a 300 μL reaction volume, including 1.93 g / L L-histidine betaine, 0.2 M phosphate buffer (pH 8.5), and 0.33 μg / mL purified enzyme (Ni-NTA purified). The reaction was carried out at room temperature for 30 min. The difference in absorbance between the sample at 30 min and 0 min was used to evaluate enzyme activity. Each reaction was tested in triplicate.

[0077] Enzyme activity unit definition: Under pH 8.5 and room temperature conditions, the amount of enzyme (mg) required to catalyze the production of 1 μmole of uric acid from L-histidine betaine per minute is defined as 1 unit (U).

[0078] Reaction time / min <![CDATA[OD 290 -CK]]> <![CDATA[OD 290 -EGT]]> 0 0.955 0.946 30 0.958 0.955

[0079] Based on the above results, the activity of EGT in catalyzing the detrimethylamine reaction of L-histidine betaine is calculated to be 0.08 U / mg. This is far less than the industrial enzyme activity standard (10 U / mg), making it necessary to improve the EGT enzyme activity through mutagenesis.

[0080] Example 4: Construction and screening of EGT saturated mutant libraries

[0081] We constructed an enzyme model of EGT using the EGT amino acid sequence as a template and performed saturation mutagenesis at 26 sites in the pocket structure. These sites are 56, 57, 61, 62, 63, 65, 66, 82, 85, 86, 143, 146, 148, 193, 197, 313, 329, 409, 277, 280, 369, 370, 379, 382, ​​385, and 386. The primer list is shown in Table 1 below.

[0082] The three forward (F) primers for each site were mixed in a ratio of NDT:VHG:TGG = 12:9:1 before PCR. PCR system: Primer 25 μL, primers F / R (10 mM) 2 μL each, H2O 20 μL, plasmid 1 μL (~50 ng / μL).

[0083] The PCR program was as follows: 1 cycle: 98℃ 30 s, 25 cycles: 98℃ 10 s, 60℃ 10 s, 72℃ 3 min 30 s, 1 cycle: 72℃ 5 min.

[0084] Table 1. Primers used in the mutation

[0085] EGT4 56F-NDT AAGCGTGGTGTAGCCGTANDTGGCTTAAACACCGGTGTTG EGT4 56F-VHG AAGCGTGGTGTAGCCGTAVHGGGCTTAAACACCGGTGTTG EGT4 56F-TGG AAGCGTGGTGTAGCCGTATGGGGCTTAAACACCGGTGTTG EGT4 57F-NDT AAGCGTGGTGTAGCCGTATACNDTTTAAACACCGGTGTTGGCT EGT4 57F-VHG AAGCGTGGTGTAGCCGTATACVHGTTAAACACCGGTGTTGGCT EGT4 57F-TGG AAGCGTGGTGTAGCCGTATACTGGTTAAACACCGGTGTTGGCT EGT4 56-57R TACGGCTACACCACGCTTGTCCAACTCAAAAAT EGT4 61F-NDT GTATACGGCTTAAACACCNDTGTTGGCTGGAATAAAGATAAGGTGGTG EGT4 61F-VHG GTATACGGCTTAAACACCVHGGTTGGCTGGAATAAAGATAAGGTGGTG EGT4 61F-TGG GTATACGGCTTAAACACCTGGGTTGGCTGGAATAAAGATAAGGTGGTG EGT4 62F-NDT GTATACGGCTTAAACACCGGTNDTGGCTGGAATAAAGATAAGGTGGTG EGT4 62F-VHG GTATACGGCTTAAACACCGGTVHGGGCTGGAATAAAGATAAGGTGGTG EGT4 62F-TGG GTATACGGCTTAAACACCGGTTGGGGCTGGAATAAAGATAAGGTGGTG EGT4 63F-NDT GTATACGGCTTAAACACCGGTGTTNDTTGGAATAAAGATAAGGTGGTG EGT4 63F-VHG GTATACGGCTTAAACACCGGTGTTVHGTGGAATAAAGATAAGGTGGTG EGT4 63F-TGG GTATACGGCTTAAACACCGGTGTTTGGTGGAATAAAGATAAGGTGGTG EGT4 65F-NDT GTATACGGCTTAAACACCGGTGTTGGCTGGNDTAAAGATAAGGTGGTGTATG EGT4 65F-VHG GTATACGGCTTAAACACCGGTGTTGGCTGGVHGAAAGATAAGGTGGTGTATG EGT4 65F-TGG GTATACGGCTTAAACACCGGTGTTGGCTGGTGGAAAGATAAGGTGGTGTATG EGT4 66F-NDT GTATACGGCTTAAACACCGGTGTTGGCTGGAATNDTGATAAGGTGGTGTATGCGGACAG EGT4 66F-VHG GTATACGGCTTAAACACCGGTGTTGGCTGGAATVHGGATAAGGTGGTGTATGCGGACAG EGT4 66F-TGG GTATACGGCTTAAACACCGGTGTTGGCTGGAATTGGGATAAGGTGGTGTATGCGGACAG EGT4 61-66R GGTGTTTAAGCCGTATACGGCTACACCACGCTTGTCC EGT4 82F-NDT GATGCGTACAATAAAAATNDTCTGCGCAGCCACATGATTG EGT4 82F-VHG GATGCGTACAATAAAAATVHGCTGCGCAGCCACATGATTG EGT4 82F-TGG GATGCGTACAATAAAAATTGGCTGCGCAGCCACATGATTG EGT4 85F-NDT GATGCGTACAATAAAAATCTGCTGCGCNDTCACATGATTGGCGTGGGCC EGT4 85F-VHG GATGCGTACAATAAAAATCTGCTGCGCVHGCACATGATTGGCGTGGGCC EGT4 85F-TGG GATGCGTACAATAAAAATCTGCTGCGCTGGCACATGATTGGCGTGGGCC EGT4 86F-NDT GATGCGTACAATAAAAATCTGCTGCGCAGCNDTATGATTGGCGTGGGCCCTG EGT4 86F-VHG GATGCGTACAATAAAAATCTGCTGCGCAGCVHGATGATTGGCGTGGGCCCTG EGT4 86F-TGG GATGCGTACAATAAAAATCTGCTGCGCAGCTGGATGATTGGCGTGGGCCCTG EGT4 82-86R ATTTTTATTGTACGCATCATATCTGTCCGCATACACC EGT4 143F-NDT ATTAAGTCTCGTGGCTCTNDTGGTGAGGCGGACATAGCCAC EGT4 143F-VHG ATTAAGTCTCGTGGCTCTVHGGGTGAGGCGGACATAGCCAC EGT4 143F-TGG ATTAAGTCTCGTGGCTCTTGGGGTGAGGCGGACATAGCCAC EGT4 146F-NDT ATTAAGTCTCGTGGCTCTGTTGGTGAGNDTGACATAGCCACTCTGCC EGT4 146F-VHG ATTAAGTCTCGTGGCTCTGTTGGTGAGVHGGACATAGCCACTCTGCC EGT4 146F-TGG ATTAAGTCTCGTGGCTCTGTTGGTGAGTGGGACATAGCCACTCTGCC EGT4 148F-NDT ATTAAGTCTCGTGGCTCTGTTGGTGAGGCGGACNDTGCCACTCTGCCGGCAAT EGT4 148F-VHG ATTAAGTCTCGTGGCTCTGTTGGTGAGGCGGACVHGGCCACTCTGCCGGCAAT EGT4 148F-TGG ATTAAGTCTCGTGGCTCTGTTGGTGAGGCGGACTGGGCCACTCTGCCGGCAAT EGT4 143-148R AGAGCCACGAGACTTAATAACCGGATGCACGCCCCGGT EGT4 193F-NDT CTGGGTCCGAAAGACGGCNDTGGCATTGTTAGCAGCAACG EGT4 193F-VHG CTGGGTCCGAAAGACGCVHGGGCATTGTTAGCAGCAACG EGT4 193F-TGG CTGGGTCCGAAAGACGGCTGGGGCATTGTTAGCAGCAACG EGT4 197F-NDT CTGGGTCCGAAAGACGGCTTGGGCATTGTTNDTAGCAACGCCCAAGGCGC EGT4 197F-VHG CTGGGTCCGAAAGACGGCTTGGGCATTGTTVHGAGCAACGCCCAAGGCGC EGT4 197F-TGG CTGGGTCCGAAAGACGGCTTGGGCATTGTTTGGAGCAACGCCCAAGGCGC EGT4 193-197R GCCGTCTTTCGGACCCAGCTTCAACGGCTCCAAACCGC EGT4 277F-NDT TGGGAAGGTCGTGCCCTGNDTGATCCGCTGTCGTTCCG EGT4 277F-VHG TGGGAAGGTCGTGCCCTGVHGGATCCGCTTGTCGTTCCG EGT4 277F-TGG TGGGAAGGTCGTGCCCTGTGGGATCCGCTGTCGTTCCG EGT4 280F-NDT TGGGAAGGTCGTGCCCTGCAGGATCCGNDTTCGTTCCGCTGCCAGAG EGT4 280F-VHG TGGGAAGGTCGTGCCCTGCAGGATCCGVHGTCGTTCCGCTGCCAGAG EGT4 280F-TGG TGGGAAGGTCGTGCCCTGCAGGATCCGTGGTCGTTCCGCTGCCAGAG EGT4 277-280R CAGGGCACGACCTTCCCACGGCTTTTCCAAATAGCTACCT EGT4 313F-NDT ATTGAACGCTACCGATGACNDTCCATGTTTGCTTCCGGAGG EGT4 313F-VHG ATTGAACGCTACCGATGACVHGCCATGTTTGCTTCCGGAGG EGT4 313F-TGG ATTGAACGCTACCGATGACTGGCCATGTTTGCTTCCGGAGG EGT4 313R GTCATCGGTAGCGTTCAATTCAACGCCCAGTT EGT4 329F-NDT ATGTGTGGTTCACCGAATNDTGAACCGTTAACGTGGGTTC EGT4 329F-VHG ATGTGTGGTTCACCGAATVHGGAACCGTTAACGTGGGTTC EGT4 329F-TGG ATGTGTGGTTCACCGAATTGGGAACCGTTAACGTGGGTTC EGT4 329R ATTCGGTGAACCACACATGCGGTCTT EGT4 369F-NDT GGTTTCACCAAACTGAACNDTTTTTTGACGCCAGCTGAGG EGT4 369F-VHG GGTTTCACCAAACTGAACNDTTTTTTGACGCCAGCTGAGG EGT4 369F-TGG GGTTTCACCAAACTGAACNDTTTTTTGACGCCAGCTGAGG EGT4 370F-NDT GGTTTCACCAAACTGAACCGCNDTTTGACGCCAGCTGAGGGC EGT4 370F-VHG GGTTTCACCAAACTGAACCGCVHGTTGACGCCAGCTGAGGGC EGT4 370F-TGG GGTTTCACCAAACTGAACCGCTGGTTGACGCCAGCTGAGGGC EGT4 369-370R GTTCAGTTTGGTGAAACCTGGATCGCCAATAC EGT4 379F-NDT CCAGCTGAGGGCGCAGTGNDTGCTTATGGTACGATCCAGAAGACCG EGT4 379F-VHG CCAGCTGAGGGCGCAGTGVHGGCTTATGGTACGATCCAGAAGACCG EGT4 379F-TGG CCAGCTGAGGGCGCAGTGTGGGCTTATGGTACGATCCAGAAGACCG EGT4 382F-NDT CCAGCTGAGGGCGCAGTGATGCTTATNDTACGATCCAGAAGACCGTTAGCT EGT4 382F-VHG CCAGCTGAGGGCGCAGTGATCGCTTATVHGACGATCCAGAAGACCGTTAGCT EGT4 382F-TGG CCAGCTGAGGGCGCAGTGATCGCTTATTGGACGATCCAGAAGACCGTTAGCT EGT4 379-382R CACTGCGCCCTCAGCTGGCGTCAAAAAGCGGTTC EGT4 385F-NDT ATCGCTTATGGTACGATCNDTAAGACCGTTAGCTACCTGGACAC EGT4 385F-VHG ATCGCTTATGGTACGATCVHGAAGACCGTTAGCTACCTGGACAC EGT4 385F-TGG ATCGCTTATGGTACGATCTGGAAGACCGTTAGCTACCTGGACAC EGT4 386F-NDT ATCGCTTATGGTACGATCCAGNDTACCGTTAGCTACCTGGACAC EGT4 386F-VHG ATCGCTTATGGTACGATCCAGVHGACCGTTAGCTACCTGGACAC EGT4 386F-TGG ATCGCTTATGGTACGATCCAGTGGACCGTTAGCTACCTGGACAC EGT4 385-386R GATCGTACCATAAGCGATCACTGCGCCCTCAGCT EGT4 409F-NDT TCCCTGGATTTTCTGTCTNDTGCGGGTCACATTGAAGATAC EGT4 409F-VHG TCCCTGGATTTTCTGTCTVHGGCGGTCACATTGAAGATAC EGT4 409F-TGG TCCCTGGATTTTCTGTCTTGGGCGGGTCACATTGAAGATAC EGT4 409R AGACAGAAAATCCAGGGAGCACGGATTTGCATACATAC

[0086] Note: The suffix "-F" in primer names indicates forward direction; "-R" indicates reverse direction.

[0087] After digesting the original template in the PCR product with DpnI, the cells were transformed into competent E. coli BL21(DE3) cells, plated onto kanamycin-resistant plates, and cultured overnight to obtain saturated mutant libraries at different sites.

[0088] Transformants from the mutant library were inoculated into 96-well deep-well plates containing 700 μL ZYM-5052 medium with 50 mg / L kanamycin and cultured overnight at 30 °C. 100 μL of the culture was added to 50 μL of 80% glycerol for preservation, and the remaining 600 μL was centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and the plate was frozen at -20 °C overnight. The next day, the plate was thawed at room temperature for 30 min, and 2 g / L L-histidine betaine was dissolved in 0.2 M phosphate buffer (pH 8.5) to obtain the substrate solution. 500 μL of substrate was added, and the cells were resuspended. After mixing, the plate was incubated at 40 °C for 30 min. After the reaction, the 96-well plate was centrifuged at 4000 rpm for 5 min, and 60 μL of the supernatant was added to 240 μL of ddH2O and transferred to a microplate. The plate was then read at 290 nm. The activity of EGT was enhanced using site-directed saturation mutagenesis. A mutant gene library with no fewer than 100 transformants was constructed, and enzyme activity was screened. The obtained mutant gene sequences showed at least 98% homology to sequence SEQ ID NO: 2. The obtained amino acid sequences showed at least 98% homology to sequence SEQ ID NO: 1.

[0089] The supernatant of mutant strains with high readings at 290 nm in 96-well deep-well plates was used for screening. Substrate preparation: 2 g / L L-histidine betaine dissolved in 0.2 M Tris-HCl (pH 9.0). Reaction conditions: 500 μL substrate + 400 μL bacterial cells, reacted at 40℃ for 30 min. After the reaction, the 96-well plates were centrifuged at 4000 rpm for 5 min, and 60 μL of the supernatant was diluted with 240 μL ddH2O and read at 290 nm. A positive mutant was obtained after screening. According to gene sequencing results, the corresponding mutation type was EGT-K386L (AAG-CTG), and the strain and mutant enzyme were named EGT-386.

[0090] Example 5: Enzyme activity assay of mutants

[0091] The mutant strain EGT-386 was fermented and the mutant enzyme protein was purified according to the method described in Example 2. The enzyme activity of the mutant was then measured.

[0092] The 300 μL reaction system included 1.93 g / L L-histidine betaine, 0.2 M phosphate buffer (pH 8.5), and 0.33 μg / mL purified enzyme (Ni-NTA purified). The reaction was carried out at room temperature for 30 min. The difference in absorbance between the test sample at 30 min and 0 min was used for enzyme activity determination. Each reaction was tested in triplicate. Comparison results with the wild-type enzyme EGT are shown in Table 2.

[0093] Table 2. Comparison of enzyme activities between mutant and wild-type EGT

[0094] mutant Mutant amino acids Vitality (U / mg) EGT -- 0.08 EGT-386 K386L 5.57

[0095] As shown in the table above, the positive mutation enhances the activity of L-histidine betaine. Compared to wild-type EGT, EGT-386 exhibits approximately 66-fold increased enzyme activity, making it the optimal mutation.

[0096] The accuracy of the enzyme activity comparison results between the mutant and the wild-type enzyme EGT was examined by HPLC.

[0097] Set up a 300 μL reaction system including 1.93 g / L L-histidine betaine, 0.2 M phosphate buffer (pH 8.5), and 33.3 μg / mL purified enzyme (Ni-NTA purified). React overnight at room temperature. Samples were then analyzed using HPLC. HPLC parameters are as follows: Figure 3 As shown in Figure A, the HPLC chromatogram is as follows: Figure 3 As shown in B.

[0098] Compared to the wild-type enzyme EGT, the mutant EGT-386 can efficiently convert L-histidine betaine to uric acid. This result is consistent with the findings based on OD... 290 The detected enzyme activity trends were consistent, confirming the accuracy of the enzyme-coupled detection method for L-histidine betaine.

[0099] The following focuses on the mutant enzyme EGT-386.

[0100] Example 6: Catalytic activity test of EGT-386 against L-histidine betaine analogues

[0101] L-histidine and N,N-dimethyl-L-histidine are common residual impurities in the synthesis of L-histidine betaine. Their deamination or dedimethylamine removal can also form uric acid, thus interfering with the reaction. To determine the specificity of EGT-386 for L-histidine betaine, 9.8 mM L-histidine betaine (1.93 g / L), 9.8 mM L-histidine, and 9.8 mM N,N-dimethyl-L-histidine were dissolved in 0.2 M phosphate buffer (pH 8.5). After adding 0.33 μg / mL EGT-386, the reaction was carried out in a 300 µL reaction system for 30 min, and the OD was measured. 290 Results. The results are shown in Table 3.

[0102] Table 3. Catalytic results of EGT-386 on L-histidine betaine, L-histidine, and N,N-dimethyl-L-histidine

[0103] deal with L-histidine betaine L-histidine N,N-Dimethyl-L-histidine <![CDATA[OD 290 Incremental 0.591 0.004 -0.001 Relative vitality 147 1 0

[0104] As shown in the table above, EGT-386 can specifically detect L-histidine betaine. The enzyme activity catalyzing the L-histidine betaine reaction is at least 147 times greater than that catalyzing L-histidine or N,N-dimethyl-L-histidine.

[0105] Example 7: Enzyme-coupled detection of L-histidine betaine based on EGT-386

[0106] A 0-39 mg / mL histidine betaine solution was prepared using 0.2 M phosphate buffer (pH 8.5). EGT-386 enzyme was added to a final concentration of 8.3 μg / mL, and a 300 µL reaction mixture was prepared. The reaction was incubated at room temperature for 30 min, followed by OD measurement. 290 With 0 minOD 290 The absorbance difference was used to prepare the L-histidine betaine standard curve.

[0107] The results are as follows Figure 4 As shown, EGT-386 can accurately detect L-histidine betaine content ranging from 2 to 39 mg / L. Its detection limit is 50 times higher than the HPLC detection limit (~100 mg / L), indicating that enzyme-coupled detection has higher sensitivity.

[0108] Example 8: EGT-386 enzyme-coupled detection of mixtures containing L-histidine betaine

[0109] To investigate the effects of L-histidine and N,N-dimethyl-L-histidine on the detection of L-histidine betaine, we set up four treatment groups. Treatment group 1 consisted of 0, 2, 5, 10, and 19 mg / L L-histidine betaine; treatment group 2 consisted of 0, 2, 5, 10, and 19 mg / L L-histidine betaine plus an equimolar mixture of L-histidine; treatment group 3 consisted of 0, 2, 5, 10, and 19 mg / L L-histidine betaine plus an equimolar mixture of N,N-dimethyl-L-histidine; and treatment group 4 consisted of 0, 2, 5, 10, and 19 mg / L L-histidine betaine plus an equimolar mixture of L-histidine and an equimolar mixture of N,N-dimethyl-L-histidine. The reaction volume was 300 µL. The substrate for each treatment group was prepared using 0.2 M phosphate buffer (pH 8.5), and the final concentration of EGT-386 enzyme in the system was 8.3 μg / mL. The reaction was carried out at room temperature for 60 min, followed by detection of OD. 290 The change in absorbance value. Compared with 0 min OD 290 The difference in absorbance values ​​is used to prepare the corresponding standard curve.

[0110] The results are as follows Figure 5As shown, there were no significant differences among the four treatment groups. L-histidine and N,N-dimethyl-L-histidine, under equimolar conditions, did not interfere with the enzyme-coupled detection of L-histidine betaine involved in this invention, demonstrating the accuracy and reliability of the enzyme-coupled detection.

[0111] It should be understood that 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 and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of ergothionase (EGT) in the detection of L-histidine betaine.

2. The application as described in claim 1, characterized in that, The ergothionase is an ergothionase derived from Blautia producta with the amino acid sequence shown in SEQ ID NO: 1 (Uniport ID: A0A6P1Z204) or a conserved variant polypeptide thereof, wherein the conserved variant polypeptide is a mutant with more than 90% homology to SEQ ID NO: 1 in amino acid sequence and with enhanced enzyme activity.

3. The application as described in claim 2, characterized in that, The conserved variant polypeptide is an ergothionein trimethylamine lyase mutant with the amino acid sequence shown in SEQ ID NO:

3.

4. A method for detecting L-histidine betaine, characterized in that, Includes the following steps: (1) Weigh the analyte that may contain L-histidine betaine, dissolve it in water or aqueous solution A, or dilute it with water or aqueous solution A to prepare a transparent aqueous solution B; (2) Adjust the pH of aqueous solution B to 8-9, preferably around pH 8.5, add 0.02-0.2 U / mL, preferably around 0.05 U / mL of ergothioneine trimethylamine lyase, and react at 20-35℃ for 15-45 minutes to allow L-histidine betaine to react and obtain reaction solution C; (3) The absorbance of reaction solution C at 290 nm was detected by spectrophotometry. The content of L-histidine betaine in the analyte was determined by using the standard curve of uric acid aqueous solution and the equimolar relationship between uric acid and L-histidine betaine.

5. The method as described in claim 4, characterized in that, In step (1), if the analyte is added to water to form a turbid liquid, a transparent aqueous solution B is obtained by centrifugation / filtration and / or decolorization with activated carbon. In step (1), the aqueous solution A is a buffer solution with pH 8-9, preferably around pH 8.5, such as phosphate buffer solution (PBS). or In step (3), the spectrophotometric method is performed using a spectrophotometer or an enzyme-linked immunosorbent assay (ELISA) reader.

6. The method as described in claim 4, characterized in that, The analyte mentioned in step (1) is a decomposition product of L-histidine betaine.

7. An ergothioneine trimethylamine lyase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

3.

8. The gene encoding the ergothioneine trimethylamine lyase mutant SEQ ID NO: 3 as described in claim 7.

9. The gene as described in claim 8, characterized in that, The nucleotide sequence of the gene encoding the ergothionein trimethylamine lyase mutant SEQ ID NO: 3 is shown in SEQ ID NO:

4.

10. A kit for detecting L-histidine betaine, which determines the L-histidine betaine content in the analyte by colorimetric method, characterized in that, The kit includes: ergothionein trimethylamine lyase as described in claim 1; a buffer or buffer solution such as phosphate buffered solution (PBS); and uric acid standards for preparing a standard curve.

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