Benzaldehyde lyase mutant and application thereof in preparation of alpha-hydroxymethyl ketone compound

Abstract

The invention provides a benzaldehyde lyase mutant and application of the benzaldehyde lyase mutant in synthesis of alpha-hydroxymethyl ketone compounds. The wild type of the benzaldehyde lyase is derived from Herbiciux sp.SALV-R1, mutant protein with mutated core amino acid related to enzyme catalytic activity is obtained through site-specific mutagenesis, the activity of catalyzing hydroxymethylation of furfural (or aromatic benzaldehyde) and formaldehyde is remarkably improved, the concentration of a conversion substrate can reach 1M, and the benzaldehyde lyase has great industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of enzyme engineering and biotechnology, and particularly relates to a benzaldehyde lyase mutant and its application in the preparation of α-hydroxymethyl ketone compounds. Background Art

[0002] α-Hydroxymethyl ketone compounds are important structural units of many natural products and drug molecules. Using such compounds, chiral amino alcohols, chiral diols, chiral amino acids, etc. with higher application values can be obtained, and they have great application potential in functional foods, medicines, pharmaceuticals, and synthetic chemistry. Traditional chemical catalysis can effectively obtain α-hydroxymethyl ketones. For example, N-heterocyclic carbene-catalyzed hydroxymethylation of aldehydes, hydrolysis of α-haloketones, oxidation of alkenes, 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 the production. (Huang, J.; Li, J.; Zheng, J.; Wu, W.; Hu, W.; Ouyang, L.; Jiang, H., Dual role of H2O2 in palladium-catalyzed dioxygenation of terminalalkenes.Organic Letters2017,19(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 ofα-haloketones toα-hydroxyketones using cesium formate.Journal of OrganometallicChemistry2009,694(21),3452-3455.; Dong, K.; Sang, R.; Soule, J.F.; Bruneau, C.; Franke, R.; Jackstell, R.; Beller, M., Efficient domino hydroformylation / benzoincondensation:Highly selective synthesis ofα-hydroxy ketones.Chemistry-AEuropean Journal2015,21(50),18033-18037.).

[0003] Biocatalysis has attracted extensive attention from researchers due to its environmentally friendly characteristics and high selectivity. Saravanakumar Shanmuganathan et al. achieved the hydroxymethylation reaction of furfural or benzaldehyde and formaldehyde using benzaldehyde lyase to obtain the target product α-hydroxymethyl ketone. However, this method requires the addition of a large amount of formaldehyde (3 equivalents), and the product concentration is only 50 mM, making it difficult to be industrially utilized (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 Xianghe et al. achieved the hydroxymethylation reaction of 50 g / L furfural by improving the soluble expression of pyruvate decarboxylase and adopting a fed-batch method. However, the substrate scope of this enzyme is relatively 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 is extremely likely to cause the denaturation or inactivation of biocatalysts. At the same time, the highly selective and controllable conversion of formaldehyde is still an important factor restricting the industrial application of such enzymes. Therefore, it is necessary to find a biocatalytic enzyme reagent for the efficient synthesis of α-hydroxymethyl ketone. Summary of the Invention

[0004] To solve the above problems, the present invention provides a mutant of benzaldehyde lyase modified by genetic engineering means. Specifically, the activity of the modified mutant of benzaldehyde lyase in synthesizing α-hydroxymethyl ketone is significantly improved.

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

[0006] Preferably, the benzaldehyde lyase mutant provided by the present invention is a benzaldehyde lyase mutant mutated at one or more sites among sites 27, 29, 395, 417, 549, 551, 552, and 553 corresponding to amino acids at positions 1-558 of SEQ ID NO. 1:

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

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

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

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

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

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

[0013] The present invention also provides the coding gene of the above mutant. Further provided are an expression vector and a recombinant cell containing the gene.

[0014] The present invention also provides the application of the benzaldehyde lyase mutant or its coding gene in the preparation of α-hydroxymethyl ketone compounds.

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

[0016] Formula 1):

[0017]

[0018] In a specific embodiment, the catalytic reaction uses the wet bacterial cells obtained by fermenting and culturing an engineered bacterium expressing the coding gene of the benzaldehyde lyase mutant as a catalyst, uses furfural or aromatic benzaldehyde and formaldehyde as substrates, and uses a buffer solution with a pH of 6.0 - 9.0 as a reaction medium, and the reaction is carried out under the condition of 25°C - 50°C.

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

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

[0021] The conversion rate of the α-hydroxymethyl ketone catalyzed by the benzaldehyde lyase mutant of the present invention is ≥50%, preferably, ≥90%, more preferably, ≥99%; far higher than the conversion rate of the wild-type benzaldehyde lyase. Therefore, the wild-type of the benzaldehyde lyase of the present invention is derived from Herbiconiux sp. SALV-R1, and a mutant protein with mutations in the core amino acids related to enzyme catalytic activity is obtained by site-directed mutagenesis. It has a significantly improved activity in catalyzing the hydroxymethylation of furfural (or aromatic benzaldehyde) and formaldehyde, and the concentration of the converted substrate can reach 1 M, which has great industrial application prospects. Description of the Drawings

[0022] Figure 1 1H NMR spectrum of the synthetic product 2-furylhydroxymethyl ketone of mutant M6.

[0023] Figure 2 13C NMR spectrum of the synthetic product 2-furylhydroxymethyl ketone of mutant M6.

[0024] Figure 3 1H NMR spectrum of the synthetic product 2-hydroxyacetophenone of mutant M6.

[0025] Figure 4 13C NMR spectrum of the synthetic product 2-hydroxyacetophenone of mutant M6. Detailed Embodiments

[0026] The present invention will be further described below through specific embodiments for better understanding, but it does not limit the present invention. Herein, 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 alanine A at the 27th position is mutated to isoleucine I, and so on.

[0027] In an embodiment of the present invention, the preparation method of the benzaldehyde lyase mutant of the present invention is as follows: Escherichia coli is used as the expression host. Specifically, the preparation method includes the following steps: (1) The gene of the corresponding mutation site of benzaldehyde lyase is constructed onto the pET-21a expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) The recombinant plasmid is transferred into host bacterial cells (preferably Escherichia coli BL21(DE3)) to obtain the corresponding engineering strain. (3) The engineering strain is inoculated into LB medium and cultured at 37 °C for 6 h, then 0.1 mM isopropylthiogalactoside (IPTG) is added, and cultured at 25 °C for 6 - 12 h. (4) The cells are collected by centrifugation.

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

[0029] The wild type of benzaldehyde lyase is derived from Herbiconiux sp. SALV-R1. 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 between the restriction enzyme 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). Positive clones are picked to obtain the recombinant expression transformant E. coli BL21(DE3) / pET-21a-HeBAL. Through previous research, a dominant mutant HeBAL A27I / V29I / G417S (M3) was obtained, which can achieve the hydroxymethylation reaction of furfural. 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 modeling with Alphafold2. Non-conserved residues within its substrate-binding pocket were selected for site-directed mutagenesis (mutated to leucine) respectively, and mutant primers were designed, using pET21a-HeBAL M3 as the template. The obtained mutant monoclonal colonies were cultured, the expressed proteins were screened for activity, and the dominant mutants were determined by gas-phase detection.

[0031] The sites for library mutagenesis 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 for them (the primer sequences are shown in Table 1). The mutants were constructed by a two-step PCR method, and the high-fidelity polymerase FastPfu-DNA was used for PCR. The PCR reaction conditions were as follows: in a 50 μL PCR reaction system, 5 μL of 10×Pfubuffer, 5 μL of dNTP (2 mM), 2 μL of MgSO4 (25 mM), 20 - 100 ng of template, 1 μL of each of a pair of mutant primers (10 μM), 1 μL of Pfu polymerase, and sterile distilled water was added to 50 μL. The reaction program for small-fragment PCR: (1) pre-denaturation at 95 °C for 2 min, (2) denaturation at 95 °C for 20 sec, (3) annealing at 55 °C for 20 sec, (4) extension at 72 °C for 12 sec, (5) final extension at 72 °C for 5 min, and steps (2) - (4) were carried out for 20 - 30 cycles. The reaction program for large-fragment PCR: (1) pre-denaturation at 95 °C for 2 min, (2) denaturation at 95 °C for 20 sec, (3) annealing at 60 °C for 50 sec, (4) extension at 72 °C for 4 min, (5) final extension at 72 °C for 5 min, and steps (2) - (4) were carried out for 20 - 30 cycles. Then the PCR products were stored at 4 °C. After the PCR products were analyzed and verified by agarose gel electrophoresis, restriction endonuclease DpnI was added and digested at 37 °C for 2 h. The digested products were transferred into E. coli BL21(DE3) competent cells and spread on plates containing ampicillin antibiotic, and were statically cultured in a 37 °C incubator for about 12 h. Single colonies were picked for sequencing, and the corresponding mutants were obtained when the sequencing was correct.

[0032] The obtained mutants were identified for activity through an analytical reaction system, and the reaction formula is as follows:

[0033]

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

[0035] Table 1 Primer sequences of mutation sites

[0036]

[0037]

[0038]

[0039] Table 2 Screening results of related mutants

[0040]

[0041]

[0042] Example 2: Induced Expression of Benzaldehyde Lyase Mutants

[0043] Single colonies of the genetically engineered bacteria of the above mutants were separately inoculated into 4 mL of LB liquid medium containing ampicillin antibiotic (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl), and cultured overnight in a shaker at 37 °C and 200 rpm to obtain seed solutions. The overnight cultured seed solutions were transferred to 50 mL of LB medium containing ampicillin antibiotic at an inoculation amount of 1%, 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 mixture was induced at 25 °C and 200 rpm for 8 - 12 h. The cells were collected by centrifugation at 4 °C and 6000 rpm. The cells were resuspended with potassium phosphate buffer (100 mM, pH 7.0), and disrupted by ultrasonic treatment or a high-pressure homogenizer. The supernatant (crude enzyme solution) was collected by centrifugation at 4 °C and 12000 rpm for subsequent SDS-PAGE and enzyme activity detection.

[0044] Example 3: Optimization of Reaction Conditions for Benzaldehyde Lyase Mutants

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

[0046] Table 3 Optimal Reaction Temperature of Benzaldehyde Lyase Mutants

[0047]

[0048] Using benzaldehyde lyase mutants M3 and M6 (50 mg / mL) as biocatalysts, 300 mM furfural, 360 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to 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 reactions were carried out on a shaker at 30 °C and 200 rpm for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by gas chromatography.

[0049] Table 4 Optimal Reaction pH of Benzaldehyde Lyase

[0050]

[0051] Example 4: Catalytic synthesis of α-hydroxymethyl ketones by benzaldehyde lyase mutants

[0052] The mutants M4_2, M5_1, M6, and M7 with relatively high activities, whose amino acid sequences are SEQ ID NO.6, 7, 9, and 10, were used respectively. After protein induction and expression according to the method of Example 2 for culturing and inducing expression, the 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 potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 50 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR. The detection results showed that the product 2-furyl hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M4_2, 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 potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 50 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR. The detection results showed that the product 2-furyl hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M5_1, 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 potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 50 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 24 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR. The detection results showed that the product 2-furyl hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, 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 added in a fed-batch manner to a potassium phosphate buffer solution containing 2.5 mM MgSO4 and 0.1 mM ThDP (pH 7.0, 100 mM), with a total reaction volume of 50 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 5 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR. The detection results were as follows: The product 2-furyl hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, and the furfural substrate conversion rate was 99%.

[0057] Using benzaldehyde lyase mutant M6 (25 mg / mL), 700 mM furfural (10% DMSO) and 840 mM formaldehyde were added in a fed-batch manner to a potassium phosphate buffer solution containing 2.5 mM MgSO4 and 0.1 mM ThDP (pH 7.0, 100 mM), with a total reaction volume of 50 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 7 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR. The detection results were as follows: The product 2-furyl hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, and the furfural substrate conversion rate was 99%.

[0058] Using benzaldehyde lyase mutant M7 (25 mg / mL), 1000 mM furfural (10% DMSO) and 1200 mM formaldehyde were added in a fed-batch manner to a potassium phosphate buffer solution containing 2.5 mM MgSO4 and 0.1 mM ThDP (pH 7.0, 100 mM), with a total reaction volume of 50 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR. The detection results were as follows: The product 2-furyl hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M7, and the furfural substrate conversion rate was 99%.

[0059] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM benzaldehyde (10% DMSO), 240 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 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR and the product was obtained by silica gel column separation and purification. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, 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 MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by separation and purification using a silica gel column. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 61%.

[0061] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-fluorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by separation and purification using a silica gel column. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 68%.

[0062] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-fluorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by separation and purification using a silica gel column. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 84%.

[0063] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-chlorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by separation and purification using a silica gel column. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 87%.

[0064] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-chlorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by silica gel column separation and purification. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 45%.

[0065] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-bromobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by silica gel column separation and purification. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 85%.

[0066] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-bromobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by silica gel column separation and purification. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 97%, and the product separation yield was 59%.

[0067] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-methylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, and the substrate conversion rate and product yield were detected by NMR, and the product was obtained by silica gel column separation and purification. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 75%.

[0068] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-methylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, the substrate conversion rate and product yield were detected by NMR, and the product was obtained by separation and purification using a silica gel column. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 88%.

[0069] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-trifluoromethylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, the substrate conversion rate and product yield were detected by NMR, and the product was obtained by separation and purification using a silica gel column. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 72%.

[0070] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-trifluoromethylbenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, the substrate conversion rate and product yield were detected by NMR, and the product was obtained by separation and purification using a silica gel column. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 46%, and the product separation yield was 33%.

[0071] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 3-methoxybenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 12 h. The reaction system was extracted with ethyl acetate, the substrate conversion rate and product yield were detected by NMR, and the product was obtained by silica gel column separation and purification. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 81%.

[0072] Using benzaldehyde lyase mutant M6 (50 mg / mL), 200 mM 4-fluoro, 3-chlorobenzaldehyde (10% DMSO), 240 mM formaldehyde, 2.5 mM MgSO4 and 0.1 mM ThDP were added to a potassium phosphate buffer solution (pH 7.0, 100 mM), and the total reaction volume was 20 mL. The reaction was carried out on a shaker at 30 °C and 200 rpm for 20 h. The reaction system was extracted with ethyl acetate, the substrate conversion rate and product yield were detected by NMR, and the product was obtained by silica gel column separation and purification. The detection results were as follows: The product α-hydroxymethyl ketone was obtained by the catalysis of benzaldehyde lyase mutant M6, the substrate conversion rate was 99%, and the product separation yield was 84%.

[0073] As shown above, benzaldehyde lyase mutants (50 mg / mL) M4_2, M5_1 and M6 can react with 300 mM furfural, and the conversion rate can reach more than 80%; benzaldehyde lyase mutant (25 mg / mL) M7 can react with up to 1000 mM furfural in a fed-batch mode, and the conversion rate can reach 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 reach more than 97%, and the product separation yield is medium to high (45%-88%).

Claims

1. A benzaldehyde lyase mutant, characterized in that The present invention is a benzaldehyde lyase mutant in which one or more of the amino acids 27, 29, 395, 417, 549, 551, 552 and 553 in the amino acids corresponding to positions 1 to 558 of SEQ ID NO. 1 are mutated.

2. The benzaldehyde lyase mutant according to claim 1, wherein The mutations are A27I; V29I; Y395L or Y395A; G417S; E549L or E549Y; V551S or V551L; I552L or I552M; M553L or M553I; Preferably, one of the following mutations is present in positions 1 to 558 of the amino acid sequence corresponding to SEQ ID NO. 1: A27I, V29I and G417S; A27I, V29I, Y395L and G417S; A27I, V29I, Y395L, G417S and I552L; A27I, V29I, G417S, I552L and M553L; A27I, V29I, G417S, E549L, I552L and M553L; A27I, V29I, G417S, E549L, V551S, I552L and M553L.

3. A gene encoding the benzaldehyde lyase mutant according to claim 1 or 2.

4. An expression vector containing a gene encoding the benzaldehyde lyase mutant according to claim 1 or 2.

5. A recombinant cell comprising a gene encoding the benzaldehyde lyase mutant according to claim 1 or 2.

6. Use of the benzaldehyde lyase mutant according to claim 1 or 2, or the gene encoding the benzaldehyde lyase mutant according to claim 3, in the preparation of α-hydroxymethyl ketone compounds.

7. A method for preparing α-hydroxymethyl ketone compounds, characterized in that: The product is obtained by carrying out a catalytic reaction using the benzaldehyde lyase mutant as claimed in claim 1 or 2 as a catalyst and furfural or aromatic benzaldehyde and formaldehyde as substrates.

8. The method according to claim 7, wherein The wet bacteria obtained by fermentation culture of an engineered bacterium expressing the gene encoding the benzaldehyde lyase mutant as claimed in claim 3 are used as catalysts, furfural or aromatic benzaldehyde and formaldehyde are used as substrates, and a buffer solution with a pH of 6.0-9.0 is used as a reaction medium. The catalytic reaction is carried out at 25°C-50°C.

9. The method according to claim 7 or 8, wherein In the catalytic reaction, the concentration of the catalytic substrate in the reaction system is 50-1200 mM, more preferably, the substrate concentration is 300-1000 mM; the amount of bacteria contained in the reaction system is 10-150 g / L, more preferably, 20-70 g / L; the pH of the reaction system is 6.0-9.0, and optimally 7.0; the reaction temperature is 25°C-50°C, and optimally 30°C.

10. The method according to claim 9, wherein ThDP and MgSO4 are also added to the reaction system, and the reaction is carried out at 150 rpm-250 rpm for 5-25 hours; more preferably, 0.1 mM ThDP and 2.5 mM MgSO4 are added, and the reaction is carried out on a shaker at 30°C and 200 rpm for 12 hours.

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