Glycol aldehyde synthetase mutant and application thereof in improving heat stability of glycolic aldehyde synthetase

Abstract

The invention discloses a glycolaldehyde synthetase mutant and application of the glycolaldehyde synthetase mutant in improving the thermal stability of glycolaldehyde synthetase, and belongs to the technical field of biological catalysis application. According to the glycolaldehyde synthetase mutant disclosed by the invention, on the basis of an amino acid sequence as shown in SEQ ID NO.1, the glycolaldehyde synthetase mutant has the following mutations: A381P / E509F, A381P / K290P, A381P, S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V, A381P / S61A or A381P / K290P / S61A. The mutant protein can catalyze formaldehyde to prepare hydroxyacetaldehyde (glycolaldehyde). In addition, the mutant protein can also improve the catalytic efficiency from formaldehyde to glycolaldehyde, and due to the improvement of the thermal stability, continuous catalysis of industrial enzyme in actual reaction is facilitated, so that the mutant has a good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biocatalysis application, and more particularly to an ethanol aldehyde synthetase mutant and application thereof in improving the thermal stability of ethanol aldehyde synthetase. BACKGROUND

[0002] Ethanol aldehyde is an important bulk product intermediate and an important raw material for organic synthesis. Using this compound as an intermediate, ethylene glycol, glycolic acid, D-erythrose, xylose and PHB can be obtained, which have great application prospects in food additives, biodegradable plastics, fine chemicals and pharmaceuticals. At present, this compound is mostly used as an important intermediate in the utilization of C1 compounds to synthesize high value-added chemicals.

[0003] Ethanol aldehyde synthetase has the function of catalyzing two molecules of formaldehyde to synthesize one molecule of ethanol aldehyde, and is a non-natural enzyme obtained by modification of benzoyl formic acid decarboxylase. Due to its unique catalytic ability, ethanol aldehyde synthetase has attracted much attention in C1 utilization. However, its low catalytic efficiency and thermal stability limit the efficiency of formaldehyde detoxification and C1 conversion.

[0004] The thermal stability of enzymes is usually improved by directed evolution, and the screening of mutant libraries is carried out in micro-well plates. In recent years, with the progress of computational methods, various computational prediction platforms have been designed, such as FireProt, PoPMuSiC, etc. Through the analysis of enzyme sequences and virtual saturation mutation, the possible sequence space is rapidly explored to realize the improvement of thermal stability, which has been verified in the improvement of thermal stability of many enzymes.

[0005] Ethylene glycol, as the simplest dihydric alcohol and important organic chemical raw material, is widely used in various fields. At present, a multi-enzyme cascade system including ethanol aldehyde synthetase has been developed to catalyze the synthesis of ethylene glycol from methanol, but the stability of the enzymes in the existing synthesis route of ethylene glycol from C1 compounds is still poor, which cannot maintain continuous catalytic reaction and is difficult to realize industrialization. Therefore, it is necessary to design and modify ethanol aldehyde synthetase with better stability and activity.

[0006] Therefore, it is necessary to provide an ethanol aldehyde synthetase mutant and application thereof in improving the thermal stability of ethanol aldehyde synthetase. SUMMARY

[0007] Therefore, the present application provides an ethanol aldehyde synthetase mutant and application thereof in improving the thermal stability of ethanol aldehyde synthetase.

[0008] The application adopts a computer-aided rational design method to explore the sequence space of the ethanol aldehyde synthetase mutant preserved in the laboratory, excavates key unit point mutants that can affect the thermal stability of the ethanol aldehyde synthetase, and significantly improves the thermal stability of the ethanol aldehyde synthetase through a combined mutation strategy. Further, the ethanol aldehyde synthetase mutant with good thermal stability and activity is applied to continuous catalysis to synthesize ethylene glycol, and the accumulation of ethylene glycol is greatly improved.

[0009] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions: The ethanol aldehyde synthetase mutant has the following mutations based on the amino acid sequence shown in SEQ ID NO. 1: A381P / E509F, A381P / K290P (GALS M5), A381P (GALS M4), S61A, A381P / K290P / E509F, A381P / C49A, A381P / T100V, A381P / S61A or A381P / K290P / S61A.

[0010] Further, the related biological material of the ethanol aldehyde synthetase mutant is a nucleic acid molecule capable of expressing the ethanol aldehyde synthetase mutant, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

[0011] Further, the application of the ethanol aldehyde synthetase mutant or the related biological material in improving the thermal stability of the ethanol aldehyde synthetase.

[0012] Further, the application of the ethanol aldehyde synthetase mutant or the related biological material in improving the specific activity of the ethanol aldehyde synthetase.

[0013] Further, the application of the ethanol aldehyde synthetase mutant in multi-enzyme cascade continuous catalysis for preparing ethylene glycol from methanol. The multi-enzyme continuous catalysis reaction is performed to obtain ethylene glycol by taking the ethanol aldehyde synthetase mutant or the related biological material, lactal reductase and methanol oxidase as catalysts and methanol as substrate.

[0014] The term "GALS" in the application refers to ethanol aldehyde synthetase, "fuco" refers to lactal reductase, "AOX" refers to methanol oxidase, and "HPLC" refers to high performance liquid chromatography.

[0015] The term "protein" and "protein" in the application can be used interchangeably.

[0016] The term "enzyme activity" (enzyme activity) in the application is also called enzyme activity, which refers to the ability of an enzyme to catalyze a certain chemical reaction; the enzyme activity in the application is expressed by specific activity, which refers to the enzyme activity per mg of enzyme protein, and the unit is U / g.

[0017] The term "Tm" as used herein refers to the melting temperature of a protein.

[0018] The term "variant" and "mutant" can be used interchangeably in the present application.

[0019] The term "TPP" as used herein refers to thiamine pyrophosphate.

[0020] The term "FAD" as used herein refers to flavin adenine dinucleotide.

[0021] The term "NADH" as used herein refers to reduced form of coenzyme I.

[0022] The term "wild type" as used herein refers to an enzyme protein translated from an amino acid or base sequence without any changes.

[0023] The detection method and instrument in the present application: (1) PTC-200 PCR instrument (MJ RESEARCH. INC. USA) was used for PCR amplification.

[0024] (2) MINI-SUB CELL GT POWER PAC 1000 type agarose gel electrophoresis instrument (Bio-Rad Company, USA) was used for detection and separation of PCR products.

[0025] (3) HPLC U3000 high performance liquid chromatograph (Thermo Fisher Scientific) was used for product determination.

[0026] (4) 10 mL specification nickel agarose gel FF affinity chromatography column filler preloaded column (bersee, China) was used for protein purification.

[0027] (5) 870 type enzyme marker (Thermo Fisher Scientific) was used for determination of the concentration of the purified protein.

[0028] Compared with the prior art, the application provides an ethanol aldehyde synthetase mutant and application thereof in improving the thermal stability of ethanol aldehyde synthetase, and the activity of the ethanol aldehyde synthetase mutant protein in catalyzing the synthesis of ethanol aldehyde from formaldehyde is 1.28 times higher than that of the ethanol aldehyde synthetase protein shown in SEQ ID NO. 1, wherein the protein melting temperature Tm can reach 63 DEG C, the catalytic activity of the ethanol aldehyde synthetase is improved, and the thermal stability is also improved. Moreover, the activity of the mutant protein does not decrease after incubation at 40 DEG C, 45 DEG C and 50 DEG C for 6 hours, and the original activity can still be maintained at 80% after incubation at 55 DEG C for 6 hours. When the ethanol aldehyde synthetase mutant is used for continuous catalysis of the synthesis of ethylene glycol from methanol through multi-enzyme cascade, the yield of ethylene glycol can reach 10.6 g / L at most, which is superior to the existing synthesis strategies. Due to the improvement of the thermal stability, the industrial enzyme can be used for continuous catalysis in actual reactions, and therefore the mutant protein has a good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the provided drawings.

[0030] Figure 1 It is a plasmid map of pET-22b(+)-GALS-M3.

[0031] Figure 2 It is the protein melting temperature Tm value (A) and specific enzyme activity (B) of 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 application.

[0032] Figure 3 It is the residual activity change of the thermal stability of GALS-M3 and three mutant proteins at 40 DEG C, 45 DEG C, 50 DEG C and 55 DEG C, respectively; wherein A: GALS-M3; B: S61A; C: A381P; D: A381P / E509F.

[0033] Figure 4Residual activity changes of four mutant proteins at 40℃, 45℃, 50℃ and 55℃, respectively; wherein A: A381P / S61A; B: A381P / T100V; C: A381P / C49A; D: A381P / K290P.

[0034] Figure 5 Residual activity changes of two mutant proteins at 40℃, 45℃, 50℃ and 55℃, respectively; wherein A: A381P / K290P / E509F; B: A381P / K290P / S61A.

[0035] Figure 6 Results of using the mutant of ethanol aldehyde synthetase for preparing ethylene glycol by multi-enzyme cascade continuous catalysis. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0037] Example 1: Obtaining of enzyme protein The gene of wild-type ethanol aldehyde synthetase GALS is derived from Pseudomonas putida, and the PDB number of the amino acid sequence of the protein is 6A50 (see patent 202411959375.0). The research basis of the present application is that the 87th threonine (T) is mutated to alanine (A), the 416th alanine (A) is mutated to threonine (T), and the 463rd tryptophan (W) is mutated to isoleucine (I) in the amino acid sequence of the wild-type ethanol aldehyde synthetase, and the mutated amino acid sequence is shown as SEQ ID NO. 1, which is referred to as ethanol aldehyde synthetase mutant M3 (GALS-M3). Without changing the amino acid sequence of GALS-M3, the codons of the GALS-M3 gene are replaced by the codons preferred by Escherichia coli (high frequency of use), and after codon optimization, the optimized GALS-M3 gene sequence is obtained (as shown in SEQ ID NO. 2).

[0038] The amino acid sequence of the ethanol aldehyde synthetase mutant M3 is shown as SEQ ID NO. 1.

[0039] MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQ ASRKPAFINLHSAAGTGNAMGALSNAR A SHSPLIVTAGQQTRAMIGVEAGETNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMA SMPAQGPVYLSVPYDDWDKDADPQSHHLFDRHVSSSVRLNDQDLDILVKALNSASNP AIVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAIS QLLEGHDVVLVIGAPVFRYVFYDPGQYLKPGTRLISVTCDPLEAARAPMGDAIVADIGA MASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVFDTLNDMAPENAIYLNESTST TAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQL T EPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGMLR I FAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK; SEQ ID NO. 1.

[0040] The optimized GALS-M3 gene sequence is shown as SEQ ID NO. 2.

[0041]

[0042] Construction of expression vector The DNA fragment between the Ndel and Xhol sites of pET-22b(+) vector was replaced by 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 ).

[0043] Construction of mutant of ethanol aldehyde synthase GALS-M3 (1) Obtaining of mutant for improving the thermal stability of GALS-M3: The amino acid sequence of GALS-M3 protein was submitted to FireProt 2.1 online web server to explore the whole sequence space of GALS-M3 to predict possible mutation sites. According to the B factor and folding free energy ΔΔG, 16 unit point mutants including K290P, C398P, T448V, D210M, S332M, A381P, E110L, T100V, A102W, N388T, D191W, S61A, G505R, C49A, D166F, and E509F were determined.

[0044] Then site-directed mutagenesis was performed on the recombinant plasmid pET-22b(+)-GALS-M3. The method of primeSTAR Max kit was used for construction. The primers with overlap overlap were used to complete the site-directed mutagenesis of the corresponding sites. The site-directed mutagenesis reaction system: ddH2O 20 μL, primeSTAR Max 25 μL, template (plasmid pET-22b(+)-GALS-M3) 1 μL, and 2 μL of each mutant primer.

[0045] The primer sequences of all site-directed mutations are as follows: K290P-F: acctgccgccgggtaccc; SEQ ID NO. 3.

[0046] K290P-R: ccggcggcaggtactgaccc; SEQ ID NO. 4.

[0047] C398P-F: ctacttcccggctgctggtggtct; SEQ ID NO. 5.

[0048] C398P-R: gcagccgggaagtagtaagaacccgg; SEQ ID NO. 6.

[0049] T448V-F: catcccggttatcttcgttatcatgaacaacggtacc; SEQ ID NO. 7.

[0050] T448V-R: cgaagataaccgggatgttgtactgagcagc; SEQ ID NO. 8.

[0051] D210M-F: ggtccgatggttgacgctgctaacg; SEQ ID NO. 9.

[0052] D210M-R: gtcaaccatcggacccagaacgatagccg; SEQ ID NO. 10.

[0053] S332M-F: tgaagaaatgtctcgtcagctgccgaccg; SEQ ID NO. 11.

[0054] S332M-R: ctgacgagacatttcttcaaccaggttagccagagc; SEQ ID NO. 12.

[0055] A381P-F: ccaccccgcagatgtggcagc; SEQ ID NO. 13.

[0056] A381P-R: catctgcggggtggtagaggtagattcg; SEQ ID NO. 14.

[0057] E110L-F: gctgggctgaccaacgttgacg; SEQ ID NO. 15.

[0058] E110L-R: ttggtcagcccagcttcaacaccgatcatag; SEQ ID NO. 16.

[0059] T100V-F: cagcaggttcgtgctatgatcggtgt; SEQ ID NO. 17.

[0060] T100V-R: gcacgaacctgctgaccagcg; SEQ ID NO. 18.

[0061] A102W-F: gacccgttggatgatcggtgttgaagc; SEQ ID NO. 19.

[0062] A102W-R: gatcatccaacgggtctgctgaccagc; SEQ ID NO. 20.

[0063] N388T-F: cgtctgaccatgcgtaacccggg; SEQ ID NO. 21.

[0064] N388T-R: cgcatggtcagacgctgccacatc; SEQ ID NO. 22.

[0065] D191W-F: gacctgtggatcctggttaaagctctgaactctg; SEQ ID NO. 23.

[0066] D191W-R: ccaggatccacaggtcctggtcgttc; SEQ ID NO. 24.

[0067] S61A-F: caggctgctcgtaaaccggctttcat; SEQ ID NO. 25.

[0068] S61A-R: gtttacgagcagcctgagcgtaaccgtc; SEQ ID NO. 26.

[0069] G505R-F: ctgaaacgttctctgcaggaagctctgtctg; SEQ ID NO. 27.

[0070] G505R-R: tgcagagaacgtttcagctgttccaggttgt; SEQ ID NO. 28.

[0071] C49A-F: ggaagctgctgttgttggtatcgctgacg; SEQ ID NO. 29.

[0072] C49A-R: caacaacagcagcttcctgcagagccag; SEQ ID NO. 30.

[0073] D166F-F: gacaaattcgctgacccgcagtctcacc; SEQ ID NO. 31.

[0074] D166F-R: gggtcagcgaatttgtcccagtcgtcg; SEQ ID NO. 32.

[0075] E509F-F: ctgcagttcgctctgtctgctaaaggtcc; SEQ ID NO. 33.

[0076] E509F-R: cagagcgaactgcagagaacctttcagc; SEQ ID NO. 34.

[0077] (2) DNA purification: FastPure Gel DNA Extraction Mini Kit from Vazyme was used for purification. 50 μL ddH2O and 500 μL GDP Buffer were added to the PCR product and mixed; poured into the chromatographic column and centrifuged at 12000 rpm for 1 min, and the filtrate was discarded; 700 μL GW Buffer was added, centrifuged at 12000 rpm for 1 min, and the filtrate was discarded; centrifuged at 12000 rpm for 2 min, and the remaining liquid was filtered and discarded; placed in a 65°C metal bath to dry the remaining ethanol; 40 μL of 65°C preheated ddH2O was added, and the filtrate was collected by centrifugation at 12000 rpm for 1 min, which was the purified DNA.

[0078] (3) Purification of DNA fragments: The circularization system was 5 μL purified DNA and 5 μL pEASY-Basic Seamless Cloning and Assembly Kit. The PCR instrument parameters were set as follows: 50°C, 15 min.

[0079] (4) GALS-M3 and the predicted mutant were subjected to shake flask expression. The circularization product was transformed into BL21(DE3) competent cells from QiaGen and cultured overnight on Amp-resistant agar plates. Single colonies were inoculated into LB medium containing 100 μg / ml Amp and cultured at 37°C for 12 h. Then, 1% of the inoculum was 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.

[0080] (5) Purification of glycolaldehyde synthetase and its mutants: The bacterial cells were collected by centrifugation at 4500 rpm, and the cells were resuspended in PBS buffer a (containing 50 mM PBS solution, 100 mM NaCl, 10 mM imidazole) with pH 7.5. After ultrasonic disruption, the supernatant was collected by centrifugation, and the crude enzyme solution was purified by Ni-NTA resin affinity column (Solarbio). The non-target proteins were eluted with 10 mL PBS buffer a and 20 mL PBS buffer b (containing 50 mM PBS solution, 100 mM NaCl, 50 mM imidazole). Then the target protein was eluted with 15 mL PBS buffer c (containing 50 mM PBS solution, 100 mM NaCl, 300 mM imidazole) and collected, and the target protein was concentrated and desalted by ultrafiltration tube to obtain a suitable concentration of pure enzyme solution. The protein concentration was calculated by applying G250 coomassie brilliant blue to react with the pure enzyme solution and detecting the absorbance at 595 nm using an enzyme marker (Thermo Fisher Scientific) model 870.

[0081] Example 4 Activity and thermal stability detection of glycolaldehyde synthetase Activity detection: Reaction system: 50 mM formaldehyde, 2 mM Mg 2+ , 0.1 mM TPP, mutant protein 100 μg, total volume of reaction system 1 mL, buffer 0.05 M, pH=8.0 PBS. After reaction at 37°C for 1 h, 50 μL of reaction solution + 950 μL of 2,4-dinitrophenylhydrazine derivative solution was taken and derivatized at 60°C for 1 h, and then the generation amount of product glycolaldehyde was detected by HPLC to calculate the relative activity of mutant protein and GALS-M3.

[0082] HPLC detection of glycolaldehyde: U3000 high performance liquid chromatograph with ultraviolet detector, Aoclaim 120C18 5 μm 4.6*250 mm column (Thermo), 35°C, 0.8 mL / min, detection wavelength 210 nm, mobile phase pure water: acetonitrile=3:7.

[0083] Relative activity is defined as the ratio of specific activity of mutant protein to specific activity of GALS-M3 protein.

[0084] Specific activity is defined as the amount of enzyme that converts 1 μmol of substrate (formaldehyde) to product (glycolic acid) in 1 min under specific conditions, so the calculation formula of specific enzyme activity is: U / g= (C 乙醇醛 (μmol) x 2 / (time (min)) / protein mass (g).

[0085] Protein melting temperature Tm detection: The purified glycolaldehyde synthetase and its mutants were controlled at a concentration of about 1 mg / ml, and a micro amount of protein sample was taken by capillary, and the melting temperature Tm of all enzymes was detected by NanoDSF instrument (NanoTemper Prometheus NT.48, Germany).

[0086] After detection, the mutants with improved activity and thermal stability were selected for two rounds of combined mutation, and finally nine mutants with improved activity and thermal stability were obtained, as shown in Table 1 and Figure 2 .

[0087] Table 1 Enzyme properties of GALS-M3 and its mutant proteins

[0088] Example 5 Determination of change in residual activity of thermal stability of glycolaldehyde synthetase mutants GALS-M3 and its mutant proteins were respectively placed in a metal bath at 40℃, 45℃, 50℃ and 55℃ for incubation, and samples were taken over time to determine the change in activity of glycolaldehyde synthetase after incubation for different times.

[0089] Reaction conditions: 5 mM Mg 2+ , 0.5 mM TPP, 30 mM formaldehyde, 100 μg of the above mutant protein, the total volume of the reaction system was 1 mL, the buffer was 0.05 M PBS with pH=8.0. After 50 μL of the reaction solution was derivatized with 950 μL of 2,4-dinitrophenylhydrazine derivative solution at 60℃ for 1 h, the generation amount of product glycolaldehyde was detected by HPLC to calculate the change trend of activity of the mutant protein before and after incubation with GALS-M3.

[0090] The thermal inactivation curves of GALS-M3 and its mutant proteins are shown in Figures 3-5 .

[0091] Example 6 Glycolaldehyde synthetase mutants and methanol dehydrogenase and lactaldehyde reductase cascade continuous catalysis of methanol to synthesize ethylene glycol Initial reaction system: 150 mM methanol, 5 mM Mg 2+, 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, 1 mg / ml lactol reductase fuco, the total volume of the reaction system was 3 mL, the buffer was 0.05 M PBS with pH=8.0. The reaction was carried out at 37°C. 50 mM methanol and 25 mM NADH were supplemented every 24 h, and the amount of ethylene glycol generated was monitored. 100 μL of the reaction solution + 100 μL of 0.2 M sulfuric acid was taken to terminate the reaction, and the amount of product ethylene glycol generated was detected by HPLC.

[0092] HPLC detection of ethylene glycol method: U3000 high performance liquid chromatograph with a differential detector, Aminex® HPX-87H organic acid column (BIO-RAD, 300x7.8mm), 65°C, 0.6 mL / min, and the mobile phase was 5 mM sulfuric acid.

[0093] The final accumulation amount of ethylene glycol can reach 10.6 g / L after continuous catalysis for 288 h, and the results are shown in Figure 6

[0094] The above description of the disclosed embodiments enables one skilled in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended 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. A glycolaldehyde synthase mutant, 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 glycolaldehyde synthase mutant according to claim 1.

3. An expression vector comprising a gene encoding the glycolaldehyde synthase mutant according to claim 1.

4. A recombinant cell comprising a gene encoding the glycolaldehyde synthase mutant according to claim 1.

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

6. Use of the glycolaldehyde synthase mutant according to claim 1 or the gene encoding the glycolaldehyde synthase mutant according to claim 2, the expression vector according to claim 3, or the recombinant cell according to claim 4 in increasing ethylene glycol production.

7. A method for continuously catalytically preparing ethylene glycol, characterized in that: The glycolaldehyde synthase mutant according to claim 1 is used as a catalyst, methanol is used as a substrate, and a multi-enzyme cascade continuous catalytic reaction is carried out with methanol oxidase AOX and lactaldehyde dehydrogenase fuco to obtain the product.

8. Use of the glycolaldehyde synthase mutant according to claim 1 or the gene encoding the glycolaldehyde synthase mutant according to claim 2, the expression vector according to claim 3, or the recombinant cell according to claim 4 in improving the thermostability of glycolaldehyde synthase.

9. Use of the glycolaldehyde synthase mutant according to claim 1 or the gene encoding the glycolaldehyde synthase mutant according to claim 2, the expression vector according to claim 3, or the recombinant cell according to claim 4 in improving the specific activity of glycolaldehyde synthase.

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