Benzaldehyde lyase mutants and use thereof in the preparation of alpha-hydroxymethyl ketones

Abstract

The application provides a benzaldehyde lyase mutant and application thereof in synthesis of alpha-hydroxymethyl ketone compounds. Herbiconiux sp. SALV-R1 The wild type of the benzaldehyde lyase is derived from Herbiconiux sp. SALV-R1 , and a mutant protein with mutation of core amino acids related to enzyme catalytic activity is obtained through site-directed mutation, which has significantly improved activity of hydroxymethylation of furfural (or aromatic benzaldehyde) and formaldehyde, and the concentration of the converted substrate can reach 1M, which has great industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of enzyme engineering and biotechnology, in particular to benzaldehyde lyase mutants and their use in the preparation of α-hydroxymethyl ketones. BACKGROUND

[0002] α-hydroxymethyl ketones are important structural units of many natural products and drug molecules. The use of such compounds can obtain chiral amino alcohols, chiral diols and chiral amino acids with higher application value, which have great application potential in functional food, medicine, pharmaceuticals and synthetic chemistry. Traditional chemical catalysis can effectively obtain α-hydroxymethyl ketones, such as N-heterocyclic carbene catalyzed hydroxymethylation of aldehydes, hydrolysis of α-haloketones, oxidation of olefins, etc. However, the metal catalysts required for chemical catalysis are toxic and expensive, in addition, the synthesis reaction conditions are harsh, the product yield is low, and it is difficult to industrialize production. (Huang, J.; Li, J.; Zheng, J.; Wu, W.; Hu, W.; Ouyang, L.; Jiang, H., Dual role of H2O2 in palladium-catalyzed dioxygenation of terminal alkenes. Organic Letters 2017, 19 (13), 3354-3357.; Wong, F. F.; Chang, P. W.; Lin, H. C.; You, B. J.; Huang, J. J.; Lin, S. K., An efficient and convenient transformation of α-haloketones to α-hydroxyketones using cesium formate. Journal of Organometallic Chemistry 2009, 694 (21), 3452-3455.; Dong, K.; Sang, R.; Soule, J. F.; Bruneau, C.; Franke, R.; Jackstell, R.; Beller, M., Efficient domino hydroformylation / benzoin condensation: Highly selective synthesis of α-hydroxy ketones. Chemistry-A European Journal 2015, 21 (50), 18033-18037.)

[0003] Biocatalysis has attracted extensive attention due to its environmentally friendly characteristics and high selectivity. Shanmuganathan et al. used benzaldehyde lyase to realize the hydroxymethylation reaction of furfural or benzaldehyde and formaldehyde to obtain the target product α-hydroxymethyl ketone, but this method needs to add a large amount of formaldehyde (3 equivalents) and the product concentration is low, only 50 mM, which is difficult to industrialize (Shanmuganathan, S.; Natalia, D.; Greiner, L.; Domínguez de María, P., Oxidation-hydroxymethylation-reduction: A one-pot three-step biocatalytic synthesis of optically active α-aryl vicinal diols. Green Chemistry. 2012, 14 (1), 94-97.). Zhang et al. improved the soluble expression of pyruvate decarboxylase, and realized the hydroxymethylation reaction of 50 g / L furfural by fed-batch method, but the substrate scope of this enzyme is narrow (Zhang, X.; Wei, H.; Wei, X.; Qi, T.; Zong, X.; Liu, Z.; Qin, J.; Gao, X.; Zheng, G.; Ma, Q., Biosynthesis of 2-furylhydroxymethylketone, an intermediate of cefuroxime, from furfural and formaldehyde using a ThDP-dependent enzyme. Green Chemistry 2023, 25 (12), 4713-4722.). In addition, formaldehyde in the reaction system can easily cause denaturation or inactivation of biological enzymes, and the high selectivity and controllable conversion of formaldehyde are still important factors limiting the industrial application of this enzyme. Therefore, it is necessary to find a high-efficiency biocatalytic enzyme reagent for synthesizing α-hydroxymethyl ketone. SUMMARY

[0004] In order to solve the above problems, the present application provides a benzaldehyde lyase mutant modified by genetic engineering, specifically, the activity of the modified benzaldehyde lyase mutant for synthesizing α-hydroxymethyl ketone is significantly improved.

[0005] Firstly, the present application provides a benzaldehyde lyase mutant, the benzaldehyde lyase mutant protein has at least 90% identity with the amino acid sequence shown in SEQ ID NO. 1, and the mutant protein has significantly improved activity for synthesizing α-hydroxymethyl ketone.

[0006] Preferably, the benzaldehyde lyase mutant provided by the present application is a mutant of benzaldehyde lyase at one or more of positions 27, 29, 395, 417, 549, 551, 552 and 553 in the amino acid sequence of positions 1-558 of SEQ ID NO. 1:

[0007] In another preferred embodiment, the tyrosine (Y) at position 395 is mutated to leucine (L) and alanine (A), preferably leucine (L).

[0008] In another preferred embodiment, the glutamic acid (E) at position 549 is mutated to leucine (L) and tyrosine (Y), preferably leucine (L).

[0009] In another preferred embodiment, the valine (V) at position 551 is mutated to serine (S) and leucine (L), preferably serine (S).

[0010] In another preferred embodiment, the isoleucine (I) at position 552 is mutated to leucine (L) and methionine (M), preferably leucine (L).

[0011] In another preferred embodiment, the methionine (M) at position 553 is mutated to leucine (L) and isoleucine (I), preferably leucine (L).

[0012] More specifically, the following combination of mutations: mutation of position 27 to isoleucine (I) and mutation of position 29 to isoleucine (I) and mutation of position 417 to serine (S) and mutation of position 549 to leucine (L) and mutation of position 551 to serine (S) and mutation of position 552 to leucine (L) and mutation of position 553 to leucine (L).

[0013] The present application also provides the encoding gene of the above-mentioned mutant. Further provided are expression vectors and recombinant cells containing the gene.

[0014] The present application also provides the use of the benzaldehyde lyase mutant or the encoding gene thereof in the preparation of α-hydroxymethyl ketone compounds.

[0015] The present application thus provides a method for preparing α-hydroxymethyl ketone compounds, characterized in that the catalytic reaction is carried out with the benzaldehyde lyase mutant as catalyst and with furfural or aromatic benzaldehyde and formaldehyde as substrates to obtain (reaction

[0016] Formula 1):

[0017]

[0018] In one embodiment, the catalytic reaction is carried out with the wet bacterial body obtained by fermenting the engineered bacteria expressing the gene encoding the benzaldehyde lyase mutant as catalyst, with furfural or aromatic benzaldehyde and formaldehyde as substrate, with a buffer solution with pH of 6.0-9.0 as reaction medium, and under the condition of 25-50℃.

[0019] In a more preferred embodiment, in the reaction, the catalytic substrate concentration in the reaction system is 50-1200mM, more preferably, the substrate concentration is 300-1000mM; the reaction system contains bacterial body amount of 10-150g / L, more preferably, 20-70g / L; the pH of the reaction system is 6.0-9.0, and the optimal pH is 7.0; the reaction temperature is 25-50℃, and the optimal temperature is 30℃.

[0020] In a specific reaction system, thiamine pyrophosphate (ThDP) and MgSO4 are also added, and the reaction is carried out under the condition of 150-250rpm, and the reaction time is 5-25h. More preferably, 0.1mM ThDP and 2.5mM MgSO4 are added, and the reaction is carried out at 30℃ on a 200rpm shaker for 12h.

[0021] The benzaldehyde lyase mutant of the present application has a conversion rate of the obtained α-hydroxymethyl ketone of ≥50%, preferably ≥90%, and more preferably ≥99%, which is much higher than that of the wild-type benzaldehyde lyase. Therefore, the wild-type benzaldehyde lyase of the present application is derived from Herbiconiux sp. SALV-R1, and the mutant protein obtained by site-directed mutation has a mutation in the core amino acid related to the catalytic activity of the enzyme, which has significantly improved the activity of hydroxymethylation of furfural (or aromatic benzaldehyde) and formaldehyde, and the concentration of the converted substrate can reach 1M, which has great industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 NMR hydrogen spectrum of the product 2-furyl hydroxymethyl ketone synthesized by the mutant M6.

[0023] Figure 2 NMR carbon spectrum of the product 2-furyl hydroxymethyl ketone synthesized by the mutant M6.

[0024] Figure 3 NMR hydrogen spectrum of the product 2-hydroxyacetophenone synthesized by the mutant M6.

[0025] Figure 4 NMR carbon spectrum of the product 2-hydroxyacetophenone synthesized by the mutant M6. DETAILED DESCRIPTION

[0026] The application is further described below by way of specific embodiments, so as to better understand the application, but does not constitute a limitation on the application. Among them, as used herein, the term "AxxB" means that the amino acid A at the xxth position is changed to amino acid B, for example, "A27I" means that the alanine A at the 27th position is mutated to isoleucine I, and so on.

[0027] In the embodiments of the application, the preparation method of the benzaldehyde lyase mutant of the application is as follows: E. coli is used as the expression host. Specifically, the preparation method comprises the following steps: (1) constructing the gene of the corresponding mutation site of the benzaldehyde lyase into the pET-21a expression vector to obtain a recombinant plasmid with the enzyme gene of interest. (2) transforming the recombinant plasmid into host bacterial cells (preferably E. coli BL21 (DE3)) to obtain a corresponding engineering strain. (3) inoculating the engineering strain into LB medium, culturing at 37°C for 6 h, adding 0.1 mM isopropyl thiogalactoside (IPTG), and culturing at 25°C for 6-12 h. (4) centrifuging to collect the bacterial cells.

[0028] Example 1: Construction and culture of benzaldehyde lyase mutants

[0029] The wild type of benzaldehyde lyase is derived from Herbiconiux sp. SALV-R1, and its amino acid sequence is shown as SEQ ID NO. 1, and the corresponding nucleotide sequence is SEQ ID NO. 2. The nucleotide sequence is fully synthesized and cloned into the restriction endonuclease sites NdeI and XhoI of the pET-21a vector to obtain the recombinant plasmid pET-21a-HeBAL, which is further transformed into the expression host E. coli BL21 (DE3). A positive clone is picked to obtain the recombinant expression transformant E. coli BL21 (DE3) / pET-21a-HeBAL. An advantageous mutant HeBAL A27I / V29I / G417S (M3) obtained through previous research can achieve the hydroxymethylation reaction of furfural, and its amino acid sequence is shown as SEQ ID NO. 3, and the corresponding nucleotide sequence is SEQ ID NO. 4 (Li, Y.; Yao, P.; Zhang, S.; Feng, J.; Su, H.; Liu, X.; Sheng, X.; Wu, Q.; Zhu, D.; Ma, Y., Creating a new benzaldehyde lyase for atom-economic synthesis of chiral 1,2,4-butanetriol and 2-aminobutane-1,4-diol from formaldehyde. Chem Catalysis 2023, 3 (1) 100467.).

[0030] The simulated protein structure of HeBALM3 was obtained by Alphafold2 modeling, and non-conserved residues in the substrate binding pocket were selected for site-directed mutagenesis (mutated to leucine), and mutation primers were designed with pET21a-HeBALM3 as a template. The obtained mutant monoclonal colonies were cultured, and the expressed protein was subjected to activity screening, and the dominant mutant was determined by gas phase detection.

[0031] The sites of the library mutation were I24, N25, G26, I27, I29, D30, T46, R47, N48, M50, T72, A73, G74, G75, G76, F77, T78, N79, A392, Y395, W397, H413, G414, Y415, G417, S418, M419, G420, G445, A446, V447, G448, Y449, W474, G475, A476, T477, H479, A480, Q481, N492, N493, P546, E548, E549, I552 and M553, and primers were designed (see Table 1 for primer sequences). The mutant was constructed by two-step PCR, and high-fidelity polymerase FastPfu-DNA was used for PCR. The PCR reaction conditions were as follows: in a total volume of 50 μL of the PCR reaction system, 5 μL of 10×Pfu buffer, 5 μL of dNTP (2 mM), 2 μL of MgSO4 (25 mM), 20-100 ng of template, 1 μL of each mutant primer (10 μM), 1 μL of Pfu polymerase, and 50 μL of sterilized distilled water were added. The small fragment PCR reaction program was as follows: (1) 95°C pre-denaturation for 2 min, (2) 95°C denaturation for 20 sec, (3) 55°C annealing for 20 sec, (4) 72°C extension for 12 sec, (5) 72°C final extension for 5 min, and steps (2)-(4) were repeated for 20-30 cycles. The PCR product was stored at 4°C. After agarose gel electrophoresis analysis, DpnI was added for digestion at 37°C for 2 h. The digestion product was transferred to E. coli BL21 (DE3) competent cells and plated on plates containing ampicillin, and incubated in a 37°C incubator for about 12 h. Single colonies were picked for sequencing, and the correct sequencing obtained the corresponding mutant.

[0032] The activity of the obtained mutant was identified by analyzing the reaction system, and the reaction formula was as follows:

[0033]

[0034] The specific method is as follows: 200 mM furfural, 300 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP are added to 100 mM potassium phosphate buffer (pH 7.0) in 50 mg / mL wet bacteria, the reaction volume is 1 mL, and the reaction system is reacted at 30 DEG C for 24 h. Then the reaction system is extracted with 1 mL of ethyl acetate, and the reaction of the mutant is detected by gas phase. Through screening, the beneficial mutant sites with improved enzyme activity are Y395 and I552; further, the saturated mutant library of Y395 and I552 sites is established by taking M3 as a template, and the dominant mutants M4_1 (A27I / V29I / Y395L / G417S) and M4_2 (A27I / V29I / G417S / I552L) are obtained, and the protein sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6; taking M4_1 and M4_2 as templates, an iterative saturated mutant library is established, and the mutation sites are I27, T46, H413, E549, V551, I552 and M553, respectively, and through screening, the mutants M5_1 (A27I / V29I / G417S / I552L / M553L) and M5_2 (A27I / V29I / Y395L / G417S / I552L) with further improved activity are obtained, and the protein sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8; then taking M5_2 as a template, the sites E549 and V551 are selected to construct a six-point combination mutant library, and the mutant M6 (A27I / V29I / G417S / E549L / I552L / M553L) with further improved activity is obtained, and the protein sequence is shown in SEQ ID NO. 9; then taking M6 as a template, the site V551 is selected to construct a seven-point combination mutant library, and the mutant M7 (A27I / V29I / G417S / E549L / V551S / I552L / M553L) with further improved activity is obtained, and the protein sequence is shown in SEQ ID NO. 10 (the specific data is shown in Table 2).

[0035] Table 1 primer sequence of mutation site

[0036]

[0037]

[0038]

[0039] Table 2 screening results of related mutants

[0040]

[0041]

[0042] Example 2: Induced expression of benzaldehyde lyase mutants

[0043] The single colony of the genetically engineered bacteria of the above mutants was inoculated into 4 mL of LB liquid medium (10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl) containing ampicillin, and cultured at 37 °C in a 200 rpm shaker overnight, which was the seed liquid. The seed liquid cultured overnight was inoculated into 50 mL of LB medium containing ampicillin at a 1% inoculation amount, and cultured at 37 °C and 200 rpm until the OD600 was about 0.6-1.0. Then, 0.5 mM IPTG was added, and the culture was induced at 25 °C and 200 rpm for 8-12 h. The bacterial cells were collected by centrifugation at 4 °C and 6000 rpm. The bacterial cells were resuspended with potassium phosphate buffer (100 mM, pH 7.0), and were broken by ultrasonic or high-pressure homogenizer. The supernatant (crude enzyme solution) was collected by centrifugation at 4 °C and 12000 rpm, and was subjected to subsequent SDS-PAGE and enzyme activity detection.

[0044] Example 3: Optimization of reaction conditions of benzaldehyde lyase mutants

[0045] Benzaldehyde lyase mutants M3 and M6 (50 mg / mL) were used as biocatalysts, 300 mM furfural, 360 mM formaldehyde, 2.5 mM MgSO4, and 0.1 mM ThDP were added into potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 1 mL. The reaction was carried out at 25 °C, 30 °C, 37 °C, 45 °C, and 200 rpm on a shaker for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by gas phase.

[0046] Table 3 Optimum reaction temperature of benzaldehyde lyase mutants

[0047]

[0048] Benzaldehyde lyase mutants M3 and M6 (50 mg / mL) were used as biocatalysts, 300 mM furfural, 360 mM formaldehyde, 2.5 mM MgSO4, and 0.1 mM ThDP were added into different potassium phosphate buffer solutions (pH 6.5, pH 7.0, pH 7.5, pH 8.5, 100 mM), and the total reaction volume was 1 mL. The reaction was carried out at 30 °C and 200 rpm on a shaker for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by gas phase.

[0049] Table 4 Optimum reaction pH of benzaldehyde lyase

[0050]

[0051] Example 4: Benzaldehyde lyase mutant catalyze synthesis of a-hydroxymethyl ketone

[0052] The amino acid sequences of the mutants M4_2, M5_1, M6, and M7 with higher activity are SEQ ID NO. 6, 7, 9, and 10, respectively. After protein induction expression according to the culture and induction expression method of Example 2, the bacterial cells were collected and used as biocatalysts.

[0053] Using benzaldehyde lyase mutant M4_2 (50 mg / mL), 300 mM furfural (10% DMSO), 360 mM formaldehyde, 2.5 mM MgSO4, and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), with a total reaction volume of 50 mL. The reaction was carried out at 30°C on a shaker at 200 rpm for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance. The detection results showed that the benzaldehyde lyase mutant M4_2 catalyzed the product 2-furanyl hydroxymethyl ketone, and the furfural substrate conversion rate was 80%.

[0054] Using benzaldehyde lyase mutant M5_1 (50 mg / mL), 300 mM furfural (10% DMSO), 360 mM formaldehyde, 2.5 mM MgSO4, and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), with a total reaction volume of 50 mL. The reaction was carried out at 30°C on a shaker at 200 rpm for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance. The detection results showed that the benzaldehyde lyase mutant M5_1 catalyzed the product 2-furanyl hydroxymethyl ketone, and the furfural substrate conversion rate was 90%.

[0055] Using benzaldehyde lyase mutant M6 (50 mg / mL), 300 mM furfural (10% DMSO), 360 mM formaldehyde, 2.5 mM MgSO4, and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), with a total reaction volume of 50 mL. The reaction was carried out at 30°C on a shaker at 200 rpm for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance. The detection results showed that the benzaldehyde lyase mutant M6 catalyzed the product 2-furanyl hydroxymethyl ketone, and the furfural substrate conversion rate was 99%.

[0056] Using benzaldehyde lyase mutant M6 (25 mg / mL), 500 mM furfural (10% DMSO) and 600 mM formaldehyde were fed in batch into a potassium phosphate buffer solution (pH 7.0, 100 mM) containing 2.5 mM MgSO4and 0.1 mM ThDP, with a total reaction volume of 50 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 5 h. The reaction mixture was extracted with ethyl acetate, and the substrate conversion and product yield were detected by NMR. The results showed that the benzaldehyde lyase mutant M6 catalyzed the product 2-furylhydroxymethyl ketone, and the substrate conversion rate of furfural was 99%.

[0057] Using benzaldehyde lyase mutant M6 (25 mg / mL), 700 mM furfural (10% DMSO) and 840 mM formaldehyde were fed in batch into a potassium phosphate buffer solution (pH 7.0, 100 mM) containing 2.5 mM MgSO4and 0.1 mM ThDP, with a total reaction volume of 50 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 7 h. The reaction mixture was extracted with ethyl acetate, and the substrate conversion and product yield were detected by NMR. The results showed that the benzaldehyde lyase mutant M6 catalyzed the product 2-furylhydroxymethyl ketone, and the substrate conversion rate of furfural was 99%.

[0058] Using benzaldehyde lyase mutant M7 (25 mg / mL), 1000 mM furfural (10% DMSO) and 1200 mM formaldehyde were fed in batch into a potassium phosphate buffer solution (pH 7.0, 100 mM) containing 2.5 mM MgSO4and 0.1 mM ThDP, with a total reaction volume of 50 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction mixture was extracted with ethyl acetate, and the substrate conversion and product yield were detected by NMR. The results showed that the benzaldehyde lyase mutant M7 catalyzed the product 2-furylhydroxymethyl ketone, and the substrate conversion rate of furfural was 99%.

[0059] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM benzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction mixture was extracted with ethyl acetate, and the substrate conversion and product yield were detected by NMR and the product was separated and purified by silica gel column. The results showed that the benzaldehyde lyase mutant M6 catalyzed the product α-hydroxymethyl ketone, and the substrate conversion rate was 99%, and the product separation yield was 81%.

[0060] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 2-fluorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product α-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 61%.

[0061] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-fluorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product α-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 68%.

[0062] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-fluorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product α-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 84%.

[0063] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-chlorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product α-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 87%.

[0064] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-chlorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 45%.

[0065] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-bromobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 85%.

[0066] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-bromobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 97%, and the product separation yield is 59%.

[0067] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-methylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 75%.

[0068] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-methylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 88%.

[0069] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-trifluoromethylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 72%.

[0070] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-trifluoromethylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgS04and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 46%, and the product separation yield is 33%.

[0071] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-methoxybenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4and 0.1 mM ThDP were added into potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 81%.

[0072] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-fluoro, 3-chlorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4and 0.1 mM ThDP were added into potassium phosphate buffer solution (pH 7.0, 100 mM) with a total reaction volume of 20 mL. The reaction was carried out at 30 °C on a shaker at 200 rpm for 20 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by nuclear magnetic resonance, and the product was obtained by silica gel column separation and purification. The detection results are as follows: the benzaldehyde lyase mutant M6 catalyzes to obtain the product a-hydroxymethyl ketone, the substrate conversion rate is 99%, and the product separation yield is 84%.

[0073] In summary, benzaldehyde lyase mutants (50 mg / mL) M4_2, M5_1 and M6 can react with 300 mM furfural, and the conversion rate can be more than 80%; benzaldehyde lyase mutant (25 mg / mL) M7 can react with 1000 mM furfural in the form of fed-batch, and the conversion rate can be more than 99%; benzaldehyde lyase mutant (50 mg / mL) M6 can react with 200 mM benzaldehyde, 2-fluorobenzaldehyde, 3-fluorobenzaldehyde, 4-fluorobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde, 3-bromobenzaldehyde, 4-bromobenzaldehyde, 3-methylbenzaldehyde, 4-methylbenzaldehyde, 3-trifluoromethylbenzaldehyde, 3-methoxybenzaldehyde or 4-fluoro, 3-chlorobenzaldehyde, and the conversion rate can be more than 97%, and the product separation yield is medium to high (45%-88%).

Claims

1. A benzaldehyde lyase mutant, characterized in that it is The following mutations exist based on the amino acid sequence shown in SEQ ID NO.1: (1) A27I / V29I / Y395L / G417S, (2) A27I / V29I / G417S / I552L, (3) A27I / V29I / G417S / I552L / M553L, (4) A27I / V29I / Y395L / G417S / I552L, (5) A27I / V29I / G417S / E549L / I552L / M553L, or (6) A27I / V29I / G417S / E549L / V551S / I552L / M553L.

2. The encoding gene of the benzaldehyde lyase mutant as described in claim 1.

3. An expression vector containing the encoding gene of the benzaldehyde lyase mutant as described in claim 1.

4. Recombinant cells containing the coding gene of the benzaldehyde lyase mutant as described in claim 1.

5. The benzaldehyde lyase mutant of claim 1, or the gene encoding the benzaldehyde lyase mutant of claim 2, in the preparation of... Applications in hydroxymethyl ketone compounds.

6. A method for preparing The method for using hydroxymethyl ketone compounds is characterized by... The product is obtained by catalytic reaction using the benzaldehyde lyase mutant as described in claim 1 as a catalyst and furfural or aromatic benzaldehyde and formaldehyde as substrates.

7. The method as described in claim 6, characterized in that, Using wet cells obtained by fermentation culture of engineered bacteria expressing the coding gene of the benzaldehyde lyase mutant as described in claim 2 as a catalyst, furfural or aromatic benzaldehyde and formaldehyde as substrates, and a buffer solution with a pH of 6.0-9.0 as the reaction medium, the catalytic reaction is carried out at 25℃-50℃.

8. The method as described in claim 6 or 7, characterized in that, In the catalytic reaction, the concentration of the catalytic substrate in the reaction system is 50-1200 mM; the amount of bacteria in the reaction system is 10-150 g / L; the pH of the reaction system is 6.0-9.0; and the reaction temperature is 25℃-50℃.

9. The method as described in claim 8, characterized in that, In the catalytic reaction, the concentration of the catalytic substrate in the reaction system is 300-1000 mM; the amount of bacteria in the reaction system is 20-70 g / L; the pH of the reaction system is 7.0; and the reaction temperature is 30℃.

10. The method as described in claim 9, characterized in that, ThDP and MgSO4 were also added to the reaction system, and the reaction was carried out at 30℃ and 150 rpm - 250 rpm for 5-25 h.

11. The method as described in claim 10, characterized in that, 0.1 mMThDP and 2.5 mMMgSO4 were also added to the reaction system, and the reaction was carried out at 30℃ and 200 rpm on a shaker for 12 h.

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