Ethanolaldehyde synthase mutants and their application in improving the thermostability of ethanolaldehyde synthase

Abstract

This invention discloses a mutant of glycolaldehyde synthase and its application in improving the thermostability of glycolaldehyde synthase, belonging to the field of biocatalysis application technology. The glycolaldehyde synthase mutant disclosed in this invention, based on the amino acid sequence shown in SEQ ID NO.1, contains the following mutations: A381P / E509F, A381P / K290P, A381P, S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V, A381P / S61A, or A381P / K290P / S61A. This mutant protein can catalyze the preparation of hydroxyacetaldehyde (glycoaldehyde) from formaldehyde. The mutant protein of this invention also improves the catalytic efficiency of the formaldehyde-to-glycoaldehyde conversion. Due to the improved thermostability, it is beneficial for continuous catalysis in actual industrial reactions, thus the mutant shows good prospects for industrial applications.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis application technology, and more specifically to a mutant of ethanolaldehyde synthase and its application in improving the thermal stability of ethanolaldehyde synthase. Background Technology

[0002] Glycoaldehyde is an important intermediate for bulk products and a crucial raw material for organic synthesis. Using this compound as an intermediate, ethylene glycol, glycolic acid, D-erythrose, xylose, and PHB can be obtained, all of which have significant application prospects in food additives, biodegradable plastics, fine chemicals, and pharmaceuticals. Currently, this compound is primarily used as an important intermediate in the utilization pathway of C1 compounds to synthesize high-value-added chemicals.

[0003] Glycoaldehyde synthase, a non-natural enzyme derived from benzoylformate decarboxylase, catalyzes the synthesis of one molecule of glycolaldehyde from two molecules of formaldehyde. Due to its unique catalytic ability, glycolaldehyde synthase has attracted considerable attention in C1 utilization. However, its low catalytic efficiency and thermal stability limit the efficiency of formaldehyde detoxification and C1 conversion.

[0004] Enzyme thermostability modification is typically achieved through directed evolution, using microplates as culture media for screening mutant libraries. In recent years, advancements in computational techniques have led to the development of various computational prediction platforms, such as FireProt and PoPMuSiC. These platforms enable rapid exploration of potential sequence spaces through enzyme sequence analysis and virtual saturation mutagenesis to modify thermostability, and their effectiveness has been validated in the thermostability modification of various enzymes.

[0005] Ethylene glycol, as the simplest diol and an important organic chemical raw material, is widely used in various fields. Currently, multi-enzyme cascade systems, including glycolaldehyde synthase, have been developed to catalyze the synthesis of ethylene glycol from methanol. However, the enzymes in existing pathways using C1 compounds as raw materials for ethylene glycol synthesis still exhibit poor stability, failing to maintain continuous catalytic reactions and hindering industrial application. Therefore, it is essential to design and modify glycolaldehyde synthases with better stability and activity.

[0006] Therefore, providing a mutant of ethanolaldehyde synthase and its application in improving the thermostability of ethanolaldehyde synthase is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a mutant of ethanolaldehyde synthase and its application in improving the thermostability of ethanolaldehyde synthase.

[0008] This invention employs a computer-aided rational design approach to explore the sequence space of laboratory-preserved glycolaldehyde synthase mutants, identifying key point mutants that may affect the thermostability of glycolaldehyde synthase. A combined mutation strategy is then used to significantly improve the thermostability of glycolaldehyde synthase. Furthermore, the thermostability and activity of the glycolaldehyde synthase mutant are applied for continuous catalysis to synthesize ethylene glycol, resulting in a substantial increase in ethylene glycol accumulation.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] Glycoaldehyde synthase mutants, based on the amino acid sequence shown in SEQ ID NO.1, contain the following mutations: A381P / E509F, A381P / K290P(GALS M5), A381P(GALS M4), S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V, A381P / S61A, or A381P / K290P / S61A.

[0011] Furthermore, the relevant biological materials for the glycolaldehyde synthase mutant are nucleic acid molecules capable of expressing the glycolaldehyde synthase mutant, or expression cassettes, recombinant vectors, recombinant bacteria, or transgenic cell lines containing the nucleic acid molecules.

[0012] Furthermore, the application of the described glycolaldehyde synthase mutant or the related biomaterials in improving the thermal stability of glycolaldehyde synthase.

[0013] Furthermore, the application of the described glycolaldehyde synthase mutant or the related biological materials in improving the specific activity of glycolaldehyde synthase.

[0014] Furthermore, the application of the described glycolaldehyde synthase mutant in the continuous catalytic synthesis of ethylene glycol from methanol using a multi-enzyme cascade. Using the described glycolaldehyde synthase mutant or the related biological materials, lactalaldehyde reductase, and methanol oxidase as catalysts, and methanol as a substrate, a continuous multi-enzyme catalytic reaction is carried out to obtain ethylene glycol.

[0015] In this invention, the terms "GALS" refer to glycolaldehyde synthase, "fuco" refers to lactalaldehyde reductase, "AOX" refers to methanol oxidase, and "HPLC" refers to high performance liquid chromatography.

[0016] In this invention, the terms "protein" and "protein protein" can be used interchangeably.

[0017] The term "enzyme activity" used in this invention refers to the ability of an enzyme to catalyze a certain chemical reaction. In this invention, enzyme activity is expressed as specific activity, which refers to the enzyme activity per mg of enzyme protein, and the unit is U / g.

[0018] In this invention, the term "Tm" refers to the melting temperature of the protein.

[0019] The terms "variant" and "mutant" used in this invention can be used interchangeably.

[0020] In this invention, the term "TPP" refers to thiamine pyrophosphate.

[0021] In this invention, the term "FAD" refers to flavin adenine dinucleotide.

[0022] In this invention, the term "NADH" refers to reduced coenzyme I.

[0023] In this invention, "wild type" refers to an enzyme protein translated and expressed without alteration of its amino acid or base sequence.

[0024] The detection method and instruments in this invention:

[0025] (1) PCR amplification was performed using a PTC-200 PCR instrument (MJRESEARCH.INC., USA).

[0026] (2) PCR products were detected and separated using a MINI-SUB CELL GT POWER PAC 1000 agarose gel electrophoresis system (Bio-Rad, USA).

[0027] (3) The product was determined using an HPLC U3000 high performance liquid chromatograph (Thermo Fisher Scientific).

[0028] (4) Protein purification was performed using a 10 mL nickel agarose gel FF affinity chromatography column pre-packed with BERSE (China).

[0029] (5) The concentration of purified protein was determined using an 870 microplate reader (Thermo Fisher Scientific).

[0030] As can be seen from the above technical solution, compared with the prior art, this invention discloses a mutant of glycolaldehyde synthase and its application in improving the thermal stability of glycolaldehyde synthase. The mutant glycolaldehyde synthase protein exhibits a 1.28-fold increase in catalytic activity for the synthesis of glycolaldehyde from formaldehyde compared to the glycolaldehyde synthase protein shown in SEQ ID NO.1, with a melting temperature (Tm) reaching 63°C. This improves both the catalytic activity and thermal stability of the glycolaldehyde synthase. Furthermore, the mutant protein does not show a decrease in activity after incubation at 40, 45, and 50°C for 6 hours, and retains 80% of its original activity after incubation at 55°C for 6 hours. When the glycolaldehyde synthase mutant is used for multi-enzyme cascade continuous catalysis of methanol to ethylene glycol, the yield of ethylene glycol can reach up to 10.6 g / L, which is superior to current synthesis strategies. Due to the improved thermal stability, it is beneficial for continuous catalysis in actual industrial reactions, thus this mutant protein shows good prospects for industrial application. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 The image shows the pET-22b(+)-GALS-M3 plasmid.

[0033] Figure 2 The protein melting temperature Tm (A) and specific enzyme activity (B) at 37°C are for GALS-M3, A381P / E509F, A381P / K290P(GALS-M5), A381P(GALS-M4), S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V, A381P / S61A, and A381P / K290P / S61A of the present invention.

[0034] Figure 3 The thermostability and residual activity changes of GALS-M3 and three mutant proteins at 40℃, 45℃, 50℃, and 55℃ are shown. Among them, A: GALS-M3; B: S61A; C: A381P; D: A381P / E509F.

[0035] Figure 4The thermostability and residual activity changes of four mutant proteins at 40℃, 45℃, 50℃, and 55℃ are shown. Among them, A: A381P / S61A; B: A381P / T100V; C: A381P / C49A; D: A381P / K290P.

[0036] Figure 5 The thermostability and residual activity changes of two mutant proteins at 40℃, 45℃, 50℃, and 55℃ are shown; where A: A381P / K290P / E509F; B: A381P / K290P / S61A.

[0037] Figure 6 The results show the preparation of ethylene glycol using a multi-enzyme cascade continuous catalysis with a mutant of ethanolaldehyde synthase. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Obtaining the enzyme protein

[0040] The wild-type glycolaldehyde synthase gene GALS originates from *Pseudomonas putida*, and its amino acid sequence PDB number is 6A50 (see patent 202411959375.0). This invention is based on a mutation in the wild-type glycolaldehyde synthase amino acid sequence: threonine (T) at position 87 is mutated to alanine (A), alanine (A) at position 416 is mutated to threonine (T), and tryptophan (W) at position 463 is mutated to isoleucine (I). The resulting amino acid sequence is shown in SEQ ID NO.1 and is abbreviated as glycolaldehyde synthase mutant M3 (GALS-M3). Without altering the GALS-M3 amino acid sequence, the codons of the GALS-M3 gene are replaced with codons preferred (frequently used) by *E. coli*. After codon optimization, the optimized GALS-M3 gene sequence is obtained (as shown in SEQ ID NO.2).

[0041] The amino acid sequence of the glycolaldehyde synthase mutant M3 is shown in SEQ ID NO.1.

[0042] MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQASRKPAFINLHSAAGTGNAMGALSNAR A SHSPLIVTAGQQTRAMIGVEAGETNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYVFYDPGQYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVFDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQL T EPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGMLR I FAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK; SEQ ID NO.1。

[0043] The optimized GALS-M3 gene sequence is shown in SEQ ID NO.2.

[0044]

[0045] Example 2: Construction of expression vector

[0046] The DNA fragment between the NdeI and XhoI restriction sites of the pET-22b(+) vector was replaced with the optimized GALS-M3 gene sequence as shown in SEQ ID NO.2 to obtain the recombinant plasmid, named pET-22b(+)-GALS-M3 (vector map see...). Figure 1 ).

[0047] Example 3 Construction of GALS-M3 mutant of glycolaldehyde synthase

[0048] (1) Obtaining mutants that enhance the thermal stability of GALS-M3: The amino acid sequence of the GALS-M3 protein was submitted to the FireProt 2.1 online web server to explore the full sequence space of GALS-M3 to predict possible mutation sites. Based on the B factor and folding free energy ΔΔG, 16 single point mutants, including K290P, C398P, T448V, D210M, S332M, A381P, E110L, T100V, A102W, N388T, D191W, S61A, G505R, C49A, D166F, and E509F, were identified.

[0049] Subsequently, site-directed mutagenesis was performed on the recombinant plasmid pET-22b(+)-GALS-M3. The plasmid was constructed using the primeSTAR Max kit. Site-directed mutagenesis at the corresponding sites was performed using primers with overlapping regions. The site-directed mutagenesis reaction mixture consisted of: 20 μL ddH2O, 25 μL primeSTAR Max, 1 μL template (plasmid pET-22b(+)-GALS-M3), and 2 μL each of the upstream and downstream primers for each mutant.

[0050] The primer sequences for all site-directed mutagenesis are as follows:

[0051] K290P-F: acctgccgccgggtaccc; SEQ ID NO. 3.

[0052] K290P-R: ccggcggcaggtactgaccc; SEQ ID NO.4.

[0053] C398P-F: ctacttcccggctgctggtggtct; SEQ ID NO.5.

[0054] C398P-R: gcagccgggaagtagtaagaacccgg; SEQ ID NO. 6.

[0055] T448V-F:catcccggttatcttcgttatcatgaacaacggtacc;SEQ ID NO.7。

[0056] T448V-R:cgaagataaccgggatgttgtactgagcagc;SEQ ID NO.8。

[0057] D210M-F:ggtccgatggttgacgctgctaacg;SEQ ID NO.9。

[0058] D210M-R:gtcaaccatcggacccagaacgatagccg;SEQ ID NO.10。

[0059] S332M-F:tgaagaaatgtctcgtcagctgccgaccg;SEQ ID NO.11。

[0060] S332M-R:ctgacgagacatttcttcaaccaggttagccagagc;SEQ ID NO.12。

[0061] A381P-F:ccaccccgcagatgtggcagc;SEQ ID NO.13。

[0062] A381P-R:catctgcggggtggtagaggtagattcg;SEQ ID NO.14。

[0063] E110L-F:gctgggctgaccaacgttgacg;SEQ ID NO.15。

[0064] E110L-R:ttggtcagcccagcttcaacaccgatcatag;SEQ ID NO.16。

[0065] T100V-F:cagcaggttcgtgctatgatcggtgt;SEQ ID NO.17。

[0066] T100V-R:gcacgaacctgctgaccagcg;SEQ ID NO.18。

[0067] A102W-F:gacccgttggatgatcggtgttgaagc;SEQ ID NO.19。

[0068] A102W-R:gatcatccaacgggtctgctgaccagc;SEQ ID NO.20。

[0069] N388T-F:cgtctgaccatgcgtaacccggg;SEQ ID NO.21。

[0070] N388T-R:cgcatggtcagacgctgccacatc;SEQ ID NO.22。

[0071] D191W-F:gacctgtggatcctggttaaagctctgaactctg;SEQ ID NO.23。

[0072] D191W-R:ccaggatccacaggtcctggtcgttc;SEQ ID NO.24。

[0073] S61A-F:caggctgctcgtaaaccggctttcat;SEQ ID NO.25。

[0074] S61A-R:gtttacgagcagcctgagcgtaaccgtc;SEQ ID NO.26。

[0075] G505R-F:ctgaaacgttctctgcaggaagctctgtctg;SEQ ID NO.27。

[0076] G505R-R:tgcagagaacgtttcagctgttccaggttgt;SEQ ID NO.28。

[0077] C49A-F:ggaagctgctgttgttggtatcgctgacg;SEQ ID NO.29。

[0078] C49A-R:caacaacagcagcttcctgcagagccag;SEQ ID NO.30。

[0079] D166F-F:gacaaattcgctgacccgcagtctcacc;SEQ ID NO.31。

[0080] D166F-R:gggtcagcgaatttgtcccagtcgtcg;SEQ ID NO.32。

[0081] E509F-F: ctgcagttcgctctgtctgctaaaggtcc; SEQ ID NO. 33.

[0082] E509F-R: cagagcgaactgcagagaacctttcagc; SEQ ID NO. 34.

[0083] (2) DNA purification: The DNA was purified using the FastPure Gel DNA Extraction Mini Kit from Vazyme. 50 μL of ddH2O and 500 μL of GDP Buffer were added to the PCR product and mixed well. The mixture was then poured into a chromatography column and centrifuged at 12000 rpm for 1 min, discarding the filtrate. 700 μL of GW Buffer was added, and the mixture was centrifuged at 12000 rpm for 1 min, discarding the filtrate. The mixture was centrifuged at 12000 rpm for 2 min, and the remaining liquid was filtered out and discarded. The remaining ethanol was dried in a 65°C metal bath. 40 μL of preheated ddH2O at 65°C was added, and the mixture was centrifuged at 12000 rpm for 1 min to collect the filtrate, which was the purified DNA.

[0084] (3) Circulation of purified DNA fragments: Circulation system: 5 μL purified DNA, 5 μL pEASY-Basic Seamless Cloning and Assembly Kit. PCR instrument parameters were set to: 50℃, 15 min.

[0085] (4) GALS-M3 and the predicted mutants were expressed in shake flasks. The cyclization product was transformed into BL21(DE3) competent cells from TransGen and cultured overnight on Amp-resistant agar plates. Single colonies were picked and inoculated into LB medium containing 100 μg / ml Amp and cultured at 37°C for 12 h. Then, 1% of the cells were inoculated into 50 ml of LB medium and cultured at 37°C. When the OD600 reached 0.6-0.8, IPTG solution with a final concentration of 0.1 mM was added and induced at 16°C for 20 h.

[0086] (5) Purification of glycolaldehyde synthase and its mutants: Cells were collected by centrifugation at 4500 rpm and resuspended in PBS buffer a (pH 7.5, containing 50 mM PBS, 100 mM NaCl, and 10 mM imidazole). After sonication, the supernatant was collected by centrifugation and purified using a Ni-NTA resin affinity column (Solarbio). Non-target proteins were eluted with 10 mL PBS buffer a and 20 mL PBS buffer b (containing 50 mM PBS, 100 mM NaCl, and 50 mM imidazole). Then, the target protein was eluted and collected with 15 mL PBS buffer c (containing 50 mM PBS, 100 mM NaCl, and 300 mM imidazole). The target protein was concentrated and desalted using an ultrafiltration tube to obtain a pure enzyme solution of appropriate concentration. The protein concentration was calculated by reacting the pure enzyme solution with G250 Coomassie Brilliant Blue and measuring the absorbance at 595 nm using a Thermo Fisher Scientific ELISA reader.

[0087] Example 4: Detection of the activity and thermal stability of glycolaldehyde synthase

[0088] Activity detection:

[0089] Reaction system: 50 mM formaldehyde, 2 mM Mg 2+ The reaction mixture consisted of 0.1 mM TPP, 100 μg of mutant protein, and a total reaction volume of 1 mL. The buffer was 0.05 M PBS (pH 8.0). After reacting at 37℃ for 1 h, 50 μL of the reaction solution was added to 950 μL of 2,4-dinitrophenylhydrazine derivatization solution, and derivatized at 60℃ for 1 h. The amount of glycolaldehyde produced was detected by HPLC, and the relative activities of the mutant protein and GALS-M3 were calculated.

[0090] HPLC method for determination of glycolaldehyde: U3000 high performance liquid chromatograph with UV detector, Aoclaim 120C18 5μm 4.6*250mm column (Thermo), 35 ℃, 0.8 mL / min, detection wavelength 210nm, mobile phase was pure water:acetonitrile = 3:7.

[0091] Relative activity is defined as the ratio of the specific activity of the mutant protein to the specific activity of the GALS-M3 protein.

[0092] Specific activity is defined as the amount of enzyme that, under specific conditions, converts 1 μmol of substrate (formaldehyde) into product (glycolic acid) within 1 minute. Therefore, the formula for calculating specific enzyme activity is:

[0093] U / g = (C 乙醇醛 (μmol) × 2 / (time (min)) / protein content (g).

[0094] Protein melting temperature (Tm) detection:

[0095] The concentration of purified glycolaldehyde synthase and its mutants was controlled at around 1 mg / ml, and a small amount of protein sample was taken using a capillary tube. The melting temperature Tm of all enzymes was detected using a NanoDSF instrument (NanoTemper Prometheus NT.48, Germany).

[0096] After testing, mutants with improved activity and thermal stability were selected and subjected to two rounds of combined mutations, ultimately yielding nine mutants with improved activity and thermal stability, as shown in Table 1 and... Figure 2 As shown.

[0097] Table 1 Enzymatic properties of GALS-M3 and its mutant proteins

[0098]

[0099] Example 5: Determination of thermal stability and residual activity changes in ethanolaldehyde synthase mutants

[0100] GALS-M3 and its mutant proteins were incubated in metal baths at 40℃, 45℃, 50℃ and 55℃, respectively. Samples were taken over time to measure the changes in the activity of glycolaldehyde synthase after different incubation times.

[0101] Reaction conditions: 5 mM Mg 2+ The reaction mixture consisted of 0.5 mM TPP, 30 mM formaldehyde, and 100 μg of the mutant protein. The total volume of the reaction system was 1 mL, and the buffer was 0.05 M PBS (pH 8.0). 50 μL of the reaction solution was added to 950 μL of 2,4-dinitrophenylhydrazine derivatization solution, and derivatized at 60℃ for 1 h. The amount of glycolaldehyde produced was detected by HPLC, and the activity change trend of the mutant protein before and after incubation with GALS-M3 was calculated.

[0102] The thermal inactivation curves of GALS-M3 and its mutant proteins are as follows: Figure 3-5 As shown.

[0103] Example 6: Continuous catalysis of methanol to ethylene glycol by a cascade of glycolaldehyde synthase mutant, methanol dehydrogenase, and lactalaldehyde reductase.

[0104] Initial reaction system: 150 mM methanol, 5 mM Mg 2+The reaction mixture consisted of 1 mM TPP, 80 mM NADH, 1 mg / ml methanol oxidase (AOX), 1 mg / ml catalase (CAT), 5 mg / ml GALS-M3-A381P / K290P mutant, and 1 mg / ml lactaldehyde reductase (fuco). The total reaction volume was 3 mL, and the buffer was 0.05 M PBS (pH 8.0). The reaction was carried out at 37 °C. 50 mM methanol and 25 mM NADH were added every 24 h, and the amount of ethylene glycol produced was monitored. The reaction was terminated by adding 100 μL of 0.2 M sulfuric acid to 100 μL of the reaction solution, and the amount of ethylene glycol produced was determined by HPLC.

[0105] HPLC method for ethylene glycol determination: U3000 high performance liquid chromatograph with differential detector, Aminex® HPX-87H organic acid column (BIO-RAD, 300×7.8mm), 65 ℃, 0.6 mL / min, mobile phase was 5 mM sulfuric acid.

[0106] The final accumulation of ethylene glycol reached 10.6 g / L after 288 h of continuous catalysis, as shown in the results. Figure 6 As shown.

[0107] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Mutant of an acetolactate synthase characterized in that, Based on the amino acid sequence shown in SEQ ID NO. 1, the following mutations exist: A381P / E509F, A381P / K290P, A381P, S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V, A381P / S61A or A381P / K290P / S61A.

2. A gene encoding the mutant glycolaldehyde synthetase of claim 1.

3. An expression vector containing the gene encoding the mutant glycolaldehyde synthetase of claim 1.

4. A recombinant cell containing the gene encoding the mutant glycolaldehyde synthetase of claim 1.

5. Use of the mutant glycolaldehyde synthetase of claim 1 or the gene encoding the mutant glycolaldehyde synthetase of claim 2 or the expression vector of claim 3 or the recombinant cell of claim 4 in catalyzing the synthesis of glycolaldehyde from formaldehyde.

6. Use of the mutant glycolaldehyde synthetase of claim 1 or the gene encoding the mutant glycolaldehyde synthetase of claim 2 or the expression vector of claim 3 or the recombinant cell of claim 4 in improving the yield of ethylene glycol.

7. A process for the continuous catalytic production of ethylene glycol, characterized in that, 7. A compound of Formula (I) obtained by a multi-enzyme cascade continuous catalytic reaction of the mutant glycolaldehyde synthetase of claim 1 as catalyst, methanol as substrate, methanol oxidase AOX and lactal dehydrogenase fuco.

8. Use of the mutant glycolaldehyde synthetase of claim 1 or the gene encoding the mutant glycolaldehyde synthetase of claim 2 or the expression vector of claim 3 or the recombinant cell of claim 4 in improving the thermal stability of glycolaldehyde synthetase.

9. Use of an glycolaldehyde synthetase mutant or a gene encoding a glycolaldehyde synthetase mutant or an expression vector containing a gene encoding a glycolaldehyde synthetase mutant or a recombinant cell containing a gene encoding a glycolaldehyde synthetase mutant for increasing the specific activity of a glycolaldehyde synthetase, characterized in that, The mutant glycolaldehyde synthetase has the following mutations based on the amino acid sequence shown in SEQ ID NO. 1: A381P / E509F, A381P / K290P, A381P, S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V or A381P / S61A.

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