Glycol aldehyde synthetase mutant protein and application thereof

Abstract

The invention provides a glycolaldehyde synthetase mutant and application thereof in ethylene glycol synthesis, the protein of the mutant can catalyze formaldehyde to prepare hydroxyacetaldehyde (glycolaldehyde), meanwhile, the mutant does not depend on divalent metal ions to maintain the catalytic activity any more, and the activity is further improved in the presence of Co < 2 + > and Fe < 2 + >. The invention further provides a cofactor replacement strategy. The mutant protein provided by the invention can effectively improve the catalytic efficiency of formaldehyde to glycolaldehyde, and can be carried out under mild conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of directed evolution transformation of enzymes and biocatalytic application, and relates to a glycolaldehyde synthase mutant protein and application thereof. Background Art

[0002] Glycolaldehyde is an important intermediate for bulk products 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 mainly synthesized by chemical methods, mainly obtained by hydrolysis of acetaldehyde after halogenation; it can also be prepared by formaldehyde formylation and hydrogenation, using copper chromite catalyst; or formaldehyde self-condensation over an alkaline catalyst. The chemical synthesis conditions are harsh and the energy consumption is high (Zhao Yicong, Jiang Yueyang, Xing Tao, Han Jia, Guo Hongyue, Wang Wei, Yu Yingmin, Li Bing, Li Qingsong. "Research Progress on the Preparation of Ethylene Glycol from Glycolaldehyde." Applied Chemical Engineering 50.12(2021): 3388-3391+3395; Zhang, J., Liu, D., Liu, Y., Chu, H., Bai, J., Cheng, J., Zhao, H., Fu, S., Liu, H., and Fu, Y. (2023). Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxide. Green Chemistry 25, 3247-3255).

[0003] Biocatalysis is not only inexpensive, but also the strains or proteins can be processed and reused, and it is also environmentally friendly. Junhui Zhou et al. used ethanolaldehyde synthase to construct a multi-enzyme cascade catalytic system, which can be used to obtain products such as ethylene glycol; Hye-Jin Jo et al. used aldolase to realize the reaction of formaldehyde condensation to produce ethanolaldehyde, and used whole-cell catalysis to realize the synthesis of ethylene glycol (Zhou, J., Tian, ​​X., Yang, Q., Zhang, Z., Chen, C., Cui, Z., Ji, Y., Schwaneberg, U., Chen, B., and Tan, T. (2022). Three multi-enzyme cascade pathways for conversion of C1 to C2 / C4 compounds. Chem Catalysis 2, 2675-2690; Jo, H.-J., Kim, J.-H., Kim, Y.-N., Seo, P.-W., Kim, C.-Y., Kim, J.-W., Yu, H.-n., Cheon, H., Lee, EY, Kim, J.-S., and Park, J.-B. (2022). Glyoxylate carboligase-based whole-cell biotransformation of formaldehyde into ethylene glycol via glycolaldehyde. Green Chemistry 24, 218-226). However, the activity of the glycolaldehyde synthase involved in this type of method is still poor, the substrate affinity is weak, and it is difficult to use it industrially. Therefore, it is very necessary to design and transform a new glycolaldehyde synthase. Summary of the invention

[0004] The purpose of the present invention is to solve the problems existing in the above-mentioned prior art and provide a mutant protein of glycolaldehyde synthase, which has a significantly improved activity in catalyzing the synthesis of glycolaldehyde from formaldehyde compared with the catalytic activity of the wild-type protein (from Pseudomonas putida) as shown in SEQ ID NO.1. When wet bacteria expressing the mutant of glycolaldehyde synthase are used to catalyze the synthesis of ethylene glycol from formaldehyde, the conversion rate of formaldehyde is high and is not limited by the concentration of formaldehyde, which is superior to the existing synthesis strategies.

[0005] To this end, the first aspect of the present invention provides an amino acid mutant that affects the enzyme activity of glycolaldehyde synthase, which includes one or more of the following mutations: Thr at position 87 mutates to Ala, Val at position 94 mutates to Ile, Gly at position 109 mutates to Thr, Ala at position 416 mutates to Thr, Trp at position 463 mutates to Ile, and Phe at position 464 mutates to Trp in the amino acid sequence shown in SEQ ID NO.1 from N-terminus to C-terminus of wild glycolaldehyde synthase.

[0006] The second aspect of the present invention provides a mutant protein of glycolaldehyde synthase, the sequence of which comprises one or more of the following mutations: Thr at position 87 mutates to Ala, Val at position 94 mutates to Ile, Gly at position 109 mutates to Thr, Ala at position 416 mutates to Thr, Trp at position 463 mutates to Ile, and Phe at position 464 mutates to Trp, located from the N-terminus to the C-terminus of the amino acid sequence of wild glycolaldehyde synthase as shown in SEQ ID NO.1.

[0007] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 1, whose sequence contains a mutation in which Thr at position 87 from the N-terminus to the C-terminus of the amino acid sequence of wild ethanolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Ala; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 1 is as shown in SEQ ID NO.2.

[0008] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 2, the sequence of which contains a mutation in which Ala at position 416 from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Thr; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 2 is as shown in SEQ ID NO.3.

[0009] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 3, whose sequence contains a mutation in which Trp at position 463 from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Ile; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 3 is as shown in SEQ ID NO.4.

[0010] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 4, the sequence of which includes a mutation of Thr at position 87 to Ala and a mutation of Ala at position 416 to Thr from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 4 is as shown in SEQ ID NO.5.

[0011] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 5, the sequence of which includes a mutation of Thr at position 87 from the N-terminus to the C-terminus to Ala and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 5 is as shown in SEQ ID NO.5.

[0012] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 6, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 6 is as shown in SEQ ID NO.7.

[0013] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 7, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Ala at position 416 to Thr, a mutation of Trp at position 463 to Ile, and a mutation of Phe at position 464 to Trp in the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 7 is as shown in SEQ ID NO.8.

[0014] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 8, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Val at position 94 to Ile, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 8 is as shown in SEQ ID NO.9.

[0015] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 9, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Val at position 94 to Ile, a mutation of Gly at position 109 to Thr, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 9 is as shown in SEQ ID NO.10.

[0016] According to the present invention, the mutant protein also includes a fusion protein obtained by connecting a tag to the N-terminus or C-terminus of the above-mentioned ethanolaldehyde synthase mutant protein; preferably, the tag includes a His tag and / or a SUMO tag; further preferably, the mutant protein also includes a fusion protein obtained by connecting a His tag or a SUMO tag to the N-terminus or C-terminus of ethanolaldehyde synthase mutant protein No. 1, ethanolaldehyde synthase mutant protein No. 2, ethanolaldehyde synthase mutant protein No. 3, ethanolaldehyde synthase mutant protein No. 4, ethanolaldehyde synthase mutant protein No. 5, ethanolaldehyde synthase mutant protein No. 6, ethanolaldehyde synthase mutant protein No. 7, ethanolaldehyde synthase mutant protein No. 8 or ethanolaldehyde synthase mutant protein No. 9.

[0017] The third aspect of the present invention provides use of the mutant protein as described in the second aspect of the present invention in catalyzing the synthesis of ethanolaldehyde from formaldehyde.

[0018] According to the present invention, the application is to carry out catalytic reaction I in a reaction system for synthesizing ethanolaldehyde consisting of catalyst I, metal cofactor I, other cofactor I, reaction medium I and substrate I to synthesize ethanolaldehyde; wherein the catalyst I is a mutant of ethanolaldehyde synthase; and / or the metal cofactor is a divalent metal ion, which includes Mg 2+ 、Co 2+ , Mn 2+ , Cu 2+ , Ca 2+ , Fe 2+ and Zn 2+ one or more of; and / or, the other cofactors include thiamine pyrophosphate TPP and / or vitamin B1 (thiamine); and / or, the substrate I is formaldehyde; and / or, the reaction medium I includes a buffer solution with a pH of 6-9.

[0019] In some embodiments of the present invention, the temperature of the catalytic reaction I is 30-50°C, preferably 30-40°C; the time of the catalytic reaction I is 1-12h; and the stirring speed of the catalytic reaction I is 400rpm.

[0020] In some embodiments of the present invention, in the above-mentioned reaction system for synthesizing ethanolaldehyde, the concentration of substrate I is 10-100mM; the concentration of the metal cofactor I is 1-6mM; the concentration of other cofactors II is 0.1-2mM; and the concentration of the ethanolaldehyde synthase mutant is 0.1-201mg / mL.

[0021] The third aspect of the present invention provides the use of the mutant protein as described in the second aspect of the present invention and lactaldehyde reductase cascade catalyzing the synthesis of ethylene glycol from formaldehyde.

[0022] According to the present invention, the application is to carry out catalytic reaction II in a reaction system for synthesizing ethylene glycol composed of catalyst II, catalyst III, metal cofactor II, other cofactor II, reaction medium II and substrate II to synthesize ethylene glycol; wherein the catalyst II is a ethanolaldehyde synthase mutant or a wet bacterial cell of a ethanolaldehyde synthase mutant obtained by fermentation culture of an engineered bacterium expressing the gene encoding the ethanolaldehyde synthase; and / or, the catalyst III is lactaldehyde reductase; and / or, the metal cofactor II is a divalent metal ion, which includes Mg 2+ 、Co 2+ 、Mn2 + , Cu 2+ , Ca 2+ , Fe 2+ and Zn 2+ One or more of; and / or, the other cofactor II includes thiamine pyrophosphate TPP and / or vitamin B1 (thiamine); and / or, the substrate II is formaldehyde; and / or, the reaction medium II includes a buffer having a pH of 6-9.

[0023] In some embodiments of the present invention, the temperature of the catalytic reaction II is 30-50°C, preferably 30-40°C; the time of the catalytic reaction II is 1-12h; and the stirring speed of the catalytic reaction II is 400rpm.

[0024] In some embodiments of the present invention, in the reaction system for synthesizing ethylene glycol, the concentration of substrate II is 10-100 mM; the concentration of the metal cofactor II is 1-6 mM; the concentration of other cofactors II is 0.1-2 mM; the concentration of the ethanolaldehyde synthase mutant is 0.1-201 mg / mL; and the dosage of the ethanolaldehyde synthase mutant wet cells is 10-50 g / L.

[0025] The present invention provides a mutant protein of ethanolaldehyde synthase, the activity of which in catalyzing the synthesis of ethanolaldehyde from formaldehyde is increased by 3 times compared with the wild-type protein shown in SEQ ID NO.1, wherein the catalytic efficiency kcat / km is increased by 16 times, which greatly improves the substrate affinity and activity of ethanolaldehyde synthase; at the same time, after replacing the metal ion cofactor, when adding Co2+ and Fe 2+ When the mutant activity was compared with Mg 2+ The addition increased by 1.57 and 2.27 times respectively; after replacing the cofactor TPP with vitamin B1, the mutant was more stable and slightly improved in activity compared with the wild-type protein, and vitamin B1 was inexpensive, which greatly reduced the industrial application cost of ethanolaldehyde synthase; when wet bacteria expressing the ethanolaldehyde synthase mutant were used to catalyze formaldehyde to synthesize ethylene glycol, the formaldehyde conversion rate was ≥60% and was not limited by formaldehyde concentration, which was superior to the existing synthesis strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0027] Figure 1 The plasmid maps of GALS-pET-22b(+), GALS-PEC and GALS-pPICZB are shown, wherein A, B and C represent the expression plasmids of GALS in Escherichia coli, Corynebacterium glutamicum and Pichia pastoris expression systems, respectively.

[0028] Figure 2 The HPLC detection results of GALS wild type and mutant are shown; wherein A is the liquid phase detection graph of GALS wild type; B is the liquid phase detection graph of GALS-T87A / A416T / W463I mutant.

[0029] Figure 3 The figure shows the SDS-PAGE gel images of GALS expressed in Escherichia coli and Pichia pastoris.

[0030] Figure 4 The kinetic parameter diagrams of the partial glycolaldehyde synthase mutant proteins are shown. A is the kinetic parameter curve of GALS-T87A; B is the kinetic parameter curve of GALS-T87A / W463I; C is the kinetic parameter curve of GALS-T87A / A416T / W463I; D is the kinetic parameter curve of GALS-T87A / A416T / W463I / F464W; E is the kinetic parameter curve of GALS-T87A / A416T / W463I / V94I; and F is the kinetic parameter curve of GALS-T87A / A416T / W463I / G109T.

[0031] Figure 5 Relative activities for replacement of different divalent metal ions are shown.

[0032] Figure 6 The relative activities of replacing different cofactors are shown.

[0033] Figure 7The results of using the mutant protein of glycolaldehyde synthase to catalyze the synthesis of ethylene glycol in whole cells are shown. A is the result of ethylene glycol synthesis under 10 mM formaldehyde; B is the result of ethylene glycol synthesis under 40 mM formaldehyde. DETAILED DESCRIPTION

[0034] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0035] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0036] I. Terminology

[0037] As used herein, the term "GALS" refers to glycolaldehyde synthase, "fuco" refers to lactaldehyde reductase, and "HPLC" refers to high performance liquid chromatography.

[0038] In the present invention, the terms "protein" and "protein" can be used interchangeably.

[0039] The term "enzyme activity" as used in the present invention is also called enzyme activity, which refers to the ability of an enzyme to catalyze a certain chemical reaction; enzyme activity in the present invention is expressed as specific activity, which refers to the enzyme activity per mg of enzyme protein, with the unit of U / g.

[0040] In the present invention, the terms "variant" and "mutant" can be used interchangeably.

[0041] The term "TPP" as used herein refers to thiamine pyrophosphate.

[0042] The term "TTC" used in the present invention refers to red tetrazolium.

[0043] In the present invention, the following nomenclature is used for amino acid substitution: original amino acid, position, substituted amino acid. The following nomenclature is used for base substitution: original base, position, substituted base.

[0044] The "wild type" mentioned in the present invention refers to the enzyme protein obtained by translation and expression without changing the amino acid or base sequence.

[0045] II. Implementation Plan

[0046] As mentioned above, the existing biocatalytic ethylene glycol synthesis systems are not satisfactory, the activity of the glycolaldehyde synthase involved is still poor, the substrate affinity is weak, and it is difficult to industrialize. Therefore, the inventors have conducted a lot of in-depth research on the modification of glycolaldehyde synthase.

[0047] In the first aspect of the present invention, the inventors have discovered amino acid mutants that affect the enzyme activity of glycolaldehyde synthase, including glycolaldehyde synthase mutants with mutations in one or more sites including sites 87, 94, 109, 416, 463 and 464 in the amino acid sequence of the wild-type glycolaldehyde synthase GALS as shown in SEQ ID NO.1 from the N-terminus to the C-terminus.

[0048] Specifically, the amino acid mutants that affect the enzyme activity of glycolaldehyde synthase include one or more of the following mutations: Thr at position 87 mutates to Ala, Val at position 94 mutates to Ile, Gly at position 109 mutates to Thr, Ala at position 416 mutates to Thr, Trp at position 463 mutates to Ile, and Phe at position 464 mutates to Trp in the amino acid sequence shown in SEQ ID NO.1 from N-terminus to C-terminus of the wild glycolaldehyde synthase.

[0049] The gene of the wild-type glycolaldehyde synthase GALS shown in SEQ ID NO.1 in the present invention is derived from Pseudomonas putida, and the PDB number of the amino acid sequence of the protein is 6A50. Under the premise of not changing the amino acid sequence of GALS, the codons of the GALS gene are replaced with codons preferred by Escherichia coli (high frequency use), and after codon optimization, the optimized gene encoding the wild-type glycolaldehyde synthase GALS is obtained (SEQ ID NO.11).

[0050] In a second aspect of the present invention, a mutant protein of glycolaldehyde synthase is provided, the sequence of which comprises one or more of the following mutations: Thr at position 87 is mutated to Ala, Val at position 94 is mutated to Ile, Gly at position 109 is mutated to Thr, Ala at position 416 is mutated to Thr, Trp at position 463 is mutated to Ile, and Phe at position 464 is mutated to Trp from N-terminus to C-terminus of the amino acid sequence shown in SEQ ID NO.1 of wild glycolaldehyde synthase.

[0051] In some embodiments of the present invention, the mutant protein is a ethanolaldehyde synthase mutant protein No. 1, whose sequence contains a mutation in which Thr at position 87 from the N-terminus to the C-terminus of the amino acid sequence of the wild ethanolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Ala, which is referred to as GALS-T87A mutant in the present invention; preferably, the sequence of the ethanolaldehyde synthase mutant protein No. 1 is as shown in SEQ ID NO.2.

[0052] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 2, whose sequence contains a mutation in which Ala at position 416 from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Thr, which is referred to as GALS-A416T mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 2 is as shown in SEQ ID NO.3.

[0053] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 3, whose sequence contains a mutation in which Trp at position 463 from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Ile, which is referred to as GALS-W463I mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 3 is shown in SEQ ID NO.4.

[0054] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 4, whose sequence includes a mutation of Thr at position 87 to Ala and a mutation of Ala at position 416 to Thr from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1, which is referred to as GALS-T87A / A416T mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 4 is as shown in SEQ ID NO.5.

[0055] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 5, whose sequence includes a mutation of Thr at position 87 from N-terminus to C-terminus to Ala and a mutation of Trp at position 463 to Ile in the amino acid sequence shown in SEQ ID NO.1 of the original ethanolaldehyde synthase, which is referred to as GALS-T87A / W463I mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 5 is shown in SEQ ID NO.6.

[0056] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 6, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1, which is referred to as GALS-T87A / A416T / W463I mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 6 is shown in SEQ ID NO.7.

[0057] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 7, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Ala at position 416 to Thr, a mutation of Trp at position 463 to Ile, and a mutation of Phe at position 464 to Trp located from the N-terminus to the C-terminus of the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1, which is referred to as GALS-T87A / A416T / W463I / F464W mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 7 is shown in SEQ ID NO.8.

[0058] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 8, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Val at position 94 to Ile, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus, which is referred to as GALS-T87A / V94I / A416T / W463I mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 8 is shown in SEQ ID NO.9.

[0059] In some embodiments of the present invention, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 9, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Val at position 94 to Ile, a mutation of Gly at position 109 to Thr, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus, which is referred to as GALS-T87A / G109T / A416T / W463I mutant in the present invention; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 9 is shown in SEQ ID NO.10.

[0060] According to the present invention, the mutant protein also includes a fusion protein obtained by connecting a tag to the N-terminus or C-terminus of the above-mentioned ethanolaldehyde synthase mutant protein; preferably, the tag includes a His tag and / or a SUMO tag; further preferably, the mutant protein also includes a fusion protein obtained by connecting a His tag or a SUMO tag to the N-terminus or C-terminus of ethanolaldehyde synthase mutant protein No. 1, ethanolaldehyde synthase mutant protein No. 2, ethanolaldehyde synthase mutant protein No. 3, ethanolaldehyde synthase mutant protein No. 4, ethanolaldehyde synthase mutant protein No. 5, ethanolaldehyde synthase mutant protein No. 6, ethanolaldehyde synthase mutant protein No. 7, ethanolaldehyde synthase mutant protein No. 8 or ethanolaldehyde synthase mutant protein No. 9.

[0061] Correspondingly, the present invention also provides a biomaterial related to glycolaldehyde synthase, which is a nucleic acid molecule capable of expressing the glycolaldehyde synthase mutant, or an expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the nucleic acid molecule.

[0062] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA.

[0063] The recombinant vector may be a recombinant expression vector or a recombinant cloning vector.

[0064] The expression cassette may consist of a promoter capable of initiating transcription of the nucleic acid molecule, the nucleic acid molecule, and a transcription termination sequence.

[0065] The third aspect of the present invention provides a use of the mutant protein as described in the second aspect of the present invention in catalyzing the synthesis of ethanolaldehyde from formaldehyde, which can be understood as a method of using the mutant protein as described in the second aspect of the present invention to catalyze the synthesis of ethanolaldehyde from formaldehyde.

[0066] According to the present invention, the application is to carry out catalytic reaction I in a reaction system for synthesizing ethanolaldehyde consisting of catalyst I, metal cofactor I, other cofactor I, reaction medium I and substrate I to synthesize ethanolaldehyde; wherein the catalyst I is a mutant of ethanolaldehyde synthase; and / or the metal cofactor is a divalent metal ion, which includes Mg 2+ 、Co 2+ , Mn 2+ , Cu 2+ , Ca 2+ , Fe 2+ and Zn 2+ one or more of; and / or, the other cofactors include thiamine pyrophosphate TPP and / or vitamin B1 (thiamine); and / or, the substrate I is formaldehyde; and / or, the reaction medium I includes a buffer solution with a pH of 6-9.

[0067] In some embodiments of the present invention, the temperature of the catalytic reaction I is 30-50°C, preferably 30-40°C; the time of the catalytic reaction I is 1-12h; and the stirring speed of the catalytic reaction I is 400rpm.

[0068] In some embodiments of the present invention, in the above-mentioned reaction system for synthesizing ethanolaldehyde, the concentration of substrate I is 10-100mM; the concentration of the metal cofactor I is 1-6mM; the concentration of other cofactors II is 0.1-2mM; and the concentration of the ethanolaldehyde synthase mutant is 0.1-201mg / mL.

[0069] In some specific embodiments of the present invention, the present invention provides a method for synthesizing glycolaldehyde by replacing a divalent metal ion cofactor, characterized in that the glycolaldehyde synthase mutant is expressed as a catalyst, Mg 2+ 、Co 2+ , Mn 2 + , Cu 2+ , Ca 2+ , Fe 2+ or Zn 2+ It is a metal cofactor, catalyzing the reaction with thiamine pyrophosphate TPP as a cofactor and formaldehyde as a substrate.

[0070] Specifically, the method for synthesizing ethanolaldehyde by replacing the divalent metal ion cofactor uses the pure enzyme obtained by separating and purifying the engineered bacteria expressing the coding gene of the ethanolaldehyde synthase mutant after fermentation culture as a catalyst, formaldehyde as a substrate, and a buffer solution with a pH of 6-9 as a reaction medium, and performs a catalytic reaction at 30-50°C and 150-250rpm.

[0071] In some preferred embodiments, in the catalytic reaction, the substrate concentration in the reaction system is 10-100 mM; the divalent metal ion concentration is 1-6 mM; the thiamine pyrophosphate TPP concentration is 0.1-2 mM; the pure enzyme concentration is 0.1-2 mg / mL; the pH of the reaction system is 6-9; the reaction temperature is 30-40°C; and the reaction time is 1-12 h.

[0072] In other embodiments of the present invention, the present invention provides a method for synthesizing ethanolaldehyde by replacing cofactors, characterized in that the ethanolaldehyde synthase mutant expressed as a catalyst, TPP or vitamin B1 (thiamine) as a cofactor, and Mg 2+ It is a metal cofactor and catalyzes the reaction using formaldehyde as substrate.

[0073] Specifically, the method for synthesizing ethanolaldehyde by replacing the cofactor uses the pure enzyme obtained by separating and purifying the engineered bacteria expressing the coding gene of the ethanolaldehyde synthase mutant after fermentation culture as a catalyst, formaldehyde as a substrate, and a buffer solution with a pH of 6-9 as a reaction medium, and the catalytic reaction is carried out at 30-50°C and 150-250rpm.

[0074] In some preferred embodiments, in the catalytic reaction, the substrate concentration in the reaction system is 10-100 mM; the divalent metal ion concentration is 1-6 mM; the thiamine pyrophosphate TPP or vitamin B1 concentration is 0.1-2 mM; the pure enzyme concentration is 0.1-2 mg / mL; the pH of the reaction system is 6-9; the reaction temperature is 30-40°C; and the reaction time is 1-12 h.

[0075] The fourth aspect of the present invention provides the use of the mutant protein as described in the second aspect of the present invention and lactaldehyde reductase cascade catalyzing the synthesis of ethylene glycol from formaldehyde, which can be understood as the method of synthesizing ethylene glycol from formaldehyde by cascade catalyzing the mutant protein as described in the second aspect of the present invention and lactaldehyde reductase.

[0076] According to the present invention, the application is to carry out catalytic reaction II in a reaction system for synthesizing ethylene glycol composed of catalyst II, catalyst III, metal cofactor II, other cofactor II, reaction medium II and substrate II to synthesize ethylene glycol; wherein the catalyst II is a ethanolaldehyde synthase mutant or a wet bacterial cell of a ethanolaldehyde synthase mutant obtained by fermentation culture of an engineered bacterium expressing the gene encoding the ethanolaldehyde synthase; and / or, the catalyst III is lactaldehyde reductase; and / or, the metal cofactor II is a divalent metal ion, which includes Mg 2+ 、Co 2+ 、Mn2 + , Cu 2+ , Ca 2+ , Fe 2+ and Zn 2+ One or more of; and / or, the other cofactor II includes thiamine pyrophosphate TPP and / or vitamin B1 (thiamine); and / or, the substrate II is formaldehyde; and / or, the reaction medium II includes a buffer having a pH of 6-9.

[0077] In some embodiments of the present invention, the temperature of the catalytic reaction II is 30-50°C, preferably 30-40°C; the time of the catalytic reaction II is 1-12h; and the stirring speed of the catalytic reaction II is 400rpm.

[0078] In some embodiments of the present invention, in the reaction system for synthesizing ethylene glycol, the concentration of substrate II is 10-100 mM; the concentration of the metal cofactor II is 1-6 mM; the concentration of other cofactors II is 0.1-2 mM; the concentration of the ethanolaldehyde synthase mutant is 0.1-201 mg / mL; and the dosage of the ethanolaldehyde synthase mutant wet cells is 10-50 g / L.

[0079] In some specific embodiments of the present invention, the present invention provides a method for preparing an ethylene glycol compound, characterized in that the ethanolaldehyde synthase mutant expressed is used as a catalyst, formaldehyde is used as a substrate, and a catalytic reaction is carried out to obtain ethanolaldehyde, which is then catalyzed by lactaldehyde reductase to obtain ethylene glycol.

[0080] Specifically, the method for preparing ethylene glycol compounds from formaldehyde uses wet bacteria obtained by fermentation culture of engineered bacteria expressing the coding gene of the ethanolaldehyde synthase mutant as a catalyst, formaldehyde as a substrate, and a buffer solution with a pH of 6-9 as a reaction medium, and performs a catalytic reaction at 30-40°C and 150-250rpm.

[0081] In a more preferred embodiment, in the catalytic reaction, the substrate concentration in the reaction system is 10-100 mM; the divalent metal ion concentration is 1-6 mM; the thiamine pyrophosphate TPP concentration is 0.1-2 mM; the amount of bacteria in the reaction system is 10-50 g / L; the pH of the reaction system is 6-9; the reaction temperature is 30-40°C; and the reaction time is 1-12 h.

[0082] The detection method and instrument in the present invention:

[0083] (1) PCR amplification was performed using a PTC-200 PCR instrument (MJ RESEARCH. INC., USA).

[0084] (2) PCR products were detected and separated using a MINI-SUB CELL GT POWER PAC 1000 agarose gel electrophoresis instrument (Bio-Rad, USA).

[0085] (3) The product was determined using HPLC U3000 (Thermo Fisher Scientific).

[0086] (4) A 10 mL nickel-agarose gel FF affinity chromatography column prepacked with filler (bersee, China) was used for protein purification.

[0087] (5) The protein concentration after purification was measured using an 870 microplate reader (Thermo Fisher Scientific).

[0088] III. Embodiment

[0089] The present invention is described in detail below through specific examples. The experimental methods described below are all routine laboratory methods unless otherwise specified. The experimental materials described below are all available from commercial channels unless otherwise specified.

[0090] In some specific embodiments of the present invention, Escherichia coli Trans10, BL21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and Pichia pastoris X33 were used as host cells (purchased from the National Strain Resource Bank) for the construction and expression of glycolaldehyde synthase and mutant proteins, respectively. The culture medium formulas involved in the following embodiments are as follows:

[0091] LB liquid medium: peptone 1%, yeast extract 0.5%, NaCl 1%;

[0092] LB solid medium: agar 1.5%, peptone 1%, yeast extract 0.5%, NaCl 1%;

[0093] LBHIS medium (g / L): peptone 5, yeast powder 2.5, NaCl 5, brain heart extract (BHI) 18.5, sorbitol 91;

[0094] YPD liquid medium: 2% peptone, 1% yeast extract, 2% glucose;

[0095] The unit in the culture medium is % (W / V);

[0096] BMGY / BMMY liquid culture medium 1L: BMGY / BMMY culture medium base 44.1g, YNB 13.4g, biotin; BMGY plus 100mL sterile 10% glycerol, BMMY plus 100mL sterile 5% methanol aqueous solution.

[0097] Example 1: Obtaining enzyme protein

[0098] In the present invention, the amino acid sequence of the ethanolaldehyde synthase shown in SEQ ID No. 1 is derived from Pseudomonas putida, and the PDB number of the protein amino acid sequence is 6A50. The ethanolaldehyde synthase GALS gene is obtained from the document (DOI number of the document: 10.1038 / s41467-019-09095) (obtained by BGI based on the document). Without changing the GALS amino acid sequence, the codons of the GALS gene are replaced with codons preferred by Escherichia coli (high frequency use), and after codon optimization, the optimized GALS gene sequence is obtained.

[0099] Example 2: Construction of expression vector

[0100] The amino acid sequence shown in SEQ ID No.1 was codon-optimized and replaced with pET-22b(+) vector and pPICZB vector (see Figure 1 ). The recombinant plasmids were obtained and named as pET-22b-GALS and pPICZB-GALS.

[0101] Example 3: Expression of wild-type and mutant proteins in Escherichia coli

[0102] (1) The E. coli expression recombinant plasmid pET-22b-GALS was transferred into E. coli BL21 (DE3) (TransGen) to obtain recombinant bacteria. Positive clones were screened using ampicillin resistance plates (Amp, 100 mg / mL) and cultured overnight at 37°C;

[0103] (2) Transfer the selected single clone into 4 mL LB liquid medium (Amp, 100 mg / mL) and culture in a constant temperature incubator at 37°C and 180 rpm for 12-16 h;

[0104] (3) The bacterial solution cultured in step (2) was inoculated into 50 mL LB liquid medium (Amp, 100 mg / mL), and cultured in a constant temperature incubator at 37°C and 150 rpm until OD600 was 0.6-0.8, and then IPTG inducer (final concentration was 0.4 mM) was added, and induced culture was carried out in a constant temperature incubator at 16°C and 150 rpm for 24 h;

[0105] (4) After the culture was completed, the cells were collected by centrifugation at 4500 rpm for 10 min at 4°C, and resuspended in 10 mL of PBS solution (0.05 M, pH = 7.5) containing 10 mM imidazole and 100 mM NaCl, and ultrasonically disrupted for 15 min;

[0106] (5) After cell disruption, centrifuge at 4°C and 4500 rpm for 20 min. The resulting supernatant is the crude enzyme solution required for the experiment.

[0107] (6) The crude enzyme solution was purified by a nickel-agarose gel FF affinity chromatography column pre-packed with seasoning to obtain purified enzyme 1; the purified solution 1 was ultrafiltered using a 30 KDa ultrafiltration membrane to obtain the final purified enzyme solution, and SDS gel electrophoresis was used to verify that the protein expression was correct ( Figure 3 and Figure 4 );

[0108] (7) G250 Coomassie Brilliant Blue was used to react with the enzyme solution, and then the absorbance at 595 nm was detected using an 870 microplate reader (Thermo Fisher Scientific) to calculate the protein concentration.

[0109] Example 4: Construction of GALS mutant of glycolaldehyde synthase

[0110] The recombinant plasmid pET-22b-GALS was subjected to site-directed saturation mutagenesis. The FastPfuFly PCR SuperMix (-dye) kit was used. The NNK degenerate primers were used to complete the construction of the mutation library. According to the structure and activity pocket of GALS, the amino acid sequence of ethanolaldehyde synthase as shown in SEQ ID NO.1 was selected from the 1st to 528th amino acid positions 87, 94, 109, 416, 463 and 464 of the N-terminal to C-terminal amino acid positions for saturation mutation.

[0111] Example 5: High-throughput screening method

[0112] The red tetrazolium (TTC) derivatization method was used to color the product of the catalytic reaction, ethanolaldehyde. The derivatization solution was prepared by preparing A and B mother solutions separately, where A solution: 24mM TTC; B solution: 1M sodium hydroxide solution. The two were mixed (A: B = 1: 3 (V: V)) to form a TTC derivatization solution; the final concentration of formaldehyde reaction was 30mM, and the reaction was carried out at 37℃ for 1h; the derivatization conditions were: 100μL reaction solution + 50μLTTC derivatization solution for 8min, and the absorbance at 510nm was detected using an 870 microplate reader (Thermo Fisher Scientific) to screen effective mutants.

[0113] Finally, mutants with absorbance increased by more than 1.18 times compared with the wild type were selected for purification and re-screening, and mutants with better activity were selected for combined mutation.

[0114] Example 6: Enzyme activity assay

[0115] Reaction system: 10 mM formaldehyde, 2 mM Mg 2+ , 0.1mM TPP, 100μg of the above wild-type protein or mutant protein, the total volume of the reaction system is 1mL, the buffer is 0.05M PBS, pH=8.0. After reacting at 30-50℃ for 1-6h, take 50μL of the reaction solution + 950μL of 2,4-dinitrophenylhydrazine derivatization solution and derivatize at 60℃ for 1h, and use HPLC to detect the amount of product ethanolaldehyde to calculate the enzyme activity.

[0116] HPLC detection method for glycolaldehyde: U3000 high performance liquid chromatograph with UV detector, Aoclaim 120C185um 4.6*250mm column (Thermo), 35℃, 0.8mL / min, detection wavelength 210nm, mobile phase pure water: acetonitrile = 3:7. The test results are as follows Figure 2 shown.

[0117] One unit of enzyme activity is defined as the amount of enzyme that converts 1 μmol of substrate (formaldehyde) to produce product (glycolic acid) within 1 minute under specific conditions. Therefore, the calculation formula for specific enzyme activity is:

[0118] U / g=(C 乙醇醛 (μmol)×2 / (time (min)) / protein amount (g)

[0119] The specific activities of wild-type GALS and its mutant proteins at 37°C for 1 h are shown in Table 1:

[0120] Table 1 Specific activities of wild-type GALS and its mutant proteins

[0121] Mutants Specific activity (U / g) GALS 55.10 T87A 41.27 A416T 89.74 W463I 64.30 T87A / A416T 73.28 T87A / W463I 86.87 T87A / A416T / W463I 115.85 T87A / A416T / W463I / F464W 105.19 T87A / V94I / A416T / W463I 133.23 T87A / G109T / A416T / W463I 173.78

[0122] Example 7: Determination of kinetic parameters of some glycolaldehyde synthase mutants

[0123] Reaction system: 5 mM Mg 2+ , 0.5 mM TPP, 2 mM NADH, 50 μg glycerol dehydrogenase, 100 μg of the above-mentioned mutant protein, the total volume of the reaction system is 1 mL, the buffer is 0.05 M, pH = 8.0 PBS. The formaldehyde concentration gradient is 20-200 mM. After reacting at 30-50°C for 20 min, the kinetic parameters are determined by measuring the absorbance change at 340 nm using an 870 microplate reader (Thermo Fisher Scientific).

[0124] The kinetic parameters of the mutant proteins of glycolaldehyde synthase are shown in Table 2 ( Figure 4 ):

[0125] Table 2 Kinetic parameters of GALS and its mutant proteins

[0126] Mutants <![CDATA[kcat(s -1 )]]> Km(mM) <![CDATA[kcat / Km(M -1 ·s -1 )]]> GALS -- 170 9.6 T87A 1.65 108.57 15.20 T87A / W463I 1.89 61.16 30.90 T87A / A416T / W463I 1.72 10.79 159.3 T87A / A416T / W463I / F464W 2.68 30.67 87.45 T87A / V94I / A416T / W463I 1.55 27.06 57.18 T87A / G109T / A416T / W463I 1.38 30.46 45.15

[0127] Example 8: Replacement of divalent metal ions

[0128] Reaction system: 10mM formaldehyde, 2mM divalent metal ions, 0.1mM TPP, 100μg of the above wild-type protein or T87A / A416T / W463I mutant protein, total reaction system volume of 1mL, buffer solution of 0.05M, pH=8.0 PBS. After reacting at 30-50℃ for 1-6h, take 50μL of reaction solution + 950μL of 2,4-dinitrophenylhydrazine derivatization solution and derivatize at 60℃ for 1h, and use HPLC to detect the amount of ethanolaldehyde produced to calculate the enzyme activity.

[0129] HPLC detection method for glycolaldehyde: U3000 high performance liquid chromatograph with UV detector, Aoclaim 120C185um 4.6*250mm column (Thermo), 35°C, 0.8mL / min, detection wavelength 210nm, mobile phase pure water:acetonitrile = 3:7.

[0130] One unit of enzyme activity is defined as the amount of enzyme that converts 1 μmol of substrate (formaldehyde) to produce product (glycolic acid) within 1 minute under specific conditions. Therefore, the calculation formula for specific enzyme activity is:

[0131] U / g=(C 乙醇醛 (μmol)×2 / (time (min)) / protein amount (g)

[0132] The specific activity of GALS and its mutant proteins at 37℃ for 1h is as follows Figure 5 shown.

[0133] Example 9: Replacement of cofactors

[0134] Reaction system: 10mM formaldehyde, 2mM Mg 2+ , 0.1mM TPP / vitamin B1, 100μg of the above wild-type protein or T87A / A416T / W463I mutant protein, the total volume of the reaction system is 1mL, the buffer is 0.05M PBS, pH=8.0. After reacting at 30-50℃ for 1-6h, take 50μL of the reaction solution + 950μL of 2,4-dinitrophenylhydrazine derivatization solution and derivatize at 60℃ for 1h, and use HPLC to detect the amount of product ethanolaldehyde to calculate the enzyme activity.

[0135] HPLC detection method for glycolaldehyde: U3000 high performance liquid chromatograph with UV detector, Aoclaim 120C185um 4.6*250mm column (Thermo), 35°C, 0.8mL / min, detection wavelength 210nm, mobile phase pure water:acetonitrile = 3:7.

[0136] One unit of enzyme activity is defined as the amount of enzyme that converts 1 μmol of substrate (formaldehyde) to produce product (glycolic acid) within 1 minute under specific conditions. Therefore, the calculation formula for specific enzyme activity is:

[0137] U / g=(C 乙醇醛 (μmol)×2 / (time (min)) / protein amount (g)

[0138] The specific activity of GALS and its mutant proteins at 37℃ for 1h is as follows Figure 6 shown.

[0139] Example 10: Synthesis of ethylene glycol by catalysis of glycolaldehyde synthase mutants and lactaldehyde reductase whole cells

[0140] Reaction system: 10 / 40 mM formaldehyde, 2 mM Mg 2+ , 1mM TPP, 10-50g / L of wet bacteria obtained by fermentation culture of the engineered bacteria expressing the above T87A / A416T / W463I mutant and the gene encoding lactaldehyde reductase fuco, the total volume of the reaction system is 3mL, the buffer is 0.05M PBS, pH=8.0. After reacting at 30-50℃ for 1-6h, take 50μL of the reaction solution + 950μL of 2,4-dinitrophenylhydrazine derivative solution and derivatize at 60℃ for 1h, and use HPLC to detect the production of substrate formaldehyde and intermediate product ethanolaldehyde; take 200μL of the reaction solution + 200μL of 0.2M sulfuric acid to terminate the reaction, and use HPLC to detect the production of product ethylene glycol.

[0141] HPLC detection method for formaldehyde and glycolaldehyde: U3000 high performance liquid chromatograph with UV detector, Aoclaim120 C18 5um 4.6*250mm column (Thermo), 35°C, 0.8mL / min, detection wavelength 210nm, mobile phase pure water: acetonitrile = 3:7.

[0142] HPLC detection method for ethylene glycol: U3000 high performance liquid chromatograph with differential detector, HPX-87H organic acid column (BIO-RAD, 300×7.8 mm), 65° C., 0.6 mL / min, and 5 mM sulfuric acid as the mobile phase.

[0143] The final conversion rate of ethylene glycol at 10 / 40mM formaldehyde concentration can reach more than 62%, and the yield is as follows Figure 7 shown.

[0144] Example 11: Expression and purification of glycolaldehyde synthase mutants in Pichia pastoris GS115

[0145] (1) The recombinant plasmid pPICZB-GALS was linearized and then electroporated into the X33 strain to obtain the recombinant strain. The positive clones were screened using a zeocin-resistant YPD plate and cultured at 30°C for 2-3 days.

[0146] (2) Pick the bacterial p and the single colony with the correct sequencing to enrich on the non-antibiotic YPD plate for 2 days, then pick the match head-sized colony to inoculate into 5 mL YPD liquid culture medium and culture it in a constant temperature incubator at 30°C and 200 rpm for 12-16 h.

[0147] (3) The bacterial solution cultured in step (2) was inoculated into 50 mL of BMGY medium, and cultured in a constant temperature incubator at 30° C. and 200 rpm until vD600 reached about 6, and then the cells were collected by centrifugation. The cells were then resuspended in 50 mL of fresh BMMY medium, and 1% methanol was added for induction, and fermented in a constant temperature incubator at 28-30° C. and 200 rpm.

[0148] (4) 1% methanol was added every 24 h to induce the expression of the target protein, and the fermentation lasted for 5 days.

[0149] (5) After the culture was completed, the yeast cells were collected by centrifugation at 4°C and 4500 rpm for 20 min, and 50 mL of PBS solution (0.05 M, pH = 7.5) containing 10 mM imidazole and 100 mM NaCl was added to resuspend the cells, and the yeast cells were broken using a high-pressure homogenizer;

[0150] (6) After cell disruption, centrifuge at 4°C and 4500 rpm for 20 min. The resulting supernatant is the crude enzyme solution required for the experiment.

[0151] (7) The crude enzyme solution was purified by a nickel-agarose gel FF affinity chromatography column pre-packed with seasoning to obtain purified enzyme 1; the purified solution 1 was ultrafiltered using a 30 KDa ultrafiltration membrane to obtain the final purified enzyme solution, and SDS gel electrophoresis was used to verify that the protein expression was correct ( Figure 3 );

[0152] (8) G250 Coomassie Brilliant Blue was used to react with the enzyme solution, and then the absorbance at 595 nm was detected using an 870 microplate reader (Thermo Fisher Scientific) to calculate the protein concentration. The final protein concentration can reach 6.4 mg / mL, and the volume is about 500 μL.

[0153] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. An amino acid mutant affecting the enzyme activity of glycolaldehyde synthase, comprising one or more of the following mutations from N-terminus to C-terminus: Thr at position 87 mutated to Ala, Val at position 94 mutated to Ile, Gly at position 109 mutated to Thr, Ala at position 416 mutated to Thr, Trp at position 463 mutated to Ile, and Phe at position 464 mutated to Trp in the amino acid sequence of wild glycolaldehyde synthase (derived from Pseudomonas putida).

2. A mutant protein of glycolaldehyde synthase, wherein the sequence comprises one or more of the following mutations: Thr at position 87 is mutated to Ala, Val at position 94 is mutated to Ile, Gly at position 109 is mutated to Thr, Ala at position 416 is mutated to Thr, Trp at position 463 is mutated to Ile, and Phe at position 464 is mutated to Trp from the N-terminus to the C-terminus of the amino acid sequence of wild glycolaldehyde synthase as shown in SEQ ID NO.

1.

3. The mutant protein of glycolaldehyde synthase according to claim 2, characterized in that The mutant protein is a No. 1 glycolaldehyde synthase mutant protein, the sequence of which contains a mutation in which Thr at position 87 from the N-terminus to the C-terminus of the amino acid sequence of the wild glycolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Ala; preferably, the sequence of the No. 1 glycolaldehyde synthase mutant protein is as shown in SEQ ID NO.2; And / or, the mutant protein is a No. 2 glycolaldehyde synthase mutant protein, whose sequence comprises a mutation in which Ala at position 416 from the N-terminus to the C-terminus of the amino acid sequence of the original glycolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Thr; preferably, the sequence of the No. 2 glycolaldehyde synthase mutant protein is shown in SEQ ID NO.3; And / or, the mutant protein is a mutant protein of glycolaldehyde synthase No. 3, whose sequence comprises a mutation in which Trp at position 463 from the N-terminus to the C-terminus of the amino acid sequence of the original glycolaldehyde synthase as shown in SEQ ID NO.1 is mutated to Ile; preferably, the sequence of the mutant protein of glycolaldehyde synthase No. 3 is shown in SEQ ID NO.4; And / or, the mutant protein is a mutant protein of glycolaldehyde synthase No. 4, whose sequence comprises a mutation of Thr at position 87 to Ala and a mutation of A1a at position 416 to Thr from the N-terminus to the C-terminus of the amino acid sequence of the original glycolaldehyde synthase as shown in SEQ ID NO.1; preferably, the sequence of the mutant protein of glycolaldehyde synthase No. 4 is shown in SEQ ID NO.5; And / or, the mutant protein is a mutant protein of glycolaldehyde synthase No. 5, whose sequence comprises a mutation of Thr at position 87 to Ala and a mutation of Trp at position 463 to Ile from the N-terminus to the C-terminus of the amino acid sequence of the original glycolaldehyde synthase as shown in SEQ ID NO.1; preferably, the sequence of the mutant protein of glycolaldehyde synthase No. 5 is shown in SEQ ID NO.5; And / or, the mutant protein is a 6-ethanolaldehyde synthase mutant protein, whose sequence comprises a mutation of Thr at position 87 to Ala, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original 6-ethanolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus; preferably, the sequence of the 6-ethanolaldehyde synthase mutant protein is shown in SEQ ID NO.7; And / or, the mutant protein is a mutant protein of glycolaldehyde synthase No. 7, whose sequence comprises a mutation of Thr at position 87 to Ala, a mutation of Ala at position 416 to Thr, a mutation of Trp at position 463 to Ile, and a mutation of Phe at position 464 to Trp in the amino acid sequence of the original glycolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus; preferably, the sequence of the mutant protein of glycolaldehyde synthase No. 7 is shown in SEQ ID NO.8; And / or, the mutant protein is a mutant protein of glycolaldehyde synthase No. 8, whose sequence comprises a mutation of Thr at position 87 to Ala, a mutation of Val at position 94 to Ile, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original glycolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus; preferably, the sequence of the mutant protein of glycolaldehyde synthase No. 8 is shown in SEQ ID NO.9; And / or, the mutant protein is a mutant protein of ethanolaldehyde synthase No. 9, whose sequence includes a mutation of Thr at position 87 to Ala, a mutation of Val at position 94 to Ile, a mutation of Gly at position 109 to Thr, a mutation of Ala at position 416 to Thr, and a mutation of Trp at position 463 to Ile in the amino acid sequence of the original ethanolaldehyde synthase as shown in SEQ ID NO.1 from the N-terminus to the C-terminus; preferably, the sequence of the mutant protein of ethanolaldehyde synthase No. 9 is as shown in SEQ ID NO.

10.

4. The mutant protein of glycolaldehyde synthase according to claim 2 or 3, characterized in that The mutant protein also includes a fusion protein obtained by connecting a tag to the N-terminus or C-terminus of the above-mentioned ethanolaldehyde synthase mutant protein; preferably, the tag includes a His tag and / or a SUMO tag; further preferably, the mutant protein also includes a fusion protein obtained by connecting a His tag or a SUMO tag to the N-terminus or C-terminus of ethanolaldehyde synthase mutant protein No. 1, ethanolaldehyde synthase mutant protein No. 2, ethanolaldehyde synthase mutant protein No. 3, ethanolaldehyde synthase mutant protein No. 4, ethanolaldehyde synthase mutant protein No. 5, ethanolaldehyde synthase mutant protein No. 6, ethanolaldehyde synthase mutant protein No. 7, ethanolaldehyde synthase mutant protein No. 8 or ethanolaldehyde synthase mutant protein No.

9.

5. Use of the mutant protein according to any one of claims 2 to 4 in catalyzing the synthesis of ethanolaldehyde from formaldehyde.

6. The use according to claim 5, characterized in that: The application is to carry out catalytic reaction I in a reaction system for synthesizing ethanolaldehyde consisting of catalyst I, metal cofactor I, other cofactor I, reaction medium I and substrate I to synthesize ethanolaldehyde; wherein the catalyst I is a mutant of ethanolaldehyde synthase; and / or the metal cofactor is a divalent metal ion, which includes Mg 2+ 、Co 2+ , Mn 2+ , Cu 2+ , Ca 2+ , Fe 2+ and Zn 2+ one or more of; and / or, the other cofactors include thiamine pyrophosphate TPP and / or vitamin B1 (thiamine); and / or, the substrate I is formaldehyde; and / or, the reaction medium I includes a buffer solution with a pH of 6-9.

7. The use according to claim 6, characterized in that: The temperature of the catalytic reaction I is 30-50° C., preferably 30-40° C.; the time of the catalytic reaction I is 1-12 h; and the stirring speed of the catalytic reaction I is 400 rpm.

8. The use according to claim 6, characterized in that: In the above reaction system for synthesizing glycolaldehyde, the concentration of substrate I is 10-100 mM; the concentration of metal cofactor I is 1-6 mM; the concentration of other cofactors II is 0.1-2 mM; and the concentration of glycolaldehyde synthase mutant is 0.1-201 mg / mL.

9. Use of the mutant protein according to any one of claims 2 to 4 and lactaldehyde reductase cascade catalyzing formaldehyde to synthesize ethylene glycol; preferably, the use is to carry out catalytic reaction II in a reaction system for synthesizing ethylene glycol consisting of catalyst II, catalyst III, metal cofactor II, other cofactor II, reaction medium II and substrate II to synthesize ethylene glycol; wherein, The catalyst II is a ethanolaldehyde synthase mutant or a wet cell of a ethanolaldehyde synthase mutant obtained by fermentation culture of an engineered bacterium expressing the gene encoding the ethanolaldehyde synthase; and / or, the catalyst III is lactaldehyde reductase; and / or, the metal cofactor II is a divalent metal ion, which includes Mg 2+ 、Co 2+ , Mn 2+ , Cu 2+ , Ca 2+ , Fe 2+ and Zn 2+ One or more of; and / or, the other cofactor II includes thiamine pyrophosphate TPP and / or vitamin B1 (thiamine); and / or, the substrate II is formaldehyde; and / or, the reaction medium II includes a buffer having a pH of 6-9.

10. The use according to claim 9, characterized in that: The temperature of the catalytic reaction II is 30-50°C, preferably 30-40°C; the time of the catalytic reaction II is 1-12h; the stirring speed of the catalytic reaction II is 400rpm; Preferably, in the reaction system for synthesizing ethylene glycol, the concentration of substrate II is 10-100 mM; the concentration of the metal cofactor II is 1-6 mM; the concentration of the other cofactor II is 0.1-2 mM; the concentration of the ethanolaldehyde synthase mutant is 0.1-201 mg / mL; and the dosage of the ethanolaldehyde synthase mutant wet bacteria is 10-50 g / L.

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