A galactose oxidase mutant and its application in preparing 2-oxo-2-furanylacetic acid

Abstract

The present invention belongs to the field of bioengineering, and specifically relates to a galactose oxidase mutant and its application in the preparation of 2-oxo-2-furanyl acetic acid. F. graminearum The galactose oxidase mutant FgGOase screened by the present invention is modified to obtain a galactose oxidase mutant with high activity. M7‑2A / A193T / E195S / W290F / S291A / Q326D / T406V has good catalytic efficiency in the preparation of 2-oxo-2-furanyl acetic acid from 2-furanyl hydroxymethyl ketone, with a catalytic efficiency of 91.84~119.96 mM ‑1 S ‑1 Meanwhile, the conversion rate was 86.7% and the yield was 104 mM in a 100 mL preparative reaction.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to a galactose oxidase mutant and application thereof in the preparation of 2-oxo-2-furanylacetic acid. Background Art

[0002] Furan ammonium salt, chemically known as (Z)-2-methoxyimino-2-furanylammonium acetate, is a key intermediate in the synthesis of cefuroxime and can be prepared by oximation of 2-oxo-2-furanylacetic acid with methoxyamine.

[0003] 2-Oxo-2-furanyl acetic acid is a key precursor for the synthesis of furan ammonium salts. It is currently mainly prepared by chemical methods. The common route is that furoic acid is chlorinated to obtain furanoyl chloride, furanoyl chloride undergoes cyanation reaction, and then hydrolyzed under acidic conditions to obtain 2-oxo-2-furanyl acetic acid. However, this process produces a large amount of toxic gas, is dangerous to operate, and is difficult to treat the three wastes.

[0004] In recent years, the bioenzymatic method has become the main research method for the industrial synthesis of 2-oxo-2-furanylacetic acid due to its advantages of safety, high efficiency, low cost, stereoselectivity and environmental friendliness. For example, patent CN116355974A reported that oxidase can catalyze 2-furanylhydroxymethyl ketone to produce 2-oxo-2-furanylacetic acid, but it did not disclose the specific enzyme species and enzyme production process.

[0005] However, it has been reported that the oxidases that can completely oxidize the substrate 2-furanylhydroxymethyl ketone to produce the oxidation product carboxylic acid have problems such as low catalytic efficiency and low product yield.

[0006] Therefore, if a biological enzyme with stronger catalytic ability can be developed for the conversion and production of 2-oxo-2-furanylacetic acid, it will greatly improve the industrial preparation efficiency of cefuroxime and have important significance for the development of the cefuroxime market. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a galactose oxidase mutant and its application in the preparation of 2-oxo-2-furanylacetic acid.

[0008] The present invention develops a galactose oxidase mutant capable of efficiently catalyzing the reaction of 2-furanylhydroxymethylketone to prepare 2-oxo-2-furanylacetic acid. In addition, the amino acid sequence and nucleotide sequence corresponding to the galactose oxidase mutant, the recombinant expression plasmid capable of inducing the expression of the galactose oxidase mutant, the genetically engineered strain and the preparation method thereof are also the focus of protection of the present invention.

[0009] The present invention provides a galactose oxidase mutant, wherein the galactose oxidase mutant is obtained by mutating the amino acid sequence of galactose oxidase SEQ ID NO: 1 (the nucleotide sequence encoding the galactose oxidase, as shown in SEQ ID NO: 2), and the galactose oxidase mutant sequence is selected from one of the following sequences:

[0010] (1) replacing the 193rd alanine in the amino acid sequence of SEQ ID NO:1 with threonine;

[0011] (2) replacing the glutamic acid at position 195 of the amino acid sequence shown in SEQ ID NO:1 with serine;

[0012] (3) replacing the tryptophan at position 290 of the amino acid sequence shown in SEQ ID NO:1 with phenylalanine;

[0013] (4) replacing the serine at position 291 of the amino acid sequence shown in SEQ ID NO:1 with alanine;

[0014] (5) replacing the glutamine at position 326 of the amino acid sequence shown in SEQ ID NO: 1 with aspartic acid;

[0015] (6) replacing the threonine at position 406 of the amino acid sequence shown in SEQ ID NO: 1 with valine;

[0016] (7) A mutant comprising the amino acid residues at at least two positions in the above (1) to (6).

[0017] Among them, the galactose oxidase mutant with the best catalytic effect provided by the present invention has an amino acid sequence of the protein as shown in SEQ ID NO:3, that is, the 193rd alanine in the amino acid sequence shown in SEQ ID NO:1 is replaced by threonine, the 195th glutamate is replaced by serine, the 290th tryptophan is replaced by phenylalanine, the 291st serine is replaced by alanine, the 326th glutamine is replaced by aspartic acid, and the 406th threonine is replaced by valine.

[0018] The nucleotide sequence of the gene encoding the above-mentioned galactose oxidase mutant is shown in SEQ ID NO:4.

[0019] In addition, the present invention also provides a method for preparing the above-mentioned galactose oxidase mutant, comprising the following steps: site-directed mutagenesis of the galactose oxidase encoding gene, construction of an expression vector, transformation of a protein expression host bacterium, and induction of protein expression.

[0020] Furthermore, the present invention uses molecular cloning technology to obtain the coding gene of the mutant, and constructs a 6×His fusion expression vector of the mutant gene and introduces it into genetically engineered bacteria. E.coli The mutant enzyme protein was obtained by inducing expression in BL21 (DE3), and the mutant was used as a catalyst to carry out an enzyme catalytic reaction under appropriate conditions to prepare 2-furanyl hydroxymethyl ketone. The results showed that the catalytic efficiency of the mutant provided by the present invention was higher than that of galactose oxidase FgGOase. M7-2A The yield of synthesized 2-oxo-2-furanylacetic acid was increased by 46.4 times to 104 mM, which has great application potential and value in the industrial production of 2-oxo-2-furanylacetic acid.

[0021] All the mutation sites and any combination of mutations mentioned above in the present invention encoding galactose oxidase mutants, including but not limited to any of the amino acids in the amino acid sequence shown above that have been replaced by one or more amino acids and are active on the substrate 2-furanylhydroxymethylketone, are the key contents to be protected by the present invention. In addition, the expression cassette, vector or recombinant bacteria containing the gene also fall within the technical scope protected by the present invention.

[0022] Similarly, the catalyst comprising the above-mentioned galactose oxidase mutant is also the technical content protected by the present invention.

[0023] Furthermore, the use of the above-mentioned galactose oxidase mutant or the catalyst containing the above-mentioned galactose oxidase mutant provided by the present invention in the preparation of 2-oxo-2-furanylacetic acid using furfural and formaldehyde as initial raw materials, specifically using furfural and formaldehyde as substrates to synthesize 2-furanyl hydroxymethyl ketone, and then catalyzing the reaction with the prepared 2-furanyl hydroxymethyl ketone substrate to synthesize 2-oxo-2-furanylacetic acid, is also the technical content protected by the present invention.

[0024] The application is specifically: using the above-mentioned galactose oxidase mutant or a catalyst containing the above-mentioned galactose oxidase mutant, taking 2-furanyl hydroxymethyl ketone as a substrate, in the presence of Cu 2+ In the presence of , catalase, horseradish peroxidase and phosphate buffer, a catalytic reaction is carried out at a reaction temperature of 20-30°C to synthesize 2-oxo-2-furanylacetic acid.

[0025] The beneficial effects of the present invention are:

[0026] (1) Through the galactose oxidase FgGOase M7-2A Through transformation, a highly active galactose oxidase mutant FgGOase was obtained. M7-2A / A193T / E195S / W290F / S291A / Q326D / T406V, catalytic efficiency is 91.84~119.96 mM -1 S -1 In the range of M7-2A , the catalytic efficiency of the galactose oxidase mutant increased by 46.4 times;

[0027] (2) The galactose oxidase mutant screened in the present invention has good catalytic activity in the reaction of catalyzing 2-furanylhydroxymethyl ketone to prepare 2-oxo-2-furanylacetic acid, with a yield of up to 104 mM and a conversion rate of 86.7%. It achieves efficient catalysis of the production of 2-oxo-2-furanylacetic acid using furfural and formaldehyde as initial raw materials, and significantly improves the production efficiency of 2-oxo-2-furanylacetic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The galactose oxidase FgGOase in Example 3 of the present invention M7-2A and galactose oxidase mutant FgGOase M7-2A / A193T / E195S / W290F / S291A / Q326D / T406V protein electrophoresis;

[0029] Figure 2 is a high performance liquid chromatogram of the catalytic product of 100 mL of the preparative reaction of the galactose oxidase mutant in Example 8 of the present invention;

[0030] Figure 3 is the concentration of each substance in the catalytic product of 100 mL of the preparation-level reaction of the galactose oxidase mutant in Example 8 of the present invention. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0032] The main reagents and consumables used in the present invention are:

[0033] Phanta Max ultra-high fidelity DNA polymerase P520 was purchased from Nanjing Novogen Biotechnology Co., Ltd.; fast DpnI DNA endonuclease was purchased from Thermo Fisher Scientific; pET28(a) plasmid is a known Escherichia coli expression vector with a vector size of 5369 bp, T7 promoter, vector tag N-6×His, and vector resistance Kanamycin (kanamycin), purchased from Novogen; E.coli DH5a competent cells, E.coliBL21 (DE3) competent cells were purchased from Ang Yu Biotechnology Co., Ltd.; agar powder was purchased from Solarbio Co., Ltd.; tryptophan and yeast extract were purchased from OXOID Co., Ltd.

[0034] LB medium

[0035] Each 100 mL of LB medium contains: 1 g tryptone, 0.5 g yeast extract and 1 g NaCl, pH 7.0-7.2.

[0036] Preparation method: Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of distilled water, do not adjust the pH value, and sterilize with high pressure steam at 121°C for 20 min. If preparing solid culture medium, add 1.8 g of agar powder to 100 mL of culture medium.

[0037] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, and reference can be made to the relevant experimental manuals or manufacturer's instructions.

[0038] In the present invention, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0039] Example 1

[0040] Construction of galactose oxidase expression strain

[0041] Galactose oxidase FgGOase was obtained through the research of Birmingham, WR et al. M7-2A The open reading frame of the gene is 1956 bp in length, and the galactose oxidase encoded by the gene consists of 652 amino acids. The amino acid sequence (652aa) is shown in SEQ ID NO:1; the nucleotide sequence encoding the galactose oxidase is shown in SEQ ID NO:2.

[0042] Our laboratory has synthesized galactose oxidase FgGOase M7-2A The above gene was synthesized by GenScript Biotech Co., Ltd. and cloned into the BamHI and XhoI restriction sites of pET28a(+) plasmid, and a 6×His tag was added to the N-terminus. M7-2A Transformation expression host Escherichia coli E.coli BL21(DE3), obtained recombinant E. coli FgGOase expressing wild enzyme M7-2A .

[0043] Example 2

[0044] High-throughput screening of galactose oxidase mutants

[0045] The high-throughput screening system for mutants included 50 mM phosphate buffer (pH = 7.0), 10 mM 2-furanylhydroxymethyl ketone, 0.1 mg / mL horseradish peroxidase, 0.1 mM 4-aminoantipyrine, 1 mM 3,5-dichloro-2-hydroxybenzenesulfonyl chloride, 20 μL crude enzyme solution, and reaction at 25°C for 10 min.

[0046] Use a microplate reader to read the OD value at 515nm at 0min and 10min, and use the difference to determine the crude enzyme activity.

[0047] Example 3

[0048] Expression and activity analysis of galactose oxidase

[0049] (1) Protein expression and purification

[0050] The obtained galactose oxidase mutant recombinant plasmid was transformed into the expression host Escherichia coli E.coli BL21(DE3), LB medium was used for induction expression of heterologous protein, the strain was collected by centrifugation, the bacteria were suspended in phosphate buffer, and then ultrasonically disrupted, and the supernatant was obtained by centrifugation, and purified by Ni column affinity chromatography, and further ultrafiltration was performed using a 30kDa ultrafiltration tube to obtain concentrated and purified galactose oxidase mutant pure enzyme. It was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as shown in the attached Figure 1 shown.

[0051] (2) Determination of pure enzyme activity

[0052] The galactose oxidase enzyme activity assay system is shown in Table 1 below.

[0053] Table 1 Enzyme activity assay system

[0054] Component Final concentration <![CDATA[Cu 2+ (5 mM)]]> 0.5 mM Substrate (20 mM) 20 mM Horseradish peroxidase (1 mg / mL) 0.01 mg / mL Catalase (5 mg / mL) 0.1 mg / mL Phosphate buffer pH = 7 50 mM Purified enzyme solution 0.02mg / mL

[0055] Add to 500 uL system, temperature 25℃, rotation speed 120 rpm, react for 10 min. After the oxidation reaction is completed, add an equal volume of 1M hydrochloric acid to terminate it, dilute 10 times and use liquid phase for determination.

[0056] The HPLC analysis was carried out under the following conditions: the instrument was Agilent HPLC 1200, the chromatographic column was C18 column, mobile phase A was water + phosphate buffer (pH = 2.8), mobile phase B was acetonitrile, flow rate was 1 mL / min, and the sample volume was 20 μL.

[0057] Example 4

[0058] Construction of a galactose oxidase mutant library

[0059] The present invention firstly obtains a large number of transformants by using an error-prone PCR method, and screens and obtains mutants that are different from FgGOase by using an established mutant flux screening method and enzyme activity assay method. M7-2A FgGOase with 2.6-fold increase in enzyme activity M7-2A / T406V mutant, as shown in Table 2.

[0060] Table 2 Mutants with enhanced enzyme activity obtained by error-prone PCR method

[0061]

[0062] By analyzing the galactose oxidase FgGOase M7-2A The three-dimensional structure of the protein was determined, and the substrate was molecularly docked by Autodock. The mutation sites of the amino acid residues within 6Å around the substrate binding pocket were selected: single-point saturation mutations were performed at positions 193, 194, 195, 227, 244, 245, 271, 290, 291, 326, 329, 330, 333, 405, 406, 407, 461, 462, 463, 464, 513, 514, and 515.

[0063] The specific operation is as follows: recombinant plasmid pET28a(+)-FgGOase M7-2A / T406V was used as a template, and a pair of primers with mutation sites were used to perform whole plasmid PCR amplification with a high-fidelity enzyme to obtain a recombinant plasmid with a specified mutation site. The amplified product was digested with DpnI enzyme at 37°C for 2 h to degrade the initial template, and the digested product was converted to E.coliBL21 (DE3), spread on LB agar plates containing 50 μg / mL kanamycin, culture at 37°C overnight, and screen 100 transformants at each site for subsequent screening.

[0064] The primer designs for each mutant are shown in Table 3 below.

[0065] Table 3 Mutant primer design

[0066] Primer Sequence (5'-3') 193-F CCGTAATGACNNKTTTGAGGGTTCCCCTGGGGGGAT 193-R AACCCTCAAAMNNGTCATTACGGTAGCTCGACCACA 194-F TAATGACGCANNKGAGGGTTCCCCTGGGGGGATCAC 194-R GGGAACCCTCMNNTGCGTCATTACGGTAGCTCGACC 195-F TGACGCATTTNNKGGTTCCCCTGGGGGGATCACCCT 195-R CAGGGGAACCMNNAAATGCGTCATTACGGTAGCTCG 227-F GCATGACATGNNKTGCCCAGGTATCAGCATGGACGG 227-R TACCTGGGCAMNNCATGTCATGCTTAGTAACTGTCA 244-F GGTTACGGGCNNKAACGATGCTAAGAAGACGAGTCT 244-R TAGCATCGTTMNNGCCCGTAACCACGATCTGCCCAT 245-F TACGGGCGGTNNKGATGCTAAGAAGACGAGTCTGTA 245-R TCTTAGCATCMNNACCGCCCGTAACCACGATCTGCC 271-F AGTAGCACGCNNKTACCAGTCCTCTGCGACTATGTC 271-R AGGACTGGTAMNNGCGTGCTACTTGCATGTCGGGGC 290-F CGGGGGGTCANNKAGTGGGGGAGTATTTGAGAAAAA 290-R CTCCCCCACTMNNTGACCCCCCGATTGTAAAGACAC 291-F GGGGTCATGGNNKGGGGGAGTATTTGAGAAAAACGG 291-R ATACTCCCCCMNNCCATGACCCCCCGATTGTAAAGA 326-F CGCCGATAAANNKGGCTTACTTTTTTCCGATAATCA 326-R AAAGTAAGCCMNNTTTATCGGCGGTCAACATCGGGT 329-F ACAAGGCTTANNKTTTTCCGATAATCATGCCTGGTT 329-R TATCGGAAAAMNNTAAGCCTTGTTTATCGGCGGTCA 330-F AGGCTTACTTNNKTCCGATAATCATGCCTGGTTATT 330-R GATTATCGGAMNNAAGTAAGCCTTGTTTATCGGCGG 333-F TTTTTCCGATNNKCATGCCTGGTTATTTGGTTGGAA 333-R AACAAGGCTTMNNTTTTTCCGATAATCATGCCTGGT 405-F TTCGCCCGACNNKACAGACAGCGATGCGACAACAAA 405-R CGCTGTCTGTMNNGTCGGGCGAACCACCAAAGGTCA 407-F CGACTACACANNKAGCGATGCGACAACAAATGCCCA 407-R TCGCATCGCTMNNTGTGTAGTCGGGCGAACCACCAA 461-F GCAGCGTCGTNNKATTCCGTTTGAAGATTCGACACC 461-R TTCAAACGGAATMNNACGACGCTGCCCACCGGTAAT 462-F GCGTCGTGGGNNKCCGTTTGAAGATTCGACACCCGT 462-R TCTTCAAACGGMNNCCCACGACGCTGCCCA 463-F GTCGTGGGATTNNKTTTGAAGATTCGACACC 463-R ATCTTCAAAMNNAATCCCACGACGCTGCCCACCGG 464-F TGGGATTCCGNNKGAAGATTCGACACCCGTTTTTAC 464-R TCGAATCTTCMNNCGGAATCCCACGACGCTGCCCAC 513-F TGGCGGCGGANNKTTGTGCGGAGATTGCACGACGAA 513-R CTCCGCACAAMNNTCCGCCGCCATTAAACACACGCC 514-F CGGCGGAGGTNNKTGCGGAGATTGCACGACGAACCA 514-R AATCTCCGCAMNNACCTCCGCCGCCATTAAACACAC 515-F CGGAGGTTTGNNKGGAGATTGCACGACGAACCACTT 515-R TGCAATCTCCMNNCAAACCTCCGCCGCCATTAAACA

[0067] Galactose oxidase FgGOase M7-2A The pET28a(+) plasmid of the gene was used as a template, and the upstream primer and downstream primer mentioned above were used to amplify the galactose oxidase mutant gene according to the following PCR system and procedure.

[0068] PCR system: Phanta Max ultra-high-fidelity DNA polymerase P520 20 μL, template plasmid 1 μL, upstream primer-F (10 μM) 1 μL, downstream primer-R (10 μM) 1 μL, ddH2O 2 μL.

[0069] The PCR program was as follows: a. 98°C pre-denaturation for 30 s; b. 98°C denaturation for 10 s, 55°C annealing for 5 s, 72°C extension for 40 s, 35 cycles; c. 72°C extension for 5 min, cooling to 4°C, using DpnI enzyme to digest the template plasmid, and digesting at 37°C for 30 min; the digestion product was transformed into DH5α competent cells, the DH5α competent cells were placed on ice, and after the cells thawed, 15 μL of plasmid solution was added, placed on ice for 30 min, heat-shocked at 42°C for 45 s, and then placed on ice for 2 min, 700 μL of sterile LB liquid culture medium was added, and cultured in a shaker at 37°C and 200 rpm for 1 h, centrifuged at 4000 rpm for 1 min, 600 μL of supernatant was discarded, and the precipitated bacteria were resuspended with the remaining supernatant and all were spread on LB plate culture medium containing kanamycin resistance (50 μg / mL), and inverted cultured at 37°C overnight. A single colony was picked from the LB plate and inoculated into LB liquid culture medium containing Kana resistance (50 μg / mL), cultured overnight at 37°C with a shaker at 200 rpm, and then sent to GeneWeichi (Suzhou, China) for sequencing to identify whether the galactose oxidase mutation was successful.

[0070] Example 5

[0071] Screening for galactose oxidase mutants with enhanced activity

[0072] The catalytic activity of the enzyme was analyzed and determined by the established mutant flux screening method, and the galactose oxidase mutants with improved catalytic activity were sequenced to analyze which amino acids the amino acids at the corresponding positions mutated to.

[0073] The mutation sites and relative activities of the mutants with improved activity are shown in Table 4. The relative activity refers to the definition of FgGOase M7-2A When the relative activity of the / T406V mutant is 1, the catalytic activity of the mutant on the same substrate under the same conditions is relative to that of FgGOase. M7-2A / T406V mutant.

[0074] Table 4 Mutation sites and relative activities of mutants with improved activity

[0075]

[0076] Table 4 shows the results of the FgGOase M7-2A The site mutants and the corresponding mutant activities obtained by single mutation using / T406V as the template. From the results in Table 4, it can be seen that the activities of the mutants obtained after mutation of the 193rd, 195th, 290th, 291st, and 326th sites are higher than those of FgGOase M7-2A / T406V increased by 1.46~3.65 times.

[0077] In particular, the mutation of the W290F site showed higher catalytic activity compared with FgGOase M7-2A / T406V increased by 3.65 times.

[0078] Example 6

[0079] Combination mutations at sites that increase enzyme activity

[0080] The present invention performs combined mutations on the above-mentioned sites with improved activity, selects the best mutant for iterative mutation, and measures the relative catalytic ability. The enzyme activity of the mutants obtained by combined mutation is shown in Table 5 below.

[0081] Table 5 Determination of enzyme activity of mutants obtained by combined mutation

[0082]

[0083] The present invention provides a combined mutation site and / or mutant that can improve the catalytic activity of 2-furanyl hydroxymethyl ketone to produce 2-oxo-2-furanyl acetic acid. After screening, it was found that the mutant with the highest enzyme activity was obtained, as shown in Table 5, that is, the mutant FgGOase provided by the present invention. M7-2A / A193T / E195S / W290F / S291A / Q326D / T406V, showing significantly improved catalytic activity.

[0084] The galactose oxidase FgGOase having the amino acid sequence shown in SEQ ID NO: 1 M7-2A The nucleotide sequence of the gene, whose A193 corresponds to the 577-579 codons, the nucleotide sequence of E195 corresponds to the 583-585 codons, the nucleotide sequence of W290 corresponds to the 868-870 codons, the nucleotide sequence of S291 corresponds to the 871-873 codons, the nucleotide sequence of Q326 corresponds to the 976-978 codons, and the nucleotide sequence of T406 corresponds to the 1216-1218 codons.

[0085] Mutant FgGOase M7-2A / A193T / E195S / W290F / S291A / Q326D / T406V is a galactose oxidase gene sequence having an amino acid sequence as shown in SEQ ID NO: 1, wherein the nucleotide sequence corresponding to A193T is that the 577-579 codons are mutated from GCA to ACT, the nucleotide sequence corresponding to E195S is that the 583-585 codons are mutated from GAG to TCG, the nucleotide sequence corresponding to W290F is that the 868-870 codons are mutated from TGG to TTT, the nucleotide sequence corresponding to S291A is that the 871-873 codons are mutated from AGT to GCG, the nucleotide sequence corresponding to Q326D is that the 976-978 codons are mutated from AAA to GAT, and the nucleotide sequence corresponding to T406V is that the 1216-1218 codons are mutated from ACA to GTG, to obtain an enzyme mutant, whose amino acid sequence is shown in SEQ ID NO: NO:3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:4.

[0086] Example 7

[0087] Characterization of the catalytic efficiency of galactose oxidase and its mutants

[0088] For mutants with improved activity:

[0089] FgGOase M7-2A The kinetic parameters of / A193T / E195S / W290F / S291A / Q326D / T406V were determined. The determination system and reaction conditions of the kinetic parameters are shown in Table 6.

[0090] Table 6 Enzyme activity assay system

[0091] Components Final concentration <![CDATA[Cu 2+ (5 mM)]]> 0.5 mM Substrate (100 mM) 20 mM Horseradish peroxidase (2 mg / mL) 0.1 mg / mL 4-Aminoantipyrine (1 mM) 0.1 mM 3-5-Dichloro-dihydroxybenzenesulfonic acid (10 mM) 1 mM Phosphate buffer pH = 7 50 mM Purification of mutant enzyme solution 0.02mg / mL

[0092] The above system was mixed and added into a 96-well plate, and the increase of absorbance at 515 nm was detected within 10 min at 25°C using an ELISA reader.

[0093] Table 7 Enzyme activities of galactose oxidase and mutants

[0094] Types of enzymes Michaelis constant (mM) <![CDATA[Conversion number (S -2 )]]> <![CDATA[Catalytic efficiency (mM -1 S -1 )]]> Relative catalytic efficiency <![CDATA[FgGOase M7-2A ]]> 3.49±0.59 7.97±0.62 2.28±0.56 1 <![CDATA[FgGOase M7-2A / A193T / E195S / W290F / S291A / Q326D / T406V]]> 0.28±0.03 29.65±0.76 105.90±14.06 46.4

[0095] The catalytic efficiency of the mutant was calculated with the wild type as the unit "1", and the obtained catalytic activity results of the galactose oxidase mutant are shown in Table 7 above.

[0096] The above results show that the galactose oxidase mutant obtained by the present invention:

[0097] FgGOase M7-2A / A193T / E195S / W290F / S291A / Q326D / T406V, its catalytic efficiency is compared with the wild-type galactose oxidase FgGOase M7-2A Greatly improved, for FgGOase M7-2A The affinity of the galactose oxidase mutant for the substrate 2-furanylhydroxymethyl ketone was greatly improved.

[0098] Example 8

[0099] Synthesis of 2-oxo-2-furanylacetic acid using galactose oxidase mutants

[0100] This example mainly uses the galactose oxidase mutant FgGOase obtained by the present invention M7-2A / A193T / E195S / W290F / S291A / Q326D / T406V synthesizes 2-oxo-2-furanylacetic acid.

[0101] 100 mL preparative-scale catalytic reaction: The substrate solution was prepared using an aqueous solution at pH 7.0.

[0102] First, according to the example of the existing patent (CN116064495B) of our laboratory, the substrates furfural and formaldehyde were converted by carboxylase to prepare 2-furanyl hydroxymethyl ketone, and the final concentration was 120 mM. Then, the prepared 2-furanyl hydroxymethyl ketone was converted by galactose oxidase mutant to produce 2-oxo-2-furanylacetic acid, wherein the reaction system was Cu 2+ The final concentration was 1 mM, the final concentration of catalase was 0.2 mg / mL, the final concentration of horseradish peroxidase was 0.02 mg / mL, the final concentration of phosphate buffer was 100 mM, the reaction temperature was 25°C, the amount of galactose oxidase mutant was 50 g / L, and after 12 h of reaction, the sample was obtained by centrifugation at 12000 rpm for 10 min.

[0103] The purity of 2-oxo-2-furanyl acetic acid in the obtained product was detected by high performance liquid chromatography (HPLC). The content of 2-oxo-2-furanyl acetic acid in the sample was determined based on the peak area. The results of HPLC are shown in the attached Figure 2 As shown, the retention time of 2-oxo-2-furanylacetic acid is 4.9 min.

[0104] Figure 3 The contents of substrate 2-furanyl hydroxymethyl ketone and product 2-oxo-2-furanyl acetic acid at different reaction times are shown in FIG. Figure 3 It can be seen that the yield of 2-oxo-2-furanylacetic acid is 104 mM and the conversion rate is 86.7%.

[0105] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A galactose oxidase mutant, characterized in that: The galactose oxidase mutant is obtained by performing the following mutations on the amino acid sequence of galactose oxidase shown in SEQ ID NO: 1: replacing the 406th threonine with valine, the 193rd alanine with threonine, the 195th glutamate with serine, the 290th tryptophan with phenylalanine, the 291st serine with alanine, and the 326th glutamine with aspartic acid.

2. A gene encoding the galactose oxidase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a nucleotide sequence encoding the amino acid sequence of the galactose oxidase mutant described in claim 1.

4. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria are obtained by transforming the recombinant expression vector described in claim 3 into a host microorganism.

5. Use of the galactose oxidase mutant according to claim 1 or the recombinant engineered bacteria according to claim 4 in preparing 2-oxo-2-furanylacetic acid using 2-furanylhydroxymethyl ketone as a substrate.

6. A method for preparing 2-oxo-2-furanyl acetic acid, characterized in that: The steps include: (1) Preparation of 2-furanyl hydroxymethyl ketone using furfural and formaldehyde as substrates; (2) Using the 2-furanylhydroxymethyl ketone prepared in (1) as a substrate, adding a catalyst to carry out a catalytic reaction to prepare 2-oxo-2-furanyl acetic acid; the catalyst comprises the galactose oxidase mutant according to claim 1 or the recombinant engineered bacteria according to claim 4.

7. The method according to claim 6, characterized in that (2), the reaction conditions of the catalytic reaction are: 2 + The reaction is carried out at 20~30℃ in the presence of 2% paraformaldehyde, catalase, horseradish peroxidase and phosphate buffer.

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