A mutant of alcohol oxidase IgAOX and its application

Abstract

This invention relates to the fields of enzyme engineering and biocatalysis, specifically to a mutant of the alcohol oxidase IgAOX and its applications. The amino acid sequence of the mutant IgAOX is as follows: The amino acid sequence shown in SEQ ID NO: 1 is modified by replacing V at position 229 with A, or S at position 271 with G, or K at position 297 with R, or K at position 297 with R, while simultaneously replacing Y at position 605 with M, or S at position 271 with G, and A at position 227 with S, or K at position 297 with R, and A at position 227 with S. This invention yields mutants with improved catalytic activity and thermal stability. These mutants enable their application in biosynthesis and biocatalysis, providing new ideas and a technological foundation for the green and environmentally friendly synthesis of chemical raw materials.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and biocatalysis, specifically to a mutant of alcohol oxidase IgAOX and its applications. Background Technology

[0002] Alcohol oxidases (AOX, EC1.1.3.13) are flavin-dependent oxidases that specifically oxidize primary alcohols to primary aldehydes, belonging to the GMC (glucose-methanol-choline) oxidoreductase superfamily. As key rate-limiting enzymes in the methanol metabolism pathway, alcohol oxidases exhibit strict substrate specificity and regioselectivity. In recent years, their application value in areas such as biocarbon fixation and environmental pollutant degradation has become increasingly prominent, making them a research hotspot in the field of industrial biotechnology. However, most naturally derived alcohol oxidases generally suffer from low soluble expression levels, poor thermal stability, and low catalytic efficiency, making it difficult to meet the needs of industrial applications.

[0003] In the prior art, molecular modification has been used to enhance the catalytic activity of alcohol oxidases or to modify the substrate, such as the method disclosed in Chinese patent CN121182763A, which uses a substrate derived from *Phragmites australis*. Gloeophyllum trabeum The catalytic activity of the alcohol oxidase GtAOX was modified, and a superior mutant with significantly improved catalytic performance was successfully obtained, demonstrating the great potential of alcohol oxidase modification. Furthermore, its catalytic product, formaldehyde, is frequently used in the biosynthesis of complex multi-carbon compounds, actively responding to the "carbon neutrality" concept and promoting future scientific research and industrial applications.

[0004] In the previous screening work for constructing a gene library of enzymes for the de novo synthesis of multi-carbon compounds from CO2, this invention identified a gene derived from *Agaricus funnelii* (…). Infundibulicybe gibba A novel alcohol oxidase, IgAOX, exhibits good catalytic activity, a high substrate preference for methanol, and can be expressed solublely in *E. coli*. However, IgAOX also suffers from low heterologous expression yield, poor thermal stability (significant inactivation above 40°C), and poor substrate adaptability, and its catalytic activity still has room for improvement. Further engineering modifications are needed to apply it to industrial production. Based on the above analysis, this invention provides a mutant of the alcohol oxidase IgAOX and its application, simultaneously enhancing both stability and catalytic activity to meet the requirements of industrial applications. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a mutant of alcohol oxidase IgAOX and its application. The aim is to modify alcohol oxidase IgAOX using a semi-rational design approach, including sequence comparison and structural analysis, to simultaneously enhance the stability and catalytic activity of IgAOX, thus meeting the needs of industrial applications.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, a mutant of the alcohol oxidase IgAOX, the amino acid sequence of which is shown in any one of (a) to (f): (a) Replace V at position 229 of the amino acid sequence shown in SEQ ID NO: 1 with A; (b) Replace the S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 with G; (c) Replace the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 with R; (d) Replace K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 with R, and replace Y at position 605 with M; the specific amino acid sequence of this mutant is shown in SEQ ID NO: 9; (e) Replace S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 with G, and replace A at position 227 with S; (f) Replace the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 with R, and replace the A at position 227 with S.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Secondly, the encoding gene encodes a mutant of the alcohol oxidase IgAOX.

[0009] Furthermore, when the V at position 229 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with A, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 3; When the S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with G, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 4; When the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with R, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 5; When the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with R, and the Y at position 605 is replaced with M, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 6. When the S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with G, and the A at position 227 is replaced with S, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 7. When the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with R, and the A at position 227 is replaced with S, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 8.

[0010] Thirdly, a recombinant expression vector containing the aforementioned coding gene.

[0011] Fourthly, a recombinant strain containing the recombinant expression vector.

[0012] Furthermore, the host strain of the recombinant strain is Escherichia coli strain BL21(DE3).

[0013] Fifthly, a recombinant alcohol oxidase catalyst, comprising a mutant of the alcohol oxidase IgAOX; or a purified enzyme solution of the mutant alcohol oxidase IgAOX obtained by purifying the lysate of the recombinant strain.

[0014] Sixthly, the application of a recombinant alcohol oxidase catalyst, wherein the recombinant alcohol oxidase catalyst is used to catalyze the oxidation of methanol to formaldehyde.

[0015] Furthermore, the reaction conditions for catalytic oxidation of methanol to formaldehyde are as follows: methanol concentration of 20-50 mM, recombinant alcohol oxidase catalyst of 0.5-1.0 mg / mL, reaction temperature of 20-30℃, pH of 7.5-8.5, and reaction time of 1-3 hours.

[0016] Furthermore, the reaction conditions for catalytic oxidation of methanol to formaldehyde also include: the amount of catalase used is 200-500 U / mL.

[0017] Furthermore, the reaction conditions for catalytic oxidation of methanol to formaldehyde were as follows: methanol concentration of 50 mM, alcohol oxidase mutant pure enzyme solution volume of 0.9 mg / mL, catalase volume of 300 U / mL, reaction temperature of 25℃, pH of 8.0, and reaction time of 2 hours; the reaction solvent for catalytic oxidation of methanol to formaldehyde was N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer.

[0018] The present invention designs the following experimental procedure: First, a recombinant expression vector encoding the wild-type alcohol oxidase IgAOX was constructed. The nucleic acid sequence encoding the wild-type alcohol oxidase IgAOX gene described in this invention was ligated downstream of the expression regulatory sequence of the pET-28a(+) empty vector plasmid to achieve inducible expression of the alcohol oxidase IgAOX. The recombinant expression plasmid described in this invention can be prepared by the following method: PCR amplification of the alcohol oxidase IgAOX gene, simultaneously using restriction endonucleases... Nde I and Xho I. The empty vector plasmid pET-28a(+) was double-digested. The amplified alcohol oxidase IgAOX DNA fragment and the digested empty vector plasmid were recovered using a nucleic acid gel. The alcohol oxidase DNA fragment and the digested empty vector plasmid were ligated using a homologous recombinase and transformed into Escherichia coli DH5α to obtain a recombinant expression plasmid containing the alcohol oxidase gene.

[0019] Secondly, a semi-rational design approach was used to modify the gene, including sequence comparison and structural analysis, to identify potential mutation sites. Primers were then designed using reverse amplification PCR to perform site-directed mutagenesis, specifically replacing amino acids at corresponding positions in the IgAOX gene sequence. Dpn I digested the template and transformed it into DH5α to obtain a recombinant expression plasmid containing the alcohol oxidase mutant gene.

[0020] This invention also provides a recombinant expression strain transformed with the aforementioned mutant recombinant expression vector. This is achieved by transforming the recombinant expression plasmid containing the alcohol oxidase mutant nucleic acid described in this invention into host cells *Escherichia coli*. E. coli It is prepared using BL21 (DE3).

[0021] This invention also provides a method for preparing an alcohol oxidase mutant catalyst, which involves purifying a cell lysate containing the alcohol oxidase mutant to obtain a pure enzyme solution. The method includes the following steps: preparing a culture medium required for the growth of the recombinant expression strain, culturing the recombinant expression strain, and obtaining the recombinant alcohol oxidase catalyst.

[0022] This invention also provides the application of the above-mentioned mutant of alcohol oxidase IgAOX. The mutant catalyst described in this invention is added to a HEPES buffer containing methanol as a substrate to catalyze the oxidation of methanol to formaldehyde, generating hydrogen peroxide during the reaction. The conversion rate is analyzed using acetylacetone spectrophotometry.

[0023] The beneficial effects of this invention are: Compared with existing technologies, this invention uses a semi-rational design method to screen and obtain mutants of several advantageous alcohol oxidases. The activity and thermal stability of these mutant alcohol oxidases are significantly improved, and they can be efficiently heterologously expressed in Escherichia coli, enabling them to catalyze the oxidation of methanol to formaldehyde more efficiently. In particular, the dual mutant K297R / Y605M has an catalytic activity that is about 51.2% higher than the parent mutant and a thermal stability that is 83% higher, which brings prospects for its application in the fields of biocatalysis and synthesis. Attached Figure Description

[0024] Figure 1 This is the recombinant plasmid map of the alcohol oxidase IgAOX of the present invention (pET28a-IgAOX). Figure 2 This is an SDS-PAGE electrophoresis image of the purified wild-type and mutant alcohol oxidase IgAOX of this invention. Figure 3 This is a standard curve of formaldehyde in this invention. Detailed Implementation

[0025] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0026] Description of the source of materials and reagents: Escherichia coli BL21(DE3) competent cells were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; PCR amplification enzyme solution 2×Phanta Unifi Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmid extraction and agarose gel DNA recovery kits were both purchased from Nanjing Novizan Biotechnology Co., Ltd.; restriction endonucleases... Nde I, Xho Both enzyme I and the homologous recombinant enzyme were purchased from New England Biolabs (NEB); commercial catalase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; methanol, acetylacetone, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The absorbance was measured using a Spectra MAX 190 microplate reader at a wavelength of 414 nm.

[0027] The standard symbols and abbreviations for common protein amino acids are as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamine (Gln, Q); Glutamic acid (Glu, E); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyr, Y); Valine (Val, V).

[0028] Example 1. IgAOX sequence comparison and structural analysis.

[0029] Potential mutation sites were identified through homologous multiple sequence comparison and protein molecular structure analysis. Three-dimensional structure analysis of IgAOX (amino acid sequence as shown in SEQ ID NO: 1; nucleotide sequence as shown in SEQ ID NO: 2) revealed multiple large aromatic amino acid residues within a 5 Å radius around its FAD domain. These residues may create steric hindrance, hindering proton transfer or substrate binding and release in the catalytic reaction. Based on this, IgAOX was compared with several previously reported alcohol oxidases with high sequence homology. Mutation sites were selected targeting amino acid sites near the substrate-binding domain, FAD-binding domain, and catalytic domain. After screening and analysis based on site conservation, 10 sites were finally selected for targeted single-point mutation: G39, V87, A227, V229, S271, K297, Q310, L600, G601, and Y605. Twenty-three mutants were designed: G39A, G39S, V87T, F101S, F101V, A227S, A227T, V229A, S271A, S271C, S271G, T273A, K297D, K297E, K297R, Q310K, R339A, R339E, R339Q, L600M, L600V, G601S, Y605F, and Y605M.

[0030] The design of mutants specifically includes the following steps: (1) Primer design: The recombinant plasmid of wild-type alcohol oxidase IgAOX was used as a template ( Figure 1 Primers were designed using reverse amplification PCR for site-directed mutagenesis. SnapGene software was used for primer design, and the main primers involved are shown in Table 1.

[0031] (2) Site-directed mutagenesis of wild-type IgAOX: Based on the primers designed in Table 1, site-directed reverse amplification mutagenesis of wild-type IgAOX was performed by PCR reaction. The PCR product after mutagenesis was recovered by gel and then subjected to restriction endonuclease. Dpn Digestion was performed for 1 hour. The site-directed mutagenesis PCR reaction system and procedure, as well as the digestion system and procedure, are shown in Tables 2, 3, and 4, respectively.

[0032] (3) Construction of IgAOX mutant engineered bacteria: The PCR product after template digestion was transformed into Escherichia coli DH5α competent cells using chemical transformation. The cells were plated on LK plates, and after single-cell growth, colony PCR and sequencing were performed to successfully obtain the IgAOX mutant plasmid. The obtained recombinant plasmid was then transformed into Escherichia coli BL21(DE3) competent cells. The specific steps are as follows: Competent cells were removed from the -80°C freezer and placed on ice. After thawing, 5 μL of digested PCR product was added, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42°C water bath for 45 s, immediately placed on ice for 2-3 min, and then 200 μL of LB medium was added. The cells were incubated at 37°C and 220 rpm for 45 min. After incubation, the cells were centrifuged at 12000 rpm to remove some of the supernatant. The competent cells transformed with the recombinant plasmid were plated on LB agar plates containing 50 μg / mL kanamycin and cultured overnight. After single colonies emerged, single clones were picked for colony PCR verification. Positive clones were sequenced to further verify the correctness of the mutation. Finally, the constructed engineered bacteria were stored at -80°C for subsequent use.

[0033] Table 1. Main Primer Design Table 2. Site-directed mutagenesis reverse amplification PCR reaction system Table 3. Site-directed mutagenesis PCR reaction procedure Table 4 Dpn I. Digestive System 2. Expression and purification of IgAOX mutant protein.

[0034] The specific steps are as follows: (1) Fermentation of IgAOX mutant: The preserved IgAOX mutant engineered expression strains were streaked for activation. Single colonies were picked and inoculated into 2–3 mL of LK liquid medium (LK liquid medium: LB liquid medium containing a final concentration of 50 μg / mL kanamycin) and cultured overnight at 37°C and 200 r / min. The overnight culture seed culture was then inoculated into 100 mL of medium at a ratio of 1:100 and cultured until OD (Organic Degradation). 600 When the concentration reaches 0.6–0.8, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.5 mM, and then induce fermentation at 18°C ​​and 200 r / min for 18–20 h to induce protein expression. After induction, collect the fermentation broth and centrifuge at 4°C and 3800 rpm for 8 min to collect the cell pellet.

[0035] (2) Purification of IgAOX mutant The bacterial cells were resuspended in 10 mL of lysis buffer (buffer: 50 mM HEPES, 300 mM NaCl, pH 8.2), and the centrifuge tubes were subjected to sonication for 20 min in an ice-water mixture. The program was set to run for 2 seconds, pause for 3 seconds, and centrifuge at 12000 r / min for 10 min to collect the supernatant. All supernatant was loaded onto a Ni nucleophilic chromatography column and incubated on ice for 30 min. Impurities were eluted sequentially with HEPES buffer containing 20 mM and 50 mM imidazole, followed by elution of the target protein with HEPES buffer containing 300 mM imidazole. The protein was then concentrated and desalted using an ultrafiltration tube. The protein solution in the ultrafiltration vessel was aliquoted and stored at -80℃ for later use. The purification results of alcohol oxidase are as follows: Figure 2 As shown.

[0036] 3. Activity detection of IgAOX mutant protein.

[0037] The reaction system for the alcohol oxidase activity assay is shown in Table 5. After 2 h of reaction, 5 µL of 10% TCA (trichloroacetic acid) was added to each tube to terminate the reaction. Formaldehyde generation was then detected using the acetylacetone spectrophotometric method. For the assay, 20 µL of acetylacetone and 200 µL of diluted reaction sample were added to each well of a 96-well plate, and the plates were incubated at 50 °C for 20 min. The absorbance (A) of the reaction product at 414 nm in the 96-well plate was measured using a microplate reader. 414 The formaldehyde concentration and conversion rate were calculated based on the formaldehyde standard curve. First, a formaldehyde standard curve needs to be plotted. Specifically: prepare the acetylacetone reagent (dissolve 7.708 g ammonium acetate, 5.4 mL 500 mM acetic acid, and 100 μL acetylacetone in 4.5 mL distilled water, and bring the volume to 20 mL), and store it at 4℃ protected from light. Take an appropriate amount of formaldehyde aqueous solution and dilute the formaldehyde test solution serially; use an ELISA reader for colorimetric detection. Add 20 µL of acetylacetone reagent to each well of a 96-well plate, then add 200 µL of the diluted sample to each well, with three replicates per group; after mixing thoroughly, incubate the 96-well plate in a 50℃ incubator for 20 min. At this time, formaldehyde and Nash reagent react fully to form a yellow complex; use an ELISA reader to detect the absorbance (A) of the reaction product at 414 nm in the 96-well plate. 414 ), and plot the formaldehyde standard curve by taking the average of the data ( Figure 3 ).

[0038] The formula for calculating the conversion rate of alcohol oxidase C is shown in equation (I): C conversion rate (%) = (∆ A bs 414 -0.0035)×n×100 / 0.1933 / 30.03 / 20 (Ⅰ); Where, ∆A bs 414 The difference in absorbance at 414 nm is represented by , n represents the dilution factor of the reaction solution, 30.03 is the relative molecular mass of formaldehyde, and the methanol substrate concentration is 20 mM.

[0039] The formula for calculating relative enzyme activity is shown in equation (II): Relative enzyme activity = mutant enzyme activity / wild-type alcohol oxidase activity (II); The results of the alcohol oxidase mutant activity assay are shown in Table 6.

[0040] Table 5. Reaction system for alcohol oxidase activity assay Table 6 Comparison of relative enzyme activities between wild-type alcohol oxidase and single-point mutants. As shown in Table 6, the activities of V229A, S271G, and K297R in the IgAOX mutants were 40-50% higher than those in the wild type.

[0041] 4. Detection of the thermal stability of IgAOX mutant protein.

[0042] To maximize the retention of residual enzyme activity in different stable assays, glycerol was added to mitigate the damage to the enzyme molecular structure caused by high temperatures. Specifically, 10% glycerol was added to the reaction system, and the enzyme solution was incubated at 40℃ and 45℃ for 15 min. The remaining activity was then determined under standard conditions. The results of the thermostability assay for the alcohol oxidase mutant are shown in Table 7.

[0043] Table 7 Comparison of relative residual enzyme activities of wild-type alcohol oxidase and dominant mutants As shown in Table 7, among the IgAOX mutants, A227S and Y605M showed the greatest improvement in thermal stability compared to the wild type.

[0044] 5. Combination mutation of dominant mutants.

[0045] Based on the general principles of enzyme engineering, the superior mutants with enhanced catalytic properties (catalytic efficiency, thermal stability) obtained through screening often exhibit a positive additive effect or synergistic enhancement when combined. Therefore, this invention considers the mutants that show a 0.2-fold increase in catalytic activity as superior mutants, namely V229A, A227S, S271G, K297R, and Y605M, as the targets for the next step of dual-combination mutation.

[0046] A second amino acid substitution mutation was introduced into the dominant single mutant using reverse amplification PCR with the primer pairs shown in Table 1. The PCR system and procedure were the same as those in Table 2 and Table 3, respectively. The obtained product was used... Dpn The template was digested for 1 h, and the digestion product was transformed into *E. coli* BL21(DE3) competent cells, plated on LK plates, and incubated at 37°C for 12 h. Single colonies were picked for colony PCR and sequencing verification. Finally, purification was performed according to the steps in Part 2 above, and catalytic activity and thermostability were tested according to the steps in Part 3 above. The results of the catalytic activity and thermostability determination of the alcohol oxidase double mutant are shown in Tables 8 and 9.

[0047] Table 8 Comparison of catalytic activity and thermal stability of wild-type alcohol oxidase and double mutant. Catalytic activity assays showed that after combining the dominant single mutation sites in two groups, the activity of most of the two-group mutants was significantly reduced compared to the wild type. Only the two-group mutants K297R / A227S and K297R / Y605M showed higher activity than the wild type, with catalytic activities increasing by 13.9% and 51.2% respectively compared to the wild type. Compared with the K297R single mutant, there was no significant increase in activity (K297R enzyme activity increased by 50%).

[0048] At 40℃, in mutants combined with Y605M, which exhibits excellent thermal stability, the thermal stability of V229A / Y605M and K297R / Y605M was improved, with remaining activities increasing by 43.2% and 83.4% respectively compared to the wild type. At 45℃, most mutants essentially lost their activity, with only K297R / Y605M retaining some activity, and its remaining activity increasing by 18.2% compared to the wild type. In mutants combined with A227S, which exhibits excellent thermal stability, at 40℃, the thermal stability of S271G / A227S and K297R / A227S was improved, with remaining activities increasing by 84.8% and 109.6% respectively compared to the wild type, but their catalytic activity was severely inhibited.

[0049] In summary, only the dual-mutant K297R / Y605M showed improved catalytic activity and thermal stability, with a 51.2% increase in catalytic activity, an 83.4% increase in thermal stability at 40℃, and an 18.2% increase in thermal stability at 45℃.

[0050] 6. The alcohol oxidase mutant K297R / Y605M catalyzes the oxidation of methanol to formaldehyde.

[0051] Under the premise of enzyme activity determination according to the catalytic reaction system shown in Table 5, the formaldehyde generation and C conversion rate were detected by an acetylacetone spectrophotometer. After 2 hours of reaction, the C conversion rate of alcohol oxidase IgAOX reached 61.6%, while the C conversion rate of the dual mutant K297R / Y605M reached over 88%. This indicates that the mutant of the present invention exhibits a significant advantage in the reaction of methanol oxidation to formaldehyde.

[0052] In summary, this invention employs a semi-rational design approach. By comparing homologous sequences of alcohol oxidase IgAOX and analyzing its three-dimensional structure, key amino acid sites around the active region are identified, and a series of mutants are designed. This results in combined mutants with improved catalytic activity and thermal stability. These mutants enable their application in biosynthesis and biocatalysis, providing new ideas and a technological foundation for the green and environmentally friendly synthesis of chemical raw materials.

[0053] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the description, without departing from the scope of the present invention, should be considered to still be within the protection scope of the present invention.

Claims

1. A mutant of the alcohol oxidase IgAOX, characterized in that, The amino acid sequences of the mutants of the alcohol oxidase IgAOX are shown in any of (a) to (f): (a) Replace V at position 229 of the amino acid sequence shown in SEQ ID NO: 1 with A; (b) Replace the S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 with G; (c) Replace the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 with R; (d) Replace the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 with R, and replace the Y at position 605 with M; (e) Replace S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 with G, and replace A at position 227 with S; (f) Replace the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 with R, and replace the A at position 227 with S.

2. A gene encoding a gene, characterized in that, The encoding gene encodes a mutant of the alcohol oxidase IgAOX as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, When the V at position 229 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with A, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 3; When the S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with G, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 4; When the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with R, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 5; When the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with R, and the Y at position 605 is replaced with M, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

6. When the S at position 271 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with G, and the A at position 227 is replaced with S, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

7. When the K at position 297 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with R, and the A at position 227 is replaced with S, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

8.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the coding gene as described in claim 2 or 3.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the recombinant expression vector as described in claim 4.

6. The recombinant strain according to claim 5, characterized in that, The host strain of the recombinant strain is Escherichia coli strain BL21.

7. A recombinant alcohol oxidase catalyst, characterized in that, This includes the mutant of alcohol oxidase IgAOX as described in claim 1; or the pure enzyme solution of the mutant alcohol oxidase IgAOX obtained by purifying the lysate of the recombinant strain as described in claim 5 or 6.

8. The application of a recombinant alcohol oxidase catalyst, characterized in that, The recombinant alcohol oxidase catalyst of claim 7 is used to catalyze the oxidation of methanol to formaldehyde.

9. The application of the recombinant alcohol oxidase catalyst according to claim 8, characterized in that, The reaction conditions for catalytic oxidation of methanol to formaldehyde are as follows: methanol concentration of 20-50 mM, recombinant alcohol oxidase catalyst of 0.5-1.0 mg / mL, reaction temperature of 20-30℃, pH of 7.5-8.5, and reaction time of 1-3 hours.

10. The application of the recombinant alcohol oxidase catalyst according to claim 9, characterized in that, The reaction conditions for catalytic oxidation of methanol to formaldehyde also include: the amount of catalase used is 200-500 U / mL.

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