Process for the production of 1,3-dihydroxyacetone from glycerol and its use

Abstract

The application discloses a method for generating 1,3-dihydroxyacetone from glycerol and application thereof. The application provides a method for generating 1,3-dihydroxyacetone from glycerol, which comprises the following steps: in the presence of 1) Escherichia coli glycerol dehydrogenase GldA, 2) Lactobacillus pentosus NADH oxidase LpNox or Streptococcus pyogenes NADH oxidase variant SpNox-K184R, and 3) NAD + The substrate glycerol is oxidized to generate 1,3-dihydroxyacetone; and the SpNox-K184R is a variant obtained by mutating the 184th amino acid residue of NADH oxidase SpNox derived from Streptococcus pyogenes from K to R. The obtained catalytic pathway and component ratio can greatly improve the catalytic efficiency of glycerol to 1,3-dihydroxyacetone, provide raw materials for the synthesis of lactic acid and / or glycolic acid, and can be carried out under mild conditions, thus having a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically to a method for generating 1,3-dihydroxyacetone from glycerol and its application. Background Technology

[0002] 1,3-Dihydroxyacetone, also known as dihydroxyacetone, is a polyhydroxy ketose and the simplest naturally occurring tricarbon ketose. It has wide applications in the pharmaceutical, chemical, food, and cosmetic industries. On one hand, it serves as a raw material for cosmetics, providing excellent skin protection. On the other hand, it can be used as a basis for the synthesis of polyester compounds. Furthermore, 1,3-Dihydroxyacetone is an important metabolic intermediate. Through microbial fermentation, it can be further converted into high-value chemicals and fuel molecules, such as ethanol, butanol, lactic acid, and succinic acid. It can also be used to synthesize other sugars with higher value, such as erythritol and sorbitol. Therefore, 1,3-Dihydroxyacetone is an important chemical synthesis intermediate widely used in cosmetics manufacturing, food research, pharmaceuticals, and chemical synthesis.

[0003] The main production methods of 1,3-dihydroxyacetone include chemical synthesis and biosynthesis. Chemical synthesis has problems such as high raw material cost, harsh reaction conditions, high equipment requirements, low product recovery rate and environmental pollution, and its application is limited to a certain extent. The advantages of bioconversion are mild reaction conditions, strong reaction specificity, low environmental pollution and high substrate utilization. At present, there are three main methods for synthesizing 1,3-dihydroxyacetone. 1. Using glycerol as substrate, 1,3-dihydroxyacetone is generated by dehydrogenation under the action of glycerol dehydrogenase. Microorganisms usually contain three types of glycerol dehydrogenases (GDH): (1) NAD-dependent. + GDH (EC 1.1.1.6), mainly found in the cytoplasm, first converts glycerol to 1,3-dihydroxyacetone, which is then further phosphorylated and enters the glycolysis and tricarboxylic acid cycle pathways. (2) Dependent on NADP + GDH (EC 1.1.1.72 and EC 1.1.1.156) is found in fungi and animal tissues and can oxidize glycerol to glyceraldehyde or 1,3-dihydroxyacetone. (3) It is independent of NAD. + It does not depend on NADP. + GDH (EC 1.1.99.22) is an enzyme located on the cell membrane of *Glucobacterium*. 2. Using methanol as a substrate, 1,3-dihydroxyacetone is formed under the action of dihydroxyacetone synthase; 3. 1,3-dihydroxyacetone is obtained by conversion using fructose as a substrate.

[0004] Due to the high costs of fed-batch processes and downstream processing, the production of 1,3-dihydroxyacetone via microbial fermentation using glycerol as a substrate, oxidizing glycerol to 1,3-dihydroxyacetone under the action of intracellular glycerol dehydrogenase, is relatively expensive. Therefore, extracellular enzyme catalysis shows promise as a good alternative for the microbial production of 1,3-dihydroxyacetone.

[0005] However, most oxidation reactions catalyzed by dehydrogenases typically require NAD(P). + As cofactors, efficient cofactor regeneration strategies are crucial for the economical conduct of biocatalytic reactions. Several methods for cofactor regeneration have been developed, including photocatalysis, chemical, electrochemical, and enzymatic methods. Among these, enzymatic pathways are the most attractive due to their high specificity and eco-friendliness. NADH oxidase (Nox) catalyzes the oxidation of NADH through two-electron reduction to H₂O₂ or four-electron reduction to H₂O. It can utilize dissolved oxygen as a substrate and produces no byproducts in aqueous solution. + Candidate enzymes for regeneration. In the current work, two related co-factor cycling enzymes, Nox and GDH, were characterized. Even at low concentrations of available NAD... + In addition, increasing GDH can also promote NAD. + The conversion to NADH makes it possible to continuously supply NADH in mixed immobilized enzyme-catalyzed reactions.

[0006] Although the glycerol conversion rate remained low due to product inhibition, Nox's highly efficient NAD... + The recycling process makes it an attractive biocatalyst for industrial applications involving the catalytic oxidation of mixed dehydrogenases. Summary of the Invention

[0007] The purpose of this invention is to provide a method for generating 1,3-dihydroxyacetone from glycerol and its application.

[0008] In a first aspect, the present invention claims a method for generating 1,3-dihydroxyacetone from glycerol.

[0009] The method for generating 1,3-dihydroxyacetone from glycerol claimed in this invention may include the following steps: [the process involves] reacting glycerol with a protein, NADH with a protein, and NAD... + In the presence of glycerol, the substrate undergoes an oxidation reaction to produce 1,3-dihydroxyacetone.

[0010] The glycerol-converting protein is a glycerol dehydrogenase GldA derived from *Escherichia coli*. The NADH-converting protein is an NADH oxidase LpNox derived from *Lactobacillus pentosus* or a NADH oxidase variant SpNox-K184R derived from *Streptococcus pyogenes*; SpNox-K184R is a variant obtained by mutating the 184th amino acid residue of the NADH oxidase SpNox from *Streptococcus pyogenes* from K to R.

[0011] Furthermore, in the reaction system of the method, the initial concentration of glycerol can be 0-1000 mM (excluding 0; such as 100 mM or 200 mM), and NAD... + The initial concentration can be 0-20 mM (excluding 0; e.g., 2 mM), the initial concentration of GldA can be 0-2420 U / mL (excluding 0; e.g., 242 U / mL), and the initial concentration of LpNox or SpNox-K184R can be 0-2420 U / mL (excluding 0; e.g., 242 U / mL).

[0012] Furthermore, in the reaction system, besides glycerol and NAD, + In addition to GldA, LpNox, or SpNox-K184R, the remainder may be a near-neutral buffer solution with pH 6-8, such as 300mM potassium phosphate buffer with pH 7.1.

[0013] In the reaction system of the method described above, because a coenzyme cycle system is constructed, NAD is added initially. + No further exogenous NAD addition is required during subsequent reaction processes. + This will enable high-yield production of 1,3-dihydroxyacetone.

[0014] Furthermore, the reaction in the method can be carried out at an enzyme-catalyzable temperature of 20-40℃ (e.g., 30℃). The reaction time is preferably more than 10 hours (e.g., 10-50 hours, specifically 14 hours).

[0015] In a specific embodiment of the present invention, the amino acid sequence of GldA is shown in SEQ ID No. 1; the amino acid sequence of LpNox is shown in SEQ ID No. 2; and the amino acid sequence of SpNox-K184R is shown in SEQ ID No. 3.

[0016] Secondly, the present invention claims a method for generating lactic acid from glycerol.

[0017] The method for generating lactic acid from glycerol, as claimed in this invention, may include the following steps:

[0018] (A1) Using the method described in the first aspect above, 1,3-dihydroxyacetone was prepared from glycerol as a substrate;

[0019] (A2) The production of lactic acid from 1,3-dihydroxyacetone is catalyzed by an alkali (earth) metal hydroxide. The alkali (earth) metal hydroxide is an alkali metal hydroxide and / or an alkaline earth metal hydroxide.

[0020] Specifically, the alkali (earth) metal hydroxide can be NaOH or KOH.

[0021] In a specific embodiment of the present invention, in step (A2), the molar ratio of 1,3-dihydroxyacetone to the alkali metal hydroxide (such as NaOH or KOH) is 1:1.25 to 1:500 (e.g., 1:50 to 1:100). The reaction conditions for step (A2) are 30°C for 8-24 hours (e.g., 8 hours or 24 hours).

[0022] Thirdly, the present invention claims a method for generating glycolic acid from glycerol.

[0023] The method for generating glycolic acid from glycerol, as claimed in this invention, may include the following steps:

[0024] (B1) Using the method described in the first aspect above, 1,3-dihydroxyacetone was prepared from glycerol as a substrate;

[0025] (B2) 1,3-dihydroxyacetone is oxidized by oxides to produce glycolic acid.

[0026] Specifically, the oxide may be NaClO2.

[0027] In a specific embodiment of the present invention, in step (B2), the concentration of the oxide (such as NaClO2) in the reaction system is 180-320 mM (4 times the concentration of the product 1,3-dihydroxyacetone or much higher). The reaction conditions for step (B2) are 50°C for 24 h.

[0028] Fourthly, this invention claims protection for any of the following substances:

[0029] P1, a complete set of proteins, consisting of protein A and protein B;

[0030] Protein A is GldA as described in the first aspect above; protein B is LpNox or SpNox-K184R as described in the first aspect above.

[0031] P2, a set of nucleic acid molecules, composed of nucleic acid molecule A and nucleic acid molecule B;

[0032] The nucleic acid molecule A is a nucleic acid molecule encoding the GldA; the nucleic acid molecule B is a nucleic acid molecule encoding the LpNox or a nucleic acid molecule encoding the SpNox-K184R.

[0033] Further, the sequence of the nucleic acid molecule encoding GldA is shown in SEQ ID No. 4; the sequence of the nucleic acid molecule encoding LpNox is shown in SEQ ID No. 5; and the sequence of the nucleic acid molecule encoding SpNox-K184R is shown in SEQ ID No. 6.

[0034] P3. A complete set of expression boxes, consisting of expression box A and expression box B;

[0035] The expression cassette A is an expression cassette containing the nucleic acid molecule A; the expression cassette B is an expression cassette containing the nucleic acid molecule B.

[0036] P4. A complete set of recombinant vectors, consisting of recombinant vector A and recombinant vector B;

[0037] The recombinant vector A is a recombinant vector containing the nucleic acid molecule A; the recombinant vector B is a recombinant vector containing the nucleic acid molecule B.

[0038] In a specific embodiment of the present invention, the recombinant vector A is a recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No. 4 into the multiple cloning site (such as BamHI and NotI) of pET21b.

[0039] In one specific embodiment of the present invention, the recombinant vector B is a recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No. 5 into the multiple cloning site (such as BamHI and XhoI) of pET28a. In another specific embodiment of the present invention, the recombinant vector B is a recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No. 6 into the multiple cloning site (such as BamHI and NcoI) of pRSFDuet-1.

[0040] P5. A complete set of transgenic cell lines, consisting of transgenic cell line A and transgenic cell line B;

[0041] The transgenic cell line A is a transgenic cell line in which nucleic acid molecule A has been introduced; the transgenic cell line B is a transgenic cell line in which nucleic acid molecule B has been introduced.

[0042] P6. A set of recombinant bacteria, consisting of recombinant bacteria A and recombinant bacteria B;

[0043] The recombinant bacterium A is a recombinant bacterium containing the nucleic acid molecule A; the recombinant bacterium B is a recombinant bacterium containing the nucleic acid molecule B.

[0044] In a specific embodiment of the present invention, the recombinant bacterium A is specifically a recombinant bacterium obtained by introducing the recombinant vector A into Escherichia coli (such as BL21 Gold(DE3)); the recombinant bacterium B is specifically a recombinant bacterium obtained by introducing the recombinant vector B into Escherichia coli (such as BL21 Gold(DE3)).

[0045] P7. Complete set of products, which refers to either complete set of product A or complete set of product B;

[0046] The complete product A is composed of the complete protein and alkaline (earth) metal hydroxide described in P1; the complete product B is composed of the complete protein and oxide described in P1. The alkaline (earth) metal hydroxide is an alkali metal hydroxide and / or an alkaline earth metal hydroxide.

[0047] In a specific embodiment of the present invention, the alkali (earth) metal hydroxide is specifically NaOH or KOH; the oxide is specifically NaClO2.

[0048] Fifthly, the present invention claims protection for any of the following applications:

[0049] Q1. The use of any of the substances described in P1 to P6 of the fourth aspect above in the production of 1,3-dihydroxyacetone from glycerol.

[0050] In this application, because a coenzyme cycle system was constructed, NAD was added initially. + No further exogenous NAD addition is required during subsequent reaction processes. + This will enable high-yield production of 1,3-dihydroxyacetone.

[0051] Q2. The application of the complete product A described on page 7 of section four above in the production of lactic acid from glycerol.

[0052] Q3. The application of the complete product B described on page 7 of section four above in the production of glycolic acid from glycerol.

[0053] This invention provides a catalytic process for the production of 1,3-dihydroxyacetone from glycerol using glycerol as a substrate, through a coupled reaction of glycerol dehydrogenase and NADH oxidase. The dual-enzyme coupled reaction of this invention can catalyze the preparation of 1,3-dihydroxyacetone from glycerol, and can further synthesize lactic acid or glycolic acid from 1,3-dihydroxyacetone. The catalytic pathway and component ratios obtained by this invention can significantly improve the catalytic efficiency of the glycerol to 1,3-dihydroxyacetone conversion, and provide raw materials for the synthesis of lactic acid and / or glycolic acid. Furthermore, the process can be carried out under mild conditions, demonstrating promising application prospects. Attached Figure Description

[0054] Figure 1 The images are SDS-PAGE images of the pure enzymes from Examples 3, 7, and 11.

[0055] Figure 2 The product 1,3-dihydroxyacetone was directly detected by HPLC in Example 13.

[0056] Figure 3 The product 1,3-dihydroxyacetone was detected by HPLC ultraviolet light in Example 13 using the derivatization method.

[0057] Figure 4 The final product, lactic acid, produced by alkali metal hydroxide catalysis was detected by HPLC in Example 14.

[0058] Figure 5 The configuration of lactic acid, the final product catalyzed by alkali metal hydroxide, was determined by HPLC in Example 15.

[0059] Figure 6 The final product, glycolic acid, generated by the oxidation of oxides, was detected by HPLC in Example 16.

[0060] Figure 7 Schematic diagram of the process of converting glycerol to 1,3-dihydroxyacetone, which is then converted to lactic acid and / or glycolic acid.

[0061] Terms and Definitions

[0062] In the context of this invention, "glycerol-converting protein" refers to a protein capable of catalyzing the conversion of glycerol to 1,3-dihydroxyacetone, as long as it has the function of catalyzing the conversion of glycerol to 1,3-dihydroxyacetone, without particular limitations on its amino acid sequence and source. By way of example and not limitation, it can be, for example, a glycerol dehydrogenase (GldA) derived from *Escherichia coli* or a glycerol dehydrogenase (GDH) derived from *Klebsiella pneumoniae*.

[0063] In the context of this invention, "NADH converting protein" refers to a protein that can catalyze the dehydrogenation of NADH to NAD. + Proteins that catalyze the conversion of NADH to NAD +The function is sufficient, and there are no particular restrictions on its amino acid sequence and source. By way of example only and not limitation, it can be, for example, NADH oxidase (LpNox) derived from Lactobacillus pentosus, NADH oxidase (SpNox) derived from Streptococcus pyogenes, or variants thereof.

[0064] In this invention, the terms “glycerol conversion protein” and “glycerol dehydrogenase” and “GldA” ​​are used interchangeably, and “conversion” or “catalysis” are used interchangeably. These expressions refer to the role of catalyzing glycerol during the conversion process.

[0065] In this invention, the terms "NADH converting protein," "NADH oxidase," "NADH Oxidase," and "nicotinamide adenine dinucleotide oxidase" are used interchangeably, and "conversion" or "catalysis" is used interchangeably. These expressions refer to the conversion of NADH to NAD by the NADH converting protein. + The process. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] Example 1: Construction of a vector containing the glycerol dehydrogenase gene

[0069] Glyceryl dehydrogenase (GldA) is derived from *Escherichia coli* (hereinafter referred to as GldA), and its nucleotide sequence is SEQ ID No. 4 (encoding the protein shown in SEQ ID No. 1). A flanking sequence *g* was added to the 5' end of this nucleotide sequence (to avoid frameshift mutations), and the stop codon *tga* was removed from the 3' end (to express the His tag). Restriction enzyme sites *BamHI* and *NotHI* were added to both ends, and the recombinant plasmid *pET21b* was ligated to the multiple cloning site region of the plasmid *pET21b* using T4 DNA ligase to construct the recombinant plasmid *pET21b-GldA*. The *pET21b-GldA* vector, verified by sequencing, was then transformed into *Escherichia coli* BL21 Gold(DE3) and cultured at 37°C until a single colony grew. This single colony was a GldA-positive bacterium.

[0070] Example 2: Expression of glycerol dehydrogenase in Escherichia coli

[0071] The GldA-positive bacteria obtained in Example 1 were picked up with an inoculation needle and inoculated into 3 mL of LB medium containing ampicillin. The medium was incubated overnight at 37°C, and then transferred to 100 mL of LB medium at a 1% (V / V) inoculation rate and incubated at 37°C and 200 rpm. When OD... 600 When the concentration reaches 0.6, 0.5 mM IPTG is added, and expression is induced at 20°C. After induction, the cultured cells are collected into centrifuge tubes, resuspended in 50 mM potassium phosphate buffer (pH 7.1), and washed. After centrifugation, the cells are stored at -80°C.

[0072] Example 3: Purification of glycerol dehydrogenase

[0073] The bacterial cells from Example 2 were removed, resuspended in pH 7.1, 50 mM potassium phosphate buffer, sonicated on ice to lyse, and the supernatant was collected by centrifugation. Using Ni... 2+ The mutants expressed above were purified by affinity chromatography using a chromatography column. After washing with imidazole, the protein was desalted using a desalting column, and the purified protein was stored in potassium phosphate buffer for later use. SDS-PAGE analysis showed that the protein purity was above 95%. Figure 1 ).

[0074] Example 4: Detection of glycerol dehydrogenase activity

[0075] The purified GldA protein from Example 3 was taken out and the final concentration was 20 μg / mL. GldA protein was mixed with 2 mM NAD. +The mixture was mixed with 500 mM glycerol and reacted at 30 °C. The absorbance was measured at 340 nm for 20 min. The enzyme activity of GldA was calculated. Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of NADH per minute under conditions of 30 °C, pH 7.1, and 300 mM potassium phosphate buffer. The calculated enzyme activity of GldA was 24.2 U / mg.

[0076] Example 5: Construction of a vector containing the LpNox gene

[0077] Nicotinamide adenine dinucleotide oxidase (Nox), derived from *Lactobacillus pentosus* (hereinafter referred to as LpNox), has the nucleotide sequence SEQ ID No. 5 (encoding the protein shown in SEQ ID No. 2). The stop codon tga (expressing a His tag) was removed from the 3' end of this nucleotide sequence, and restriction enzyme sites BamHⅠ and XhoⅠ were added to both ends. The recombinant plasmid pET28a-LpNox was constructed by ligating it to the multiple cloning site region of plasmid pET28a using T4 DNA ligase. The pET28a-LpNox vector, verified by sequencing, was then transformed into *Escherichia coli* BL21 Gold(DE3) and cultured at 37°C until a single colony grew. This single colony was an LpNox-positive bacterium.

[0078] Example 6: Expression of LpNox in Escherichia coli

[0079] The LpNox-positive bacteria obtained in Example 5 were picked up with an inoculation needle and inoculated into 3 mL of LB medium containing kanamycin sulfate. The medium was incubated overnight at 37°C, and then transferred to 100 mL of LB medium at a 1% (V / V) inoculation rate and incubated at 37°C and 200 rpm. When OD... 600 When the concentration reaches 0.6, 0.5 mM IPTG is added, and expression is induced at 20°C. After induction, the cultured cells are collected into centrifuge tubes, resuspended in potassium phosphate buffer (pH 7.1, 300 mM), and washed. After centrifugation, the cells are stored at -80°C.

[0080] Example 7: Purification of LpNox protein

[0081] The bacterial cells from Example 6 were taken out, resuspended in 50 mM potassium phosphate buffer (pH 7.1), and sonicated on ice to lyse the bacteria. The supernatant was collected by centrifugation. Using Ni... 2+The mutant protein was purified by affinity chromatography using a chromatography column. After elution with imidazole, it was desalted using a desalting column, and the purified protein was stored in potassium phosphate buffer for later use. SDS-PAGE analysis showed that the protein purity was above 95%. Figure 1 ).

[0082] Example 8: Activity detection of LpNox

[0083] The purified LpNox protein from Example 7 was taken and mixed with 2 mM NADH at a final concentration of 25 μg / mL. The mixture was incubated at 30°C and the reaction was monitored at 340 nm for 20 min. The enzyme activity of LpNox was calculated. Enzyme activity is defined as the amount of enzyme required to consume 1 μmol of NADH per minute under the conditions of 30°C, pH 7.1, and 300 mM potassium phosphate buffer. The enzyme activity of LpNox was calculated to be 29.93 U / mg.

[0084] Example 9: Construction of a vector containing the SpNox-K184R gene

[0085] Nicotinamide adenine dinucleotide oxidase (Nox) derived from Streptococcus pyogenes (a mutant of it used in this invention, with lysine at position 184 changed to arginine, hereinafter referred to as SpNox-K184R), has the nucleotide sequence SEQ ID No. 6 (encoding the protein shown in SEQ ID No. 3). A flanking sequence 5'-gc was added to the 3' end of this nucleotide sequence (to avoid frameshift mutation), and a flanking sequence 5'-ctcgagcaccaccaccaccaccactga (expressing a His tag) was added to the 5' end. Restriction enzyme sites NcoⅠ and BamHI were added to both ends, and the recombinant plasmid pRSFDuet-1 was ligated to the multiple cloning site region of the plasmid pRSFDuet-1 using T4 DNA ligase to construct the recombinant plasmid pRSFDuet-SpNox-K184R. The pRSFDuet-SpNox-K184R vector, which was verified to be correct by sequencing, was then transformed into Escherichia coli BL21 Gold(DE3) and cultured at 37°C until a single colony grew. This single colony was a SpNox-K184R positive bacterium.

[0086] Example 10: Expression of SpNox-K184R in Escherichia coli

[0087] The SpNox-K184R positive bacteria obtained in Example 9 were picked up with an inoculation needle and inoculated into 3 mL of LB medium containing kanamycin sulfate. The culture was incubated overnight at 37°C, and then transferred to 100 mL of LB medium at a 1% (v / v) inoculation rate and incubated at 37°C and 200 rpm. When OD... 600 When the concentration reaches 0.6, 0.5 mM IPTG is added, and expression is induced at 20°C. After induction, the cultured cells are collected into centrifuge tubes, resuspended in 50 mM potassium phosphate buffer (pH 7.1), and washed. After centrifugation, the cells are stored at -80°C.

[0088] Example 11: Purification of SpNox-K184R protein

[0089] The bacterial cells from Example 10 were taken out, resuspended in 50 mM potassium phosphate buffer (pH 7.1), and sonicated on ice to lyse the bacteria. The supernatant was collected by centrifugation. Using Ni... 2+ The mutant protein was purified by affinity chromatography using a chromatography column. After elution with imidazole, it was desalted using a desalting column, and the purified protein was stored in potassium phosphate buffer for later use. SDS-PAGE analysis showed that the protein purity was above 95%. Figure 1 ).

[0090] Example 12: Activity detection of SpNox-K184R protein

[0091] The purified SpNox-K184R protein from Example 11 was taken and mixed with 2 mM NADH at a final concentration of 25 μg / mL. The mixture was incubated at 30°C and the reaction was monitored at 340 nm for 20 min. The enzyme activity of SpNox-K184R was calculated. Enzyme activity is defined as the amount of enzyme required to consume 1 μmol of NADH per minute at 30°C, pH 7.1, and 300 mM potassium phosphate buffer. The calculated enzyme activity of SpNox-K184R was 34.62 U / mg.

[0092] Example 13: Constructing a coenzyme cycle using GldA and LpNox / SpNox-K184R to catalyze the production of 1,3-dihydroxyacetone from glycerol.

[0093] GldA from Example 3 was diluted with LpNox from Example 7 or SpNox-K184R from Example 11 using 300mM potassium phosphate buffer at pH 7.1, and the protein concentration was determined. Based on the enzyme activity of GldA, the enzyme concentration was calculated according to the LpNox / SpNox-K184R enzyme activity ratio, resulting in a 1:1 ratio of GldA to LpNox / SpNox-K184R enzyme activity. Glycerol (see Table 2) and 2mM NAD were then added at a series of concentrations. +In the reaction system, the reaction was carried out at 30°C for 14 hours. After that, the enzyme in the reaction system was removed by centrifugation using an ultrafiltration tube. Some of the reaction samples were directly detected by HPLC, while others were derivatized and then detected by HPLC.

[0094] Direct HPLC detection method is used to detect the target product 1,3-dihydroxyacetone and the substrate glycerol. Detection conditions: chromatographic column: Aminex HPX-87H, 300mm × 7.8mm (Dewater); mobile phase: 5mM H2SO4 solution; UV absorption wavelength: 210nm; flow rate: 0.5mL / min; column temperature: 35℃; injection volume: 20μL.

[0095] Sample preparation for HPLC detection in the derivatization method: 30 μL of reaction solution (after enzyme removal) was added to 170 μL of 20 mg / mL 2,3,4,5,6-pentafluorbenzoxylamine hydrochloride (PFBHA) solution. The mixture was reacted at 30℃ for 30 min, followed by the addition of 200 μL acetonitrile and 100 μL water. The solution was filtered through a 0.22 μm organic filter before HPLC detection of the target product, 1,3-dihydroxyacetone. HPLC detection conditions after derivatization: Column: Ultimate XB-C18, 4.6 × 250 mm, 5 μm; Mobile phase: water, acetonitrile; UV absorption wavelength: 263 nm; Flow rate: 1.2 mL / min; Column temperature: 30℃; Injection volume: 20 μL. Elution conditions are shown in Table 1.

[0096] Table 1. Elution conditions for DHA derivatization by HPLC (% represents volume percentage)

[0097]

[0098] When the glycerol concentration is 100 mM and the enzyme concentration is 10 mg / mL (equivalent to 242 U / mL), the HPLC detection results of the product are as follows: Figure 2 , Figure 3 As shown in the figure, analysis revealed that GldA and LpNox / SpNox-K184R can catalyze the formation of 1,3-dihydroxyacetone from glycerol, and this was detected using two different detection methods.

[0099] The conversion rates (%) of glycerol to 1,3-dihydroxyacetone catalyzed by the coupling of GldA and LpNox / SpNox-K184R were calculated and are shown in Table 2. It can be seen that when the glycerol concentration is 100 mM, the GldA enzyme concentration is 10 mg / mL (equivalent to 242 U / mL), and SpNox-K184R with equivalent enzyme activity is used, and the reaction system contains 2 mM NAD... +At this time, the conversion rate from glycerol to 1,3-dihydroxyacetone is the highest, reaching 92.3%.

[0100] Table 2. Conversion rate (%) of glycerol to 1,3-dihydroxyacetone catalyzed by GldA and LpNox / SpNox-K184R.

[0101]

[0102]

[0103] Example 14: Conversion of glycerol to lactic acid via intermediate 1,3-dihydroxyacetone.

[0104] The enzyme in the reaction solution of 10 mg / mL (equivalent to 242 U / mL) GldA enzyme, 100 mM and 200 mM glycerol was removed by centrifugation using an ultrafiltration tube. 500 μL of the solution was slowly added to 500 μL of 5M NaOH solution, and the reaction was carried out at 30°C for 24 h. After the reaction, dilute sulfuric acid was added to adjust the pH of the solution to between 1.0 and 2.0. The solution was filtered through a 0.22 μm organic filter membrane, and the target product, lactic acid, was detected by HPLC. HPLC detection conditions: column: Aminex HPX-87H, 300 mm × 7.8 mm (Dewater); mobile phase: 5 mM H2SO4; differential detector; flow rate: 0.5 mL / min; column temperature: 35°C; injection volume: 20 μL.

[0105] HPLC detection results are as follows Figure 4 As shown in the diagram, analysis reveals that alkali metal hydroxides (sodium hydroxide) can further catalyze the conversion of 1,3-dihydroxyacetone produced from glycerol in the GldA / LpNox / SpNox-K184R coupling system to lactic acid (the process of converting glycerol to 1,3-dihydroxyacetone and subsequently to lactic acid is illustrated in the diagram). Figure 7 (As shown in the table). The conversion rate calculation results are shown in Table 3. It can be seen that the conversion rate from 1,3-dihydroxyacetone to lactic acid is approximately 78%, and the conversion rate from glycerol to lactic acid is 68.1-72.3%.

[0106] Table 3. Amount of products (1,3-dihydroxyacetone and lactic acid) generated during the glycerol to lactic acid conversion process versus substrate (glycerol) conversion rate

[0107]

[0108]

[0109] Furthermore, 1,3-dihydroxyacetone was reacted with alkali metal hydroxide (sodium hydroxide) at different ratios of 1:1.25-1:500 (molar ratio) at 30℃ for 24 h. After the reaction, dilute sulfuric acid was added to adjust the pH of the solution to between 1.0 and 2.0. The solution was filtered through a 0.22 μm organic filter membrane, and the target product, lactic acid, was detected by HPLC. The HPLC detection conditions were as follows: column: Aminex HPX-87H, 300 mm × 7.8 mm (Dewater); mobile phase: 5 mM H2SO4; differential detector; flow rate: 0.5 mL / min; column temperature: 35℃; injection volume: 20 μL. The conversion results are shown in Table 4.

[0110] Table 4. Formation rate of lactic acid in different ratios of 1,3-dihydroxyacetone with alkali (earth) metal hydroxides

[0111]

[0112] Example 15: Proportion of lactic acid configuration in the conversion of intermediate 1,3-dihydroxyacetone to lactic acid

[0113] 500 μL of a 100 mM 1,3-dihydroxyacetone solution was slowly added to 500 μL of a 250 mM-10 M alkali metal hydroxide (sodium hydroxide) solution. The reaction was carried out at 30 °C for 8 h. After the reaction, dilute sulfuric acid was added to adjust the pH of the solution to between 4.0 and 7.0. The solution was filtered through a 0.22 μm organic filter membrane, and the target products D-lactic acid and L-lactic acid were detected by HPLC. The HPLC detection conditions were as follows: column: Phenom enex Chirex 3126(D)-penicillami (4.6 mm id × 250 mm L, 5 μm); mobile phase: 2% isopropanol aqueous solution containing 2 mM CuSO4; UV detector: 230 nm; flow rate: 0.9 mL / mL; temperature: 35 °C; injection volume: 10 μL.

[0114] HPLC detection results are as follows Figure 5 As shown in the table, analysis reveals that alkali metal hydroxides (sodium hydroxide) can catalyze the conversion of 1,3-dihydroxyacetone to D-lactic acid and L-lactic acid. The calculated proportions of D and L-lactic acid configurations are shown in Table 5. Of the lactic acid produced, D-lactic acid accounted for 57-64%, and L-lactic acid accounted for 36-43%, with an ee% of 14%-28%. This result, compared to other reported production of racemic lactic acid (equal proportions of D-lactic acid and L-lactic acid), is more conducive to the separation and purification of D-lactic acid.

[0115] Table 5. Configuration ratios of lactic acid in different proportions of 1,3-dihydroxyacetone and alkali (earth) metal hydroxides

[0116]

[0117]

[0118] Note: The calculation method for ee% is: [(D-LA percentage) - (L-LA percentage)] / [(D-LA percentage) + (L-LA percentage)]. Because lactic acid configurations are racemic in equal proportions, the excess enantiomer is represented by D-LA. ee% refers to this portion of unracemic D-LA. A high ee% indicates a high proportion of a particular lactic acid configuration (here, D-lactic acid) among all lactic acid, i.e., high purity.

[0119] Example 16: Conversion of glycerol to glycolic acid via intermediate 1,3-dihydroxyacetone.

[0120] The enzyme in the reaction solution of 10 mg / mL (equivalent to 242 U / mL) GldA enzyme catalyzing 100 mM and 200 mM glycerol was removed by centrifugation using an ultrafiltration tube. 500 μL of the solution was added to 500 μL of 360 mM (100 mM glycerol as substrate) / 640 mM (200 mM glycerol as substrate) NaClO2 solution, and reacted at 50 °C for 24 h. After the reaction, the solution was filtered through a 0.22 μm organic filter membrane, and the target product glycolic acid was detected by HPLC. HPLC detection conditions: column: Aminex HPX-87H, 300 mm × 7.8 mm (Dewater); mobile phase: 5 mM H2SO4; differential detector; flow rate: 0.5 mL / min; column temperature: 35 °C; injection volume: 20 μL.

[0121] HPLC detection results are as follows Figure 6 As shown in the diagram, analysis reveals that the oxide (sodium chlorite) can further catalyze the conversion of 1,3-dihydroxyacetone, produced from glycerol by the co-catalysis of GldA and LpNox / SpNox-K184R, to glycolic acid (the process of converting glycerol to 1,3-dihydroxyacetone and subsequently to glycolic acid is illustrated in the diagram). Figure 7 (As shown in the table). The conversion rate calculation results are shown in Table 6. It can be seen that the conversion rate from 1,3-dihydroxyacetone to glycolic acid is 77.1-84.7%, and the conversion rate from glycerol to glycolic acid is 67.0-78.2%.

[0122] Table 6. Amount of products (1,3-dihydroxyacetone and glycolic acid) generated in the process of glycerol to glycolic acid versus conversion of substrate (glycerol)

[0123] 1,3-Dihydroxyacetone (mM) 92.3 173.5 Glycolic acid (mM) 78.2 133.9 Conversion rate (%) from 1,3-dihydroxyacetone to glycolic acid 84.7 77.1 Conversion rate (%) from glycerol to glycolic acid 78.2 67.0

[0124] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Methods for producing 1,3-dihydroxyacetone from glycerol and their applications <130> GNCLN220982 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 367 <212> PRT <213> Escherichia coli <400> 1 Met Asp Arg Ile Ile Gln Ser Pro Gly Lys Tyr Ile Gln Gly Ala Asp 1 5 10 15 Val Ile Asn Arg Leu Gly Glu Tyr Leu Lys Pro Leu Ala Glu Arg Trp 20 25 30 Leu Val Val Gly Asp Lys Phe Val Leu Gly Phe Ala Gln Ser Thr Val 35 40 45 Glu Lys Ser Phe Lys Asp Ala Gly Leu Val Val Glu Ile Ala Pro Phe 50 55 60 Gly Gly Glu Cys Ser Gln Asn Glu Ile Asp Arg Leu Arg Gly Ile Ala 65 70 75 80 Glu Thr Ala Gln Cys Gly Ala Ile Leu Gly Ile Gly Gly Gly Lys Thr 85 90 95 Leu Asp Thr Ala Lys Ala Leu Ala His Phe Met Gly Val Pro Val Ala 100 105 110 Ile Ala Pro Thr Ile Ala Ser Thr Asp Ala Pro Cys Ser Ala Leu Ser 115 120 125 Val Ile Tyr Thr Asp Glu Gly Glu Phe Asp Arg Tyr Leu Leu Leu Pro 130 135 140 Asn Asn Pro Asn Met Val Ile Val Asp Thr Lys Ile Val Ala Gly Ala 145 150 155 160 Pro Ala Arg Leu Leu Ala Ala Gly Ile Gly Asp Ala Leu Ala Thr Trp 165 170 175 Phe Glu Ala Arg Ala Cys Ser Arg Ser Gly Ala Thr Thr Met Ala Gly 180 185 190 Gly Lys Cys Thr Gln Ala Ala Leu Ala Leu Ala Glu Leu Cys Tyr Asn 195 200 205 Thr Leu Leu Glu Glu Gly Glu Lys Ala Met Leu Ala Ala Glu Gln His 210 215 220 Val Val Thr Pro Ala Leu Glu Arg Val Ile Glu Ala Asn Thr Tyr Leu 225 230 235 240 Ser Gly Val Gly Phe Glu Ser Gly Gly Leu Ala Ala Ala His Ala Val 245 250 255 His Asn Gly Leu Thr Ala Ile Pro Asp Ala His His Tyr Tyr His Gly 260 265 270 Glu Lys Val Ala Phe Gly Thr Leu Thr Gln Leu Val Leu Glu Asn Ala 275 280 285 Pro Val Glu Glu Ile Glu Thr Val Ala Ala Leu Ser His Ala Val Gly 290 295 300 Leu Pro Ile Thr Leu Ala Gln Leu Asp Ile Lys Glu Asp Val Pro Ala 305 310 315 320 Lys Met Arg Ile Val Ala Glu Ala Ala Cys Ala Glu Gly Glu Thr Ile 325 330 335 His Asn Met Pro Gly Gly Ala Thr Pro Asp Gln Val Tyr Ala Ala Leu 340 345 350 Leu Val Ala Asp Gln Tyr Gly Gln Arg Phe Leu Gln Glu Trp Glu 355 360 365 <210> 2 <211> 450 <212> PRT <213> Lactobacillus pentosus <400> 2 Met Lys Val Ile Val Ile Gly Cys Thr His Ala Gly Thr Ala Ala Val 1 5 10 15 Asn Gln Ile Leu Ala Ser Asn Pro Glu Thr Asp Val Thr Ile Tyr Glu 20 25 30 Arg Asn Asp Asn Val Ser Phe Leu Ser Cys Gly Ile Ala Leu Tyr Leu 35 40 45 Gly Gly Glu Val Ala Asp Pro Gln Gly Leu Phe Tyr Ser Ser Pro Glu 50 55 60 Gln Leu Ala Lys Leu Gly Ala Asn Val His Met Gln His Asp Val Thr 65 70 75 80 Asp Val Asp Thr Glu Asn His Glu Ile Thr Val Thr Asp Leu Lys Thr 85 90 95 Gly Glu Ser Lys Lys Asp Tyr Tyr Asp Lys Leu Val Val Thr Thr Gly 100 105 110 Ser Trp Pro Val Ile Pro Pro Ile Asp Gly Ile Asp Ser Pro Asn Val 115 120 125 Tyr Leu Cys Lys Asn Trp Thr His Ala Gln Ser Leu Trp Glu Ala Ala 130 135 140 Lys Pro Ala Lys Arg Val Ile Val Ile Gly Gly Gly Tyr Ile Gly Thr 145 150 155 160 Glu Leu Val Glu Ala Tyr Gln Lys Gln Gly Lys Glu Val Thr Leu Ile 165 170 175 Asp Gly Leu Pro Arg Ile Leu Asn Lys Tyr Leu Asp Lys Gly Phe Thr 180 185 190 Asp Arg Val Glu Lys Asp Phe Val Asp His Gly Ile Lys Met Ala Leu 195 200 205 Asn Gln Met Val Lys Gly Phe Ser Asp Asp Gly Lys Glu Val Thr Val 210 215 220 Lys Thr Asp Lys Gly Ser Tyr Thr Ala Asp Met Ala Ile Leu Cys Val 225 230 235 240 Gly Phe Arg Pro Asn Thr Ser Leu Leu Lys Gly Lys Val Asp Met Asn 245 250 255 Pro Asn Gly Ser Ile Lys Thr Asn Asp Tyr Met Gln Thr Ser Asp Pro 260 265 270 Asp Ile Tyr Gly Ala Gly Asp Ser Val Ala Val His Tyr Asn Pro Thr 275 280 285 Lys Lys Asp Ala Tyr Ile Pro Leu Ala Thr Asn Ala Val Arg Gln Gly 290 295 300 Thr Leu Val Gly Leu Asn Ile Phe Lys Pro Thr Arg Lys Tyr Met Gly 305 310 315 320 Thr Gln Ser Thr Ser Gly Leu Met Leu Phe Gly Lys Thr Ile Val Ser 325 330 335 Ser Gly Met Thr Leu Glu His Ala Gln Ala Glu Lys Val Pro Ala Glu 340 345 350 Ala Val Thr Phe Glu Asp Asn Tyr Arg Pro Glu Phe Met Pro Thr Thr 355 360 365 Lys Pro Val Leu Met Gln Leu Val Tyr Asn Pro Glu Thr Arg Glu Ile 370 375 380 Leu Gly Ala Gln Phe Met Ser Glu His Asp Val Ser Gln Ser Ala Asn 385 390 395 400 Val Ile Ser Val Met Ile Gln Asn His Asn Thr Ile Asp Asp Leu Gly 405 410 415 Phe Val Asp Met Phe Phe Gln Pro Ile Tyr Asp Arg Pro Phe Asn Tyr 420 425 430 Leu Asn Leu Leu Gly Gln Ala Ala Ile Ala His Ala Ala Glu Lys Val 435 440 445 Thr Glu 450 <210> 3 <211> 456 <212> PRT <213> Artificial sequence <400> 3 Met Ser Lys Ile Val Val Val Gly Ala Asn His Ala Gly Thr Ala Cys 1 5 10 15 Ile Lys Thr Met Leu Thr Asn Tyr Gly Asp Ala Asn Glu Ile Val Val 20 25 30 Phe Asp Gln Asn Ser Asn Ile Ser Phe Leu Gly Cys Gly Met Ala Leu 35 40 45 Trp Ile Gly Glu Gln Ile Ala Gly Pro Glu Gly Leu Phe Tyr Ser Asp 50 55 60 Lys Glu Glu Leu Glu Ser Leu Gly Ala Lys Val Tyr Met Glu Ser Pro 65 70 75 80 Val Gln Ser Ile Asp Tyr Asp Ala Lys Thr Val Thr Ala Leu Val Asp 85 90 95 Gly Lys Asn His Val Glu Thr Tyr Asp Lys Leu Ile Phe Ala Thr Gly 100 105 110 Ser Gln Pro Ile Leu Pro Pro Ile Lys Gly Ala Glu Ile Lys Glu Gly 115 120 125 Ser Leu Glu Phe Glu Ala Thr Leu Glu Asn Leu Gln Phe Val Lys Leu 130 135 140 Tyr Gln Asn Ser Ala Asp Val Ile Ala Lys Leu Glu Asn Lys Asp Ile 145 150 155 160 Lys Arg Val Ala Val Val Gly Ala Gly Tyr Ile Gly Val Glu Leu Ala 165 170 175 Glu Ala Phe Gln Arg Lys Gly Arg Glu Val Val Leu Ile Asp Val Val 180 185 190 Asp Thr Cys Leu Ala Gly Tyr Tyr Asp Arg Asp Leu Thr Asp Leu Met 195 200 205 Ala Lys Asn Met Glu Glu His Gly Ile Gln Leu Ala Phe Gly Glu Thr 210 215 220 Val Lys Glu Val Ala Gly Asn Gly Lys Val Glu Lys Ile Ile Thr Asp 225 230 235 240 Lys Asn Glu Tyr Asp Val Asp Met Val Ile Leu Ala Val Gly Phe Arg 245 250 255 Pro Asn Thr Thr Leu Gly Asn Gly Lys Ile Asp Leu Phe Arg Asn Gly 260 265 270 Ala Phe Leu Val Asn Lys Arg Gln Glu Thr Ser Ile Pro Gly Val Tyr 275 280 285 Ala Ile Gly Asp Cys Ala Thr Ile Tyr Asp Asn Ala Thr Arg Asp Thr 290 295 300 Asn Tyr Ile Ala Leu Ala Ser Asn Ala Val Arg Thr Gly Ile Val Ala 305 310 315 320 Ala His Asn Ala Cys Gly Thr Asp Leu Glu Gly Ile Gly Val Gln Gly 325 330 335 Ser Ile Asn Gly Ile Ser Ile Tyr Gly Leu His Met Val Ser Thr Gly Leu 340 345 350 Thr Leu Glu Lys Ala Lys Arg Leu Gly Phe Asp Ala Ala Val Thr Glu 355 360 365 Tyr Thr Asp Asn Gln Lys Pro Glu Phe Ile Glu His Gly Asn Phe Pro 370 375 380 Val Thr Ile Lys Ile Val Tyr Asp Lys Asp Ser Arg Arg Ile Leu Gly 385 390 395 400 Ala Gln Met Ala Ala Arg Glu Asp Val Ser Met Gly Ile His Met Phe 405 410 415 Ser Leu Allele Gln Glu Gly Val Thr and Glu Lys Allele Leu Thr 420 425 430 Asp Ile Phe Leu Pro His Phe Asn Lys Pro Tyr Asn Tyr Ile Thr 435 440 445 Met Ala Ala Leu Gly Ala Lys Asp 450,455 <210> 4 <211> 1104 <212> DNA <213> Artificial Sequence <400> 4 atggaccgca ttattcaatc accgggtaaa tacatccagg gcgctgatgt gattaatcgt 60 ctgggcgaat acctgaagcc gctggcagaa cgctggttag tggtgggtga caaatttgtt 120 ttaggttttg ctcaatccac tgtcgagaaa agctttaaag atgctggact ggtagtagaa 180 attgcgccgt ttggcggtga atgttcgcaa aatgagatcg accgtctgcg tggcatcgcg 240 gagactgcgc agtgtggcgc aattctcggt atcggtggcg gaaaaaccct cgatactgcc 300 aaagcactgg cacatttcat gggtgttccg gtagcgatcg caccgactat cgcctctacc 360 gatgcaccgt gcagcgcatt gtctgttatc tacaccgatg agggtgagtt tgaccgctat 420 ctgctgttgc caaataaccc gaatatggtc attgtcgaca ccaaaatcgt cgctggcgca 480 cctgcacgtc tgttagcggc gggtatcggc gatgcgctgg caacctggtt tgaagcgcgt 540 gcctgctctc gtagcggcgc gaccaccatg gcgggcggca agtgcaccca ggctgcgctg 600 gcactggctg aactgtgcta caacaccctg ctggaagaag gcgaaaaagc gatgcttgct 660 gccgaacagc atgtagtgac tccggcgctg gagcgcgtga ttgaagcgaa cacctatttg 720 agcggtgttg gttttgaaag tggtggtctg gctgcggcgc acgcagtgca taacggcctg 780 accgctatcc cggacgcgca tcacttat cacggtgaaa aagtggcatt cggtacgctg 840 acgcagctgg ttctggaaaa tgcgccggtg gaggaatcg aaaccgtagc tgcccttagc 900 catgcggtag gtttgccaat aactctcgct caacggata ttaaagaga tgtcccggcg 960 aaaatgcgaa ttgtggcaga agcggcatgt gcagaggtg aaaccattca caacatgcct 1020 ggcggcgcga cgccagatca gttttacgcc gctctgctgg tagccgacca gtacggtcag 1080 cgttttcctgc aagagtggga atga 1104 <210> 5 <211> 1353 <212> DNA <213> Artificial Sequence <400> 5 atgaaagtta tcgtaattgg ttgtactcat gccggaactg ctgctgtaa tcaatcttg 60 gcgtcaaatc cagaaacaga cgtcacgatt tatgaacgga atgacaatgt gtcattctctc 120 tcctgtggga ttgccctcta tcttggtggc gaagttgccg atccacagg gctcttctat 180 tccagtccag aacattagc caattaggc gcgaatgttc atatgcaaca tgatgtgacc 240 gacgtggata ccgaaaatca tgaaattacc gttactgatt tgaagaccgg cgaatccaag 300 aaagattat acgacaaatt agttgtcaca actggttcat ggcctgtaat tccaccaatc 360 gatggtatcg acagcccgaa cgtttacctc tgcaagaact ggacgcatgc ccaaagttta 420 tgggaagctg ccaagccagc taagcgcgtc atcgttatcg gtggggcta cattgggact 480 gaattagtcg aagcttatca gaagcaaggt aaaggaagtta ccttaattga tggcttacca 540 cggattttaa acaagtattt agaacaaggc ttcactgacc gggtcgaaaa agacttcgtt 600 gaccatggca tcaagatggc cttaaatcag atggttaaag gcttcagtga tgatggcaag 660 720 ggtttccggc caaacaccag cctattaaag ggcaaagttg acatgaaccc gaacggctct 780 attaagacaa atgactacat gcaaacatct gaccctgata tctacggtgc tggtgattcc 840 gttgcggttc actacaaccc aactagaag gatgcctaca ttccattagc cactaacgcg 900 gttcgccaag ggactttagt tggtttgaac atcttcaagc caacccggaa gtacatgggg 960 acgcaatcaa cttctggttt aatgttattc ggcaagacga tcgtttcttc tgggatgacc 1020 ttggaacatg ctcaagctga aaagtacct gcagaagccg ttaccttga agatactac 1080 cgtccagaat ttgccaac cacgaacca gttctgatgc aattggttta caacccagag 1140 acgcgtgaaa tcttaggggc ccaattcatg agtgaacatg acgtttcaca atcggctaac 1200 gtgatctcag tgatgattca aaatcacaac acgatcgatg acttaggctt tgttgacatg 1260 ttcttccagc caatctatga ccgtccattc aactacttga acttattagg ccaagcagcc 1320 atcgctcatg cggctgaaaa agtgacggaa tga 1353 <210> 6 <211> 1368 <212> DNA <213> Artificial Sequence <400> 6 atgagtaaaa tcgttgttgt tggtgcaac catgctggta cggcctgtat cacactatg 60 ttaacaact acggtgatgc taatgagat gttgtatttg accaaactc aaatatttca 120 tttttaggct gtggtaggc actggatt ggtgagcaaa tgctggacc agaaggactt 180 ttctattcag attaagaga attagagtct ttaggggcta aagtttacat ggaatcacct 240 gttcaatcaa tcgactacga tgccaaaaca gtgacagcgc ttgttgatgg taaaaaccac 300 gtggaaactt acgacaagtt gattttttgca actggctcac aacctatctt accaccgatt 360 aaaggtgccg aaatcaaaga aggatcactt gaatttgaag caactcttga aaatcttcaa 420 tttgtgaagt tataccaaaa ctcagctgat gtgattgcaa agcttgaaaa caaagacatt 480 aaacgtgtag ccgtagttgg tgctggttac attggtgttg agttagctga agctttccaa 540 cgcaaaggca gagaagtggt tctgattgat gtcgtggaca cttgcttggc aggttattac 600 gaccgtgatt tgactgactt aatggctaaa aacatggaag aacatggtat tcaactagcc 660 tttggtgaaa cagttaaaga agtagctggt aatggtaagg ttgaaaagat cattactgac 720 aaaaatgagt acgatgtgga tatggttatc ctcgccgttg gtttccgtcc aaacacaact 780 cttggaaatg gtaagattga tcttttccgt aatggtgctt tccttgtcaa taaacgccaa 840 gaaacttcta ttccaggtgt ttatgctatc ggtgactgtg caactatcta cgataatgct 900 actcgcgata caaactacat tgctttagct tcaaatgccg tccgtacagg aattgtagca 960 gcacataacg cttgtggtac agaccttgaa ggtattggcg ttcaaggctc aaacggtatt 1020 tccatttacg gattgcacat ggtttcaact ggtttgacac ttgaaaaagc aaaacgtctt 1080 ggttttgatg ctgcagtgac tgagtatact gataaccaaa aacctgaatt tatcgaacac 1140 ggtaacttcc cagataaccat taagattgtt tacgataaag actcacgtcg tatcttgggt 1200 gctcaaatgg cagcccgtga agatgtgtca atgggaattc acatgttctc acttgctatc 1260 caagaaggcg taaccattga aaagttggca ttaactgata ttttcttctt accacatttc 1320 aacaaacctt acaactacat cacaatggca gcacttggtg ccaaagac 1368

Claims

1. A method for generating 1,3-dihydroxyacetone from glycerol, comprising the following steps: [the steps are described in the original text, which are not directly related to the preceding paragraph and can be omitted.] + In the presence of glycerol, the substrate undergoes oxidation to produce 1,3-dihydroxyacetone; The glycerol-converting protein is a glycerol dehydrogenase GldA derived from Escherichia coli; The NADH converting protein is either LpNox, an NADH oxidase derived from Lactobacillus pentosus, or SpNox-K184R, a variant of NADH oxidase derived from Streptococcus pyogenes; SpNox-K184R is a variant obtained by mutating the 184th amino acid residue of SpNox, an NADH oxidase derived from Streptococcus pyogenes, from K to R. In the reaction system of the method, the initial concentration of glycerol is 100-200 mM, and the concentration of NAD is... + The initial concentration of the substance is 2 mM, the initial concentration of GldA is 121-242 U / mL, and the initial concentration of LpNox or SpNox-K184R is 121-242 U / mL. The enzyme activity ratio of GldA to LpNox / SpNox-K184R is 1:1; The amino acid sequence of GldA is shown in SEQ ID No. 1; The amino acid sequence of the LpNox is shown in SEQ ID No. 2; The amino acid sequence of SpNox-K184R is shown in SEQ ID No.

3.

2. The method according to claim 1, characterized in that: In the reaction system, besides glycerol and NAD, + Apart from GldA, LpNox, or SpNox-K184R, the remainder is a buffer solution with pH 6-8.

3. The method according to claim 1 or 2, characterized in that: In the reaction system described in this method, there is no need to add NAD exogenously again. + .

4. The method according to claim 1 or 2, characterized in that: The reaction in the method is carried out at 20-40°C.

5. A method for generating lactic acid from glycerol, comprising the following steps: (A1) 1,3-Dihydroxyacetone is prepared using glycerol as a substrate by any of the methods described in claims 1-4; (A2) Utilizing alkali metal hydroxides and / or alkaline earth metal hydroxides to catalyze the production of lactic acid from 1,3-dihydroxyacetone.

6. A method for generating glycolic acid from glycerol, comprising the following steps: (B1) 1,3-Dihydroxyacetone is prepared using glycerol as a substrate by any of the methods described in claims 1-4; (B2) 1,3-dihydroxyacetone is oxidized by oxides to produce glycolic acid.

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