A biosynthetic method for 1,4-dihydroxy-2-butanone compound

Abstract

The invention belongs to the field of biocatalysis technology, and discloses a biosynthetic method of a 1,4-dihydroxy-2-butanone compound, wherein benzaldehyde lyase is used as a catalyst to catalyze the hydroxymethylation reaction of 3-hydroxypropanal and formaldehyde to generate 1,4-dihydroxy-2-butanone. The reaction of the invention utilizes cheap and readily available aldehydes as substrates to synthesize 1,4-dihydroxy-2-butanone, providing a green and sustainable method with important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and relates to a method for preparing 1,4-dihydroxy-2-butanone by using benzaldehyde lyase as a catalyst to catalyze the hydroxymethylation reaction of 3-hydroxypropionaldehyde and formaldehyde. Background Art

[0002] 1,4-dihydroxy-2-butanone is an important natural product and drug molecule intermediate. The compound can be used to obtain chiral amino alcohols and polyhydroxy alcohols, which have great application potential in functional foods, medicines, drugs and synthetic chemistry. At present, the compound is mainly synthesized by chemical method, but the chemical catalytic synthesis not only has low yield, but also has high price of metal catalysts, which is difficult to industrialize. In addition, the chemical synthesis reaction conditions are harsh (Chung, K. et al. Chemoselective Pd-catalyzed oxidation of polyols: synthetic scope and mechanistic studies. J Am Chem Soc 135, 7593-7602; De Crisci, AG, Chung, K., Oliver, AG, Solis-Ibarra, D. & Waymouth, RM Chemoselective Oxidation of Polyols with Chiral Palladium Catalysts. Organometallics 32, 2257-2266). Biocatalysis is not only cheap, reusable, but also environmentally friendly. Christine Guérard-Hélaine et al. used aldolase to achieve the condensation reaction of acetol and formaldehyde to obtain the target product 1,4-dihydroxy-2-butanone, but the synthesis of the substrate acetol required by this method is relatively difficult and difficult to industrialize (Guérard-Hélaine, C. et al. Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist's Toolbox. ChemCatChem 7, 1871-1879). Therefore, it is very necessary to develop a method for synthesizing 1,4-dihydroxy-2-butanone from a simple and readily available substrate. Summary of the invention

[0003] The present invention provides a method for synthesizing a 1,4-dihydroxy-2-butanone compound by catalyzing benzaldehyde lyase, that is, using benzaldehyde lyase (BAL) as a catalyst and 3-hydroxypropionaldehyde (3-HPA) and formaldehyde as substrates to prepare a 1,4-dihydroxy-2-butanone compound (Formula I).

[0004]

[0005] The benzaldehyde lyase described in the present invention refers to the benzoin aldolase in the enzyme classification number EC 4.1.2.38, which is also called benzaldehyde lyase in the literature (Demir, AS, Ayhan, P., Igdir, AC & Duygu, AN Enzyme Catalyzed Hydroxymethylation of Aromatic Aldehydes with Formaldehyde. Synthesis of Hydroxyacetophenones and (S)-Benzoins. Tetrahedron 60, 6509-6512). The enzyme has the activity of catalyzing the condensation of benzaldehyde to form aromatic hydroxyketones.

[0006] The present invention mines 21 benzaldehyde lyases by functional retrieval with benzaldehyde lyase (GenBank: AAA50176.1) in the literature as a template, and the specific information thereof is shown in Table 1. The benzaldehyde lyases have the ability to convert 3-hydroxypropionaldehyde (3-HPA) and formaldehyde into 1,4-dihydroxy-2-butanone. The benzaldehyde lyase is derived from Actinomadura sp.KC345, Polymorphobacterarshaanensis, Streptomyces sp.BK335, Streptomyces umbrinus, Nocardia miyunensis, Herbiconiux sp.SALV-R1, Streptomycesmirabilis, Sphingomonasendophytica, Gordonia sp.LAM0048, Arthrobacter sp.SLBN-100, Marinitenerisporasediminis, Streptomyces sp.NBRC 110028, Streptomyceshokutonensis, Cnuibacterphyscomitrellae, Frankia, Gordonia terrae, Mycolicibacteriumchitae, Altererythrobacterrrhizovicinus, SphingobiumczechenseLL01, Variovoraxboronicumulans, Pseudomonas fluorescens. More preferably, the amino acid sequence of the benzaldehyde lyase is as shown in SEQ ID NO: 1-19, or a homologous enzyme from the same species with an identity of more than 90%, more than 95%, more than 98%, preferably more than 99%. Taking HeBAL as an example, H28, E49 and Q112 are highly conserved amino acid residues in the catalytic active center.

[0007] The vector used by the genetic engineering bacteria producing benzaldehyde lyase of the present invention is a pET series plasmid.

[0008] The genetically engineered bacteria producing benzaldehyde lyase of the present invention are Escherichia coli BL21 series.

[0009] The benzaldehyde lyase used to prepare 1,4-dihydroxy-2-butanone can be the culture of the benzaldehyde lyase mutant genetically engineered bacteria, or can be bacterial cells obtained by centrifuging the culture medium or a processed product thereof. The processed product refers to an extract obtained from the bacterial body, a crushed liquid, or a separated product obtained by separating and / or purifying the benzaldehyde lyase mutant and the carbonyl reductase in the extract. The most commonly used in the present invention is the bacterial cell obtained by centrifuging the fermentation broth.

[0010] Table 1 Benzaldehyde lyase information

[0011]

[0012]

[0013] Table 2 Enzyme identity comparison

[0014]

[0015] The present invention relates to a method for converting 3-hydroxypropanal and formaldehyde into corresponding 1,4-dihydroxy-2-butanone by using whole cells for catalysis.

[0016] Preferably, the method uses wet bacteria obtained by fermentation culture of engineered bacteria expressing the gene encoding the benzaldehyde lyase as a catalyst; preferably, it is whole cells, and the dosage is 5-100g / L, preferably 100g / L.

[0017] In a specific embodiment, a buffer solution with a pH of 6.0-11.0 is used as the reaction medium, and the reaction is carried out at 20° C.-50° C. Preferably, the buffer solution used in the reaction is an aqueous solution with a pH of 6.0-8.0, and a temperature of 20-40° C., preferably a pH of 7.0, and a temperature of 30° C. More preferably, a potassium phosphate buffer solution with a pH of 7.0 is used as the reaction medium, and the reaction is carried out at 25° C.-40° C.

[0018] Further preferably, magnesium ions and ThDP are also added to the reaction system. Preferably, the magnesium ions are specifically achieved by adding MgSO4; more preferably, the added amount of MgSO4 is 0.5mM-10.0mM.

[0019] In a specific embodiment, the concentrations of the two substrates are 0.1 mM-10 mM, preferably 4-6 mM. Considering the substrate conversion rate and product yield, the optimal substrate concentration is 5 mM.

[0020] Furthermore, the method further includes the step of purifying the 1,4-dihydroxy-2-butanone compound, specifically, after the reaction is completed, the protein is denatured with hydrochloric acid, the protein is removed by centrifugation, the supernatant is evaporated to remove water, and then the product is purified by silica gel column chromatography.

[0021] The method described in a specific example is: the genetically engineered bacteria of the benzaldehyde lyase are cultured in a seed culture medium, inoculated into a fermentation medium in a certain proportion, and after a certain period of culture, the inducer IPTG is added for a period of culture, and the bacteria are collected by centrifugation. 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP (thiamine pyrophosphate) are added to the wet bacteria of the benzaldehyde lyase, and 100mM potassium phosphate is used as a buffer solution at 30°C for 24h. After the reaction is completed, the protein is denatured with hydrochloric acid, the protein is removed by centrifugation, and the supernatant is evaporated to remove water and then the product is purified by silica gel column chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Proton spectrum of 1,4-dihydroxy-2-butanone. DETAILED DESCRIPTION

[0023] The invention is further described below by means of specific examples, the purpose of which is to provide a better understanding of the invention, but these examples do not constitute a limitation to the invention.

[0024] Example 1: Construction and cultivation of genetically engineered bacteria

[0025] The specific construction and cultivation method of the benzaldehyde lyase-producing mutant genetic engineering bacteria is as follows: the benzaldehyde lyase-producing mutant genetic engineering bacteria are derived from Actinomadura sp.KC345, Polymorphobacterarshaanensis, Streptomyces sp.BK335, Streptomyces umbrinus, Nocardia miyunensis, Herbiconiux sp.SALV-R1, Streptomycesmirabilis, Sphingomonasendophytica, Gordonia sp.LAM0048, Arthrobacter sp.SLBN-100, Mariniterisporasediminis, Streptomyces sp.NBRC 110028, Streptomyceshokutonensis, Cnuibacterphyscomitrellae, Frankia, Gordonia terrae, Mycolicibacteriumchitae, Altererythrobacterrhizovicinus, SphingobiumczechenseLL01, Variovoraxboronicumulans, Pseudomonasfluorescens benzaldehyde lyase nucleic acid sequence SEQ ID NO: 1-19 were synthesized, constructed into pET series vectors, and heterologously expressed in the host bacterium Escherichia coli. The strains stored at -80°C were thawed, streaked on the plates, and cultured overnight in a 37°C constant temperature incubator. A single colony on the plate was selected and inoculated into 20 mL of LB medium containing the corresponding antibiotics, and cultured for about 12 hours as a seed solution. The inoculation amount was inoculated into 700 mL of LB medium containing the corresponding antibiotics according to 1%, and cultured on a shaker at 37°C and 200 rpm until OD 600 The p-value was about 0.6-0.8, and IPTG with a final concentration of 0.1 mmol / L was added for induction at 25°C for 12 h, and the bacteria were collected by centrifugation at 6000 rpm.

[0026] Example 2: Benzaldehyde lyase (AsBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0027] Using benzaldehyde lyase (AsBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derived with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 76.3%. The product NMR results can be found in Figure 1 .

[0028] Example 3: Benzaldehyde lyase (PaBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0029] Using benzaldehyde lyase (PaBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 73.6%.

[0030] Example 4: Benzaldehyde lyase (SsBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0031] Using benzaldehyde lyase (SsBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 35.2%.

[0032] Example 5: Benzaldehyde lyase (SuBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0033] Using benzaldehyde lyase (SuBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 80.5%.

[0034] Example 6: Benzaldehyde lyase (NoBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0035] Using benzaldehyde lyase (NoBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 74.8%.

[0036] Example 7: Benzaldehyde lyase (HeBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0037] Using benzaldehyde lyase (HeBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 69.3%.

[0038] Example 8: Benzaldehyde lyase (SmBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0039] Using benzaldehyde lyase (SmBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 61.6%.

[0040] Example 9: Benzaldehyde lyase (SeBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0041] Using benzaldehyde lyase (SeBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 63.8%. .

[0042] Example 10: Benzaldehyde lyase (GoBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0043] Using benzaldehyde lyase (GoBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 72.8%.

[0044] Example 11: Benzaldehyde lyase (ArBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0045] Using benzaldehyde lyase (ArBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 81.5%.

[0046] Example 12: Benzaldehyde lyase (MsBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde.

[0047] Using benzaldehyde lyase (MsBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 67.4%.

[0048] Example 13: Benzaldehyde lyase (StBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0049] Using benzaldehyde lyase (StBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 76.1%.

[0050] Example 14: Benzaldehyde lyase (ShBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0051] Using benzaldehyde lyase (ShBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 78.3%.

[0052] Example 15: Benzaldehyde lyase (CpBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0053] Using benzaldehyde lyase (CpBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 45.7%.

[0054] Example 16: Benzaldehyde lyase (FrBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0055] Using benzaldehyde lyase (FrBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 46.4%.

[0056] Example 17: Benzaldehyde lyase (GtBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0057] Using benzaldehyde lyase (GtBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 82.3%.

[0058] Example 18: Benzaldehyde lyase (MyBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0059] Using benzaldehyde lyase (MyBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 56.5%.

[0060] Example 19: Benzaldehyde lyase (AlBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0061] Using benzaldehyde lyase (AlBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 24h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 45.8%.

[0062] Example 20: Preparation of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde catalyzed by benzaldehyde lyase (ScBAL)

[0063] Using benzaldehyde lyase (ScBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 38.2%.

[0064] Example 21: Benzaldehyde lyase (VbBAL) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0065] Using benzaldehyde lyase (VbBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 50h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 19.4%.

[0066] Example 22: Benzaldehyde lyase (PfBAL_2) catalyzes the production of 1,4-dihydroxy-2-butanone from 3-hydroxypropanal and formaldehyde

[0067] Using benzaldehyde lyase (PfBAL, 100 mg / mL), 5mM 3-HPA, 5mM formaldehyde, 2.5mM MgSO4 and 0.1mM ThDP were added to 100mM potassium phosphate buffer solution, and the total reaction volume was 20mL at 30°C for 48h. The reaction system was derivatized with benzyloxyamine hydrochloride, and the substrate conversion rate and product yield were detected by liquid phase. The test results showed that the product 1,4-dihydroxy-2-butanone was obtained by benzaldehyde lyase catalysis, and the 3-HPA substrate conversion rate was 23.7%.

Claims

1. A method for preparing 1,4-dihydroxy-2-butanone compounds by biocatalysis, characterized in that: The biocatalyst used is benzaldehyde lyase, and the reaction substrates are 3-hydroxypropanal and formaldehyde to form a reaction system to catalyze and obtain 1,4-dihydroxy-2-butanone compound. The benzaldehyde lyase refers to benzoin aldolase in the enzyme classification number EC 4.1.2.38; the registration number of the benzaldehyde lyase is WP_004020769.1; Magnesium ions and ThDP are also added into the reaction system.

2. The method according to claim 1, characterized in that The benzaldehyde lyase is selected from the culture of recombinant genetically engineered bacteria producing benzaldehyde lyase, or bacterial cells obtained after centrifuging the culture medium or a processed product thereof.

3. The method according to claim 2, characterized in that The method uses wet bacteria obtained by fermentation culture of engineered bacteria expressing the coding gene of the benzaldehyde lyase as a catalyst.

4. The method according to claim 3, characterized in that The catalyst is a whole cell catalyst, and its usage is 5-100 g / L.

5. The method according to any one of claims 1 to 4, characterized in that: A buffer solution with a pH of 6.0-11.0 is used as a reaction medium, and the reaction is carried out at 20°C-50°C.

6. The method according to claim 5, characterized in that Potassium phosphate buffer with a pH of 7.0 was used as the reaction medium, and the reaction was carried out at 25°C-40°C.

7. The method according to claim 1, characterized in that The magnesium ion is achieved by adding MgSO4, and the added amount of MgSO4 is 0.5 mM-10.0 mM.

8. The method according to any one of claims 1 to 4, characterized in that The concentrations of the 3-hydroxypropanal and formaldehyde are 0.1 mM-10 mM, respectively.

9. The method according to claim 8, characterized in that The concentrations of the 3-hydroxypropionaldehyde and formaldehyde are 4-6 mM, respectively.

10. The method according to any one of claims 1 to 4, characterized in that: The method also includes a step of purifying the 1,4-dihydroxy-2-butanone compound.

11. The method according to claim 10, characterized in that The purification is to denature the protein with hydrochloric acid after the reaction is completed, remove the protein by centrifugation, remove water from the supernatant by rotary evaporation, and then purify the product by silica gel column chromatography.

12. The method according to claim 3, characterized in that The vector used by the genetic engineering bacteria producing benzaldehyde lyase is a pET series plasmid; the genetic engineering bacteria producing benzaldehyde lyase is an Escherichia coli BL21 series.

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