A process for the synthesis of furfuryl alcohol by a two-enzyme cascade

Abstract

The application discloses a method for synthesizing high-concentration furfuryl alcohol by using a double-enzyme coupling method, and the method comprises the following steps: mixing alcohol dehydrogenase freeze-dried bacterial powder and glucose dehydrogenase freeze-dried bacterial powder to serve as a catalyst, using furfural as a substrate, using glucose as a co-substrate, using NADP + or NAD + as a coenzyme, using a buffer solution with a pH value of 4-9 as a reaction medium to form a reaction system, and reacting completely under the condition of 20-50 DEG C and 400 rpm, then separating and purifying the reaction liquid to obtain furfuryl alcohol. The biological catalytic system is suitable for furfural with a concentration higher than 500 mM, and 100% conversion of 500 mM can be realized within 10 hours. The substrate continuous flow process is further used to eliminate the inhibition of aldehyde substrates on the biological catalyst, the concentration of the product can reach 1500 mM, and the highest space-time yield can reach 235 g / (L.d).

Description

(I) Technical Field

[0001] This invention belongs to the field of biocatalysis and relates to a method for synthesizing furfuryl alcohol by dual-enzyme coupling catalysis using furfural as a substrate. (II) Background Technology

[0002] Furfuryl alcohol is an important raw material for fine chemical organic synthesis, with its most widespread application in the production of foundry resins. It is also widely used in the synthesis of cold-resistant plasticizers, additives, rocket fuels, synthetic fibers, and rubber. Furthermore, furfuryl alcohol is an intermediate in the production of fragrances, flavorings, pharmaceuticals, and pesticides, such as in the synthesis of lysine, vitamin C, and levulinic acid. Furfuryl alcohol can be converted from furfural, which is mainly derived from biomass materials, including corn cobs, bagasse, corn stalks, sunflower hulls, oat hulls, cottonseed hulls, and rice hulls. Currently, the industrial production process for furfural is relatively mature, with abundant sources and low prices.

[0003] The production of furfural alcohol from furfural primarily utilizes chemical methods. However, these methods suffer from complex catalysts, require harsh conditions such as high temperature and pressure, involve organic solvents and toxic chemicals, and produce highly toxic and corrosive waste that is difficult to treat, causing serious environmental problems. Furthermore, furfural's C=C form is more easily reduced than its C=O form, making selective hydrogenation and minimizing byproduct production extremely challenging for chemical methods. Biological methods, with their superior chemoselectivity, can effectively address this challenge. Compared to chemical synthesis, enzymatic biosynthesis offers advantages such as high selectivity, mild reaction conditions, and high efficiency. In summary, the biocatalytic production of furfural alcohol from furfural has become a research hotspot, but research on enzymatic biosynthesis of furfural alcohol remains immature. Existing biocatalysts often fail to meet industrial application requirements due to low catalytic efficiency, low applicable substrate concentrations, and low coenzyme cycling efficiency.

[0004] To improve the atom economy of biocatalytic synthesis, the reaction design incorporates a coenzyme cycle system: the alcohol dehydrogenase YahK utilizes NADPH to catalyze the hydrogenation of furfural to produce furfuryl alcohol and NADP. + glucose dehydrogenase BmGDH M6 It then plays a role in driving the coenzyme cycle, utilizing glucose and NADP. + Gluconic acid and NADPH are produced. To improve the synthesis efficiency of furfuryl alcohol, various alcohol dehydrogenases (YsADH, AdhP, YahK, YjgB) and various glucose dehydrogenases (BmGDH) are used. M6 The optimal formulations (BsGDH, EsGDH) were selected. This biocatalytic system can tolerate high concentrations of furfural (≥500 mM) and achieves 100% conversion within 10 h. To further alleviate the inhibition of the biocatalyst by high concentrations of aldehyde substrates, a continuous constant-rate feeding process was adopted, resulting in higher space-time yield. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a method for the synthesis of furfuryl alcohol by a dual-enzyme coupling process, using furfural as a substrate and glucose as a co-substrate. The preferred alcohol dehydrogenase, YahK, utilizes NADPH to catalyze the hydrogenation of furfural to produce furfuryl alcohol and NADP. + The preferred glucose dehydrogenase BmGDH M6 It then plays a role in driving the coenzyme cycle, utilizing glucose and NADP. + The biocatalytic system produces gluconic acid and NADPH. It can tolerate high concentrations of furfural (≥500 mM) and achieves 100% conversion. A constant-rate continuous feed process further alleviates the inhibition of the biocatalyst by high-concentration aldehyde substrates, resulting in a product concentration of up to 1500 mM and a space-time yield of up to 235 g / (L·d). This method offers advantages such as high atom economy, high catalytic efficiency, applicability to high substrate concentrations, and high coenzyme cycling efficiency.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a method for the reduction of furfural to furfuryl alcohol using a dual-enzyme coupled catalytic process. The method comprises: inducing expression of engineered alcohol dehydrogenase and glucose dehydrogenase bacteria, respectively, and freeze-drying the wet bacterial cells; mixing the obtained freeze-dried alcohol dehydrogenase and glucose dehydrogenase bacterial powders as a catalyst; using furfural as a substrate, glucose as a co-substrate, and NADP... + or NAD + Using furfural as a coenzyme, a reaction system was constructed using a buffer solution with a pH of 4–9 as the reaction medium. After the reaction was completed at 20–50°C and 400 rpm, the reaction solution was separated and purified to obtain furfuryl alcohol. This biocatalytic system can tolerate high concentrations of furfural (≥500 mM) and achieves 100% conversion. During the reaction process, an automatic titration system was used to maintain a constant pH, and the alkaline solution used for titration was a 1 M NaOH aqueous solution.

[0008] Furthermore, the alcohol dehydrogenase genetically engineered bacteria are constructed by introducing alcohol dehydrogenase genes into Escherichia coli. These alcohol dehydrogenase genes include, but are not limited to, the alcohol dehydrogenase YsADH gene (nucleotide and amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively), the alcohol dehydrogenase AdhP gene (nucleotide and amino acid sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively), the alcohol dehydrogenase YahK gene (nucleotide and amino acid sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively), and the alcohol dehydrogenase YjgB gene (nucleotide and amino acid sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively).

[0009] Furthermore, the preferred alcohol dehydrogenase gene is the alcohol dehydrogenase YahK gene; the alcohol dehydrogenase YahK gene is derived from *Escherichia coli*, GenBank accession number WP_128491393.1. The *E. coli* genome was obtained using a TaKaLa genomic DNA purification kit, and the alcohol dehydrogenase YahK encoding gene was obtained from the *E. coli* genome using primers YahK-F and YahK-R. The method for constructing the engineered alcohol dehydrogenase strain is as follows: the alcohol dehydrogenase YahK gene shown in SEQ ID NO.5 is inserted into the BamHI and XhoI restriction enzyme sites of the pET28a vector to obtain the recombinant vector pET28a-YahK; the recombinant vector pET28a-YahK is introduced into the host cell *E. coli* BL21(DE3) to obtain the recombinant engineered strain *E. coli* BL21(DE3) / pET28a-YahK.

[0010] Furthermore, the glucose dehydrogenase genetically engineered bacteria were constructed by introducing the glucose dehydrogenase gene into *Escherichia coli*; the glucose dehydrogenase gene includes, but is not limited to, the glucose dehydrogenase BmGDH with the nucleotide sequence shown in SEQ ID NO. 9. M6 The nucleotide sequences of glucose dehydrogenase BsGDH (SEQ ID NO. 11) and glucose dehydrogenase EsGDH (SEQ ID NO. 13) are shown.

[0011] Furthermore, the glucose dehydrogenase gene is preferably glucose dehydrogenase BmGDH. M6 Gene; the glucose dehydrogenase BmGDH M6 A mutant with the gene BmGDH, derived from Bacillus megaterium (GenBank accession number AAA22475). M6 The nucleotide and amino acid sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively, and the gene synthesis services were provided by Hangzhou Qingke Biotechnology Co., Ltd.

[0012] Furthermore, the glucose dehydrogenase BmGDH... M6 The genetically engineered bacteria are those that produce glucose dehydrogenase BmGDH. M6 The gene was introduced into *E. coli* to construct the gene. Specifically, the glucose dehydrogenase BmGDH shown in SEQ ID NO.9 was introduced into the gene. M6 The coding gene was inserted into the BamHI and XhoI restriction sites of the pET28a vector to obtain the recombinant vector pET28a-BmGDH. M6 The recombinant vector pET28a-BmGDH M6Recombinant genetically engineered bacteria E. coli BL21(DE3) / pET28a-BmGDH were obtained by introducing the bacteria into host cells E. coli BL21(DE3) / pET28a-BmGDH. M6 .

[0013] Furthermore, the lyophilized alcohol dehydrogenase powder and the lyophilized glucose dehydrogenase powder are mixed at a mass ratio of 0.2-5:1, preferably 1:1.

[0014] Furthermore, in the reaction system, the catalyst addition amount is 20-40 g / L (preferably 30 g / L); the final concentration of the substrate furfural is 500-1500 mM (preferably 500 mM), and the furfural to glucose concentration ratio is 1:0.5-2.5 (preferably 1:1); the final concentration of the coenzyme is 0-0.5 mM (preferably 0.2 mM), and the coenzyme is preferably NADP. + .

[0015] Furthermore, the reaction time is 6 to 30 hours, the reaction temperature is preferably 30°C, and the reaction medium is preferably a 50 mM Tris-HCl buffer solution with pH 7.0.

[0016] When the substrate addition amount is no higher than 900 mM (preferably 500–900 mM), the substrate and co-substrate are added to the reaction system at once. When the substrate addition amount is higher than 900 mM (preferably 1000–1500 mM), in order to further alleviate the inhibition of the biocatalyst by high concentrations of aldehyde substrates and obtain higher space-time yields, the substrate and co-substrate are added in a constant-rate continuous feeding manner. The fed substrate is furfural stock solution, and the fed co-substrate is a 1.2–1.8 M glucose aqueous solution. The feeding time is 1–15 h, and the reaction continues for 0–15 h after the feeding is completed. They are fed at the same rate, so that the concentration ratio of substrate to co-substrate in the reaction system is 1:0.5–2.5 (preferably 1:1), and the cumulative substrate concentration is 500–1500 mM. Preferably, the substrate is fed at a rate of 16.7–501.0 μmol / min.

[0017] Furthermore, in a 10 mL reaction system, the constant flow rate of furfural and glucose was 16.7 μmol / min, the flow time was 15 h, and the reaction continued for another 15 h after the flow was completed; in a 300 mL reaction system, the constant flow rate of furfural and glucose was 501.0 μmol / min, and the reaction was stopped after 10 h of flow.

[0018] Furthermore, the lyophilized bacterial powders of alcohol dehydrogenase and glucose dehydrogenase were prepared as follows: engineered bacteria of alcohol dehydrogenase (preferably E. coli BL21(DE3) / pET28a-YahK) and engineered bacteria of glucose dehydrogenase (preferably E. coli BL21(DE3) / pET28a-BmGDH) were prepared respectively. M6 The cells were inoculated into LB liquid medium containing a final concentration of 100 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. Then, they were transferred at a volume concentration of 2% to LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm until the bacterial concentration reached OD500. 600 To obtain an induction culture solution, add IPTG to the culture at a final concentration of 0-0.6 mM (preferably 0.2 mM) to a final concentration of 0.6-0.8, and induce culture at 12-26℃ (preferably 24℃) for 12 h. Centrifuge the induction culture solution at 4℃ and 8000 rpm for 10 min, discarding the supernatant. Resuspend the bacterial cells in 50 mM Tris-HCl buffer (pH 8.0), centrifuge at 4℃ and 8000 rpm for 10 min, discard the supernatant, and collect the wet bacterial cells. Pre-freeze the obtained wet bacterial cells at -20℃ for two days, then freeze-dry them at -40℃ for 48 h in a freeze dryer to obtain lyophilized alcohol dehydrogenase powder and lyophilized glucose dehydrogenase powder (preferably lyophilized alcohol dehydrogenase YahK powder and glucose dehydrogenase BmGDH powder). M6 (Freeze-dried bacterial powder).

[0019] Furthermore, the reaction solution separation and purification method is as follows: the reaction solution is centrifuged at 12000 rpm for 10 min, the supernatant is collected, 4 times the volume of ethyl acetate is added, and the mixture is extracted at 200 rpm and 30℃ for 1 h. After extraction, the mixture is centrifuged at 12000 rpm for 10 min, and the upper organic phase is collected. Since the reaction conversion rate is >99%, there is almost no substrate residue. The upper organic phase is subjected to vacuum rotary evaporation to remove ethyl acetate, and the product furfuryl alcohol is obtained directly.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following: The present invention provides a method for the synthesis of furfuryl alcohol by dual-enzyme coupling catalysis, using furfural as a substrate and glucose as a co-substrate, utilizing alcohol dehydrogenase YahK to catalyze the hydrogenation of furfural with NADPH to generate furfuryl alcohol and NADP. + glucose dehydrogenase BmGDH M6 It then plays a role in driving the coenzyme cycle, utilizing glucose and NADP. + The production of gluconic acid and NADPH ( Figure 1When the substrate is added in a single step, this reaction system is suitable for furfural concentrations above 500 mM, with 100% conversion achieved within 10 hours at 500 mM substrate. A constant-rate continuous feed process further alleviates the inhibition of the biocatalyst by the aldehyde substrate, resulting in a cumulative product concentration of up to 1500 mM and a space-time yield of up to 235 g / (L·d). No byproducts were detected in the reaction system, indicating that the established reaction system exhibits excellent chemoselectivity. (iv) Description of the attached drawings

[0021] Figure 1 This is a schematic diagram of a two-enzyme coupled catalytic synthesis method for furfural alcohol using furfural as the substrate and glucose as the co-substrate.

[0022] Figure 2 SDS-PAGE gel images of the culture medium of the genetically engineered bacteria before and after induction in Example 3; from left to right, lane M, Blue plus II protein marker; lane 1, induced genetically engineered bacteria E. coli BL21(DE3) / pET28a-AdhP, the thick band corresponds to AdhP, with a molecular weight of 35 kDa; lane 2, induced genetically engineered bacteria E. coli BL21(DE3) / pET28a-YahK, the thick band corresponds to YahK, with a molecular weight of 38 kDa; lane 3, induced genetically engineered bacteria E. coli BL21(DE3) / pET28a-YsADH, the thick band corresponds to YsADH, with a molecular weight of 36 kDa; lane 4, induced genetically engineered bacteria E. coli BL21(DE3) / pET28a-YjgB, the bold band corresponds to YjgB, with a molecular weight of 37 kDa; Lane 5, uninduced E. coli BL21(DE3) / pET28a-YahK; Lane 6, induced E. coli BL21(DE3) / pET28a-BsGDH, the bold band corresponds to BsGDH, with a molecular weight of 28 kDa; Lane 7, induced E. coli BL21(DE3) / pET28a-EsGDH, the bold band corresponds to EsGDH, with a molecular weight of 28 kDa; Lane 8, induced E. coli BL21(DE3) / pET28a-BmGDH M6 The bolded band corresponds to BmGDH. M6 Its molecular weight is 28 kDa.

[0023] Figure 3 This is the standard curve for measuring protein concentration using the BCA method in Example 4.

[0024] Figure 4The gas chromatogram is shown in step 1 of Example 5; the standard samples are furfuryl alcohol (8.4 min) and furfural (9.0 min).

[0025] Figure 5 This is a bar graph showing the conversion rates of furfuryl alcohol synthesized by different alcohol dehydrogenases in step 1 of Example 5.

[0026] Figure 6 This is a bar graph showing the conversion rate of furfuryl alcohol synthesized under different glucose dehydrogenase and coenzyme conditions in step 2 of Example 5.

[0027] Figure 7 This is a graph showing the conversion rate of alcohol dehydrogenase YahK at different induction temperatures in Example 6.

[0028] Figure 8 This is a graph showing the conversion rate of alcohol dehydrogenase YahK under different amounts of inducer added in Example 7.

[0029] Figure 9 The conversion curves for the synthesis of furfuryl alcohol using furfural as a substrate via dual-enzyme coupling catalysis at different temperatures are shown in Example 8.

[0030] Figure 10 The conversion curves for the synthesis of furfural alcohol based on coenzyme self-circulation using furfural as a substrate are shown in Example 9 at different pH values.

[0031] Figure 11 Example 10: Different coenzymes NADP + Conversion curve of furfural-based dual-enzyme coupled synthesis of furfuryl alcohol at different addition amounts.

[0032] Figure 12 The conversion curves for the synthesis of furfuryl alcohol by dual-enzyme coupling catalysis with furfuryl as substrate under different furfuryl and glucose concentration ratios are shown in Example 11.

[0033] Figure 13 Example 12: Different alcohol dehydrogenases YahK and glucose dehydrogenase BmGDH M6 Conversion curve of furfural-based dual-enzyme coupled synthesis of furfuryl alcohol at a mass ratio.

[0034] Figure 14 This is a graph showing the reaction progress at different substrate concentrations during the dual-enzyme coupled catalytic synthesis of furfuryl alcohol in Example 13.

[0035] Figure 15 This is a flow chart of the feedstock process for the synthesis of furfuryl alcohol via dual-enzyme coupling catalysis in Example 14.

[0036] Figure 16 The following are the gas phase results for the reaction solution and possible by-product standards of Example 14: (a) reaction solution; (b) furoic acid standard; (c) tetrahydrofurfuryl alcohol standard.

[0037] Figure 17 This is the product accumulation process of the scaled-up reaction in Example 15.

[0038] Figure 18 The images show the gas chromatography-mass spectra of the substrate furfural (a) and the product furfuryl alcohol (b) from Example 15. (V) Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0040] LB liquid medium composition: tryptic peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, water as solvent, pH adjusted to 7.0–7.5 with 1M NaOH. Autoclave at 121℃ for 20 min, store at 4℃.

[0041] Example 1: Obtaining the gene encoding alcohol dehydrogenase

[0042] 1. Obtaining the gene encoding alcohol dehydrogenase YsADH

[0043] The artificially synthesized YsADH encoding gene of alcohol dehydrogenase derived from Yorkshire (Yokenella sp. WZY002) has been disclosed in patent application CN201310188883.9, with the nucleotide and amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0044] 2. Obtaining the gene encoding alcohol dehydrogenase AdhP

[0045] The genome of *E. coli* BL21(DE3) was obtained using a TaKaLa genomic DNA small-scale purification kit. The gene encoding the alcohol dehydrogenase AdhP was obtained from the *E. coli* genome using primers AdhP-F and AdhP-R (GenBank accession number EFJ66826.1). The nucleotide and amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The primers were as follows: AdhP-F: 5'-CAAATGGGTCGCGGATCCATGAAGGCTGCAGTTGTTACGAA-3'; AdhP-R: 5'-GGTGGTGGTGGTGCTCGAGGTGACGGAAATCAATCACCATG-3'.

[0046] 3. Obtaining the gene encoding alcohol dehydrogenase YahK

[0047] The genome of *E. coli* BL21(DE3) was obtained using a small-scale purification kit for TaKaLa genomic DNA. The gene encoding the alcohol dehydrogenase YahK was obtained from the *E. coli* genome using primers YahK-F and YahK-R (GenBank accession number WP_128491393.1). The nucleotide and amino acid sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The primers were as follows: YahK-F: 5'-CAAATGGGTCGCGGATCCATGAAGATCAAAGCTGTTGGTGC-3'; YahK-R: 5'-GGTGGTGGTGGTGCTCGAGTCAGTCTGTTAGTGTGCGATTATCG-3'.

[0048] SEQ ID NO.6

[0049] MKIKAVGAYSAKQPLEPMDITRREPGPNDVKIEIAYCGVCHSDLHQVRSEWAGTVYPCVPGHEIVGRVVAVGDQVEKYAPGDLVGVGCIVDSCKHCEECEDGLENYCDHMTGTYNSPTPDEPGHTLGGYSQQIVVHERYVLRIRHPQEQLAAVAPLLCAGITTYSPLRHWQAGPG KKVGVVGIGGLGHMGIKLAHAMGAHVVAFTTSEAKREAAKALGADEVVNSRNADEMAAHLKSFDFILNTVAAPHNLDDFTTLLKRDGTMTLVGAPATPHKSPEVFNLIMKRRAIAGSMIGGIPETQEMLDFCAEHGIVADIEMIRADQINEAYERMLRGDVKYRFVIDNRTLTD&.

[0050] 4. Obtaining the gene encoding the alcohol dehydrogenase YjgB

[0051] The genome of *E. coli* BL21(DE3) was obtained using a TaKaLa genomic DNA small-scale purification kit. The gene encoding the alcohol dehydrogenase YjgB was obtained from the *E. coli* genome using primers YjgB-F and YjgB-R. The gene has GenBank accession number AAA97166.1, and its nucleotide and amino acid sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The primers are as follows: YjgB-F: 5'-CAAATGGGTCGCGGATCCATGTCGATGATAAAAAGCTACGCC-3'; YjgB-R: 5'-GGTGGTGGTGGTGCTCGAGTCAGAAATCGGCTTTCAGCAC-3'.

[0052] Example 2: Obtaining the glucose dehydrogenase encoding gene

[0053] 1. Glucose dehydrogenase BmGDH M6 Acquisition of coding genes

[0054] The glucose dehydrogenase BmGDH from Bacillus megaterium has a GenBank accession number of AAA22475. A mutant BmGDH was obtained by performing multiple substitutions at Q252L / E170K / S100P / K166R / V72I / K137R on the amino acid sequence of glucose dehydrogenase BmGDH. M6 (Disclosed in patent application 2020103075429). Glucose dehydrogenase mutant BmGDH M6 The coding gene was artificially synthesized after codon optimization (gene synthesis services were provided by Hangzhou Qingke Biotechnology Co., Ltd.), and the nucleotide and amino acid sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0055] SEQ ID NO.10

[0056] MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDIINLVQSAIKEFGKLDVMINNAGLENPVPSHEMSLSDWNKVIDTNLTGAFLGSREAIKYF VENDIRGTVINMSSVHEKIPWPLFVHYAASKGGMRLMTKTLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG&.

[0057] 2. Obtaining the gene encoding glucose dehydrogenase BsGDH

[0058] The glucose dehydrogenase BsGDH encoding gene, derived from Bacillus subtilis (GenBank accession number AFQ56330.1), was artificially synthesized after codon optimization (gene synthesis services were provided by Hangzhou Qingke Biotechnology Co., Ltd.). The nucleotide and amino acid sequences are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0059] 3. Obtaining the gene encoding glucose dehydrogenase EsGDH

[0060] The glucose dehydrogenase EsGDH encoding gene, derived from Escherichia coli (Exiguobacterium sibiricum), with GenBank accession number KM817194.1, was artificially synthesized after codon optimization (gene synthesis services were provided by Hangzhou Qingke Biotechnology Co., Ltd.). The nucleotide and amino acid sequences are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0061] Example 3: Preparation of wet cells and lyophilized bacterial powders of engineered bacteria expressing alcohol dehydrogenase and glucose dehydrogenase

[0062] 1. Construction of genetically engineered bacteria expressing alcohol dehydrogenase and glucose dehydrogenase

[0063] Engineered bacteria expressing alcohol dehydrogenase genes: The genes encoding alcohol dehydrogenase YsADH, AdhP, YahK, and YjgB were inserted between the BamH I and XhoI sites on plasmid pET28a, respectively, to obtain recombinant plasmids pET28a-YsADH, pET28a-AdhP, pET28a-YahK, and pET28a-YjgB. These recombinant plasmids were then introduced into competent E. coli BL21(DE3) cells to obtain engineered bacteria E. coli BL21(DE3) / pET28a-YsADH, E. coli BL21(DE3) / pET28a-AdhP, E. coli BL21(DE3) / pET28a-YahK, and E. coli BL21(DE3) / pET28a-YjgB.

[0064] Genetically engineered bacteria expressing glucose dehydrogenase: BmGDH glucose dehydrogenase... M6The encoding genes for glucose dehydrogenase (BsGDH) and glucose dehydrogenase (EsGDH) were inserted between the BamHI and XhoI sites on plasmid pET28a, respectively, to obtain the recombinant plasmid pET28a-BmGDH. M6 pET28a-BsGDH and pET28a-EsGDH were used to introduce recombinant plasmids into competent E. coli BL21(DE3) cells to obtain engineered E. coli BL21(DE3) / pET28a-BmGDH. M6 , E.coli BL21(DE3) / pET28a-BsGDH, E.coli BL21(DE3) / pET28a-EsGDH.

[0065] Sequencing of the extracted plasmids from the above-mentioned engineered bacteria showed that the genes for each alcohol dehydrogenase and glucose dehydrogenase were correctly inserted.

[0066] 2. Preparation of wet cell culture of engineered bacteria expressing alcohol dehydrogenase and engineered bacteria expressing glucose dehydrogenase

[0067] The wet bacterial cells were prepared as follows: The genetically engineered bacteria E. coli BL21(DE3) / pET28a-YsADH, E. coli BL21(DE3) / pET28a-AdhP, E. coli BL21(DE3) / pET28a-YahK, E. coli BL21(DE3) / pET28a-YjgB, and E. coli BL21(DE3) / pET28a-BmGDH were used. M6 E. coli BL21(DE3) / pET28a-BsGDH and E. coli BL21(DE3) / pET28a-EsGDH were inoculated into LB liquid medium containing a final concentration of 100 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. Then, they were transferred at a volume concentration of 2% to LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm until the bacterial concentration reached OD500. 600 To obtain an induction culture medium, add IPTG to a final concentration of 0.2 mM to a pH of 0.6–0.8, and incubate at 24 °C for 12 h. Under the same conditions, use the culture medium without added IPTG as the uninduced control culture medium. Centrifuge the induction culture medium at 4 °C and 8000 rpm for 10 min, discarding the supernatant. Resuspend the bacterial cells in 50 mM Tris-HCl buffer (pH 8.0), centrifuge at 4 °C and 8000 rpm for 10 min, discard the supernatant, and collect the wet bacterial cells as a biocatalyst, storing them at -20 °C for later use.

[0068] Preparation of SDS-PAGE samples: Take 1 mL each of the uninduced control culture medium and the induced culture medium, centrifuge at 12000 rpm for 1 min, discard the supernatant, and retain the bacterial cells. Add 100 μL of ultrapure water to each bacterial cell to resuspend the bacterial weight. Then, take 20 μL of each bacterial suspension, add 4 μL of 6x Protein Loading Buffer, mix well, and boil for 10 min. After boiling, centrifuge at 12000 rpm for 1 min, and take 15 μL of the supernatant from each for SDS-PAGE detection. The protein marker is BluePlus Protein Marker (14-120 kDa). Figure 2 As shown, SDS-PAGE analysis revealed the presence of alcohol dehydrogenases YsADH, AdhP, YahK, YjgB, and BmGDH. M6 Both glucose dehydrogenase BsGDH and glucose dehydrogenase EsGDH were successfully expressed in Escherichia coli.

[0069] 3. Preparation of freeze-dried bacterial powders of engineered bacteria expressing alcohol dehydrogenase and engineered bacteria expressing glucose dehydrogenase

[0070] The wet bacterial cells obtained in step 2 were pre-frozen at -20℃ for two days, and then freeze-dried at -40℃ for 48 hours in a freeze dryer to obtain freeze-dried bacterial powders expressing alcohol dehydrogenases (specifically, alcohol dehydrogenases YsADH, AdhP, YahK, and YjgB) and glucose dehydrogenases (specifically, glucose dehydrogenase BmGDH). M6 (glucose dehydrogenase BsGDH, glucose dehydrogenase EsGDH).

[0071] Example 4: Specific enzyme activity assay of alcohol dehydrogenase

[0072] 1. Preparation of crude enzyme solution of alcohol dehydrogenase

[0073] 10 mL of 50 mM Tris-HCl buffer (pH 7.0) was added to every 0.06 g of the lyophilized bacterial powder expressing alcohol dehydrogenase prepared in Example 3. The mixture was stirred with a glass rod to form a bacterial suspension. The suspension was then sonicated for 15 min at 0°C on ice, with a 2-second sonication interval followed by a 4-second pause, at a power of 210 W. The sonicated bacterial suspension was then centrifuged at 8000 rpm at 4°C for 10 min. The resulting supernatant was the crude enzyme solution and was stored at 4°C for later use.

[0074] 2. Determination of the volumetric activity of alcohol dehydrogenase

[0075] The enzyme activity of alcohol dehydrogenase was calculated by measuring the change in absorbance of NADPH at 340 nm using a single-factor kinetic method with an ELISA reader. The enzyme activity assay system added to the ELISA plate consisted of: 10 mM furfural, 0.1 mM NADPH, 15 μL of crude enzyme solution, and 300 μL of 50 mM Tris-HCl buffer (pH 7.0). After incubation at 30°C for 5 min, the absorbance at 340 nm was measured using an ELISA reader. The enzyme activity unit U was defined as the conversion of 1 μmol of NADPH to NADP per minute at 30°C. + Required enzyme amount. Perform three parallel experiments each time, and calculate the mean and standard error. The volumetric enzyme activity of alcohol dehydrogenase is calculated as shown in Formula 1.

[0076] Formula 1:

[0077] D: Dilution factor, 1; A1: Sample absorbance; A2: Blank control absorbance; V t The total reaction volume is 300 μL.

[0078] e: molar absorptivity, a constant of 6220; V s : Enzyme solution volume, 15 μL; d: Optical path length, 1 cm.

[0079] 3. Determination of protein concentration of alcohol dehydrogenase

[0080] A protein concentration standard curve was plotted using the BCA method protein concentration assay kit, with protein concentration on the x-axis and absorbance on the y-axis. Figure 3 As shown, the measured linear relationship formula is y = 0.0011x + 0.1648, where y is the absorbance value at 562 nm, x is the protein concentration of the BSA solution (mg / mL), and the standard deviation is R. 2 =0.999.

[0081] When measuring the protein concentration of crude alcohol dehydrogenase solutions using the BCA protein concentration assay kit, three parallel experiments were performed each time, and the mean and standard error were calculated. The protein concentrations of crude alcohol dehydrogenase YahK solution were determined to be 5.90 mg / mL; crude alcohol dehydrogenase YsADH solution was 3.84 mg / mL; crude alcohol dehydrogenase AdhP solution was 4.83 mg / mL; and crude alcohol dehydrogenase YjgB solution was 5.33 mg / mL.

[0082] 4. Compared with enzyme activity

[0083] The specific enzyme activity can be obtained by calculating the ratio of enzyme activity to protein concentration. The specific enzyme activity of crude alcohol dehydrogenase YsADH was calculated to be 433.45 U / g, that of crude alcohol dehydrogenase AdhP was 399.61 U / g, that of crude alcohol dehydrogenase YahK was 526.92 U / g, and that of crude alcohol dehydrogenase YjgB was 297.83 U / g.

[0084] Example 5: Construction of the initial reaction system for the synthesis of furfuryl alcohol by dual-enzyme coupling catalysis using furfural as a substrate.

[0085] 1. Comparison of different alcohol dehydrogenases catalyzing the synthesis of furfuryl alcohol

[0086] In Example 3, the lyophilized bacterial powders of alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK, and alcohol dehydrogenase YjgB were respectively mixed with glucose dehydrogenase BmGDH. M6 The lyophilized bacterial powder was mixed in a 1:1 mass ratio as a catalyst and added to a 50mM Tris-HCl buffer solution at pH 7.0. Furfural was used as the substrate, glucose as the co-substrate, and NADP coenzyme was added. + The total reaction volume was 10 mL. During the reaction, an automatic titration system was used to maintain a constant pH, and the alkali solution used for titration was a 1 M NaOH aqueous solution.

[0087] The initial 10 mL reaction system without optimized conditions was as follows: final concentration of furfural substrate 500 mM, final concentration of glucose co-substrate 1250 mM, and coenzyme NADP. + Final concentration 0.2 mM, lyophilized bacterial powder containing alcohol dehydrogenase (YsADH, AdhP, YahK, or YjgB) and glucose dehydrogenase (BmGDH). M6 The amount of dried bacterial powder added was 15 g / L. The lyophilized alcohol dehydrogenase bacterial powder and glucose dehydrogenase lyophilized bacterial powder were mixed at a mass ratio of 1:1 (0.15 g: 0.15 g) and added to 50 mM Tris-HCl buffer at pH 7.0. The mixture was reacted at pH 7.0, 400 rpm, and 30 °C for 6 h. Under the same conditions, uninduced lyophilized alcohol dehydrogenase bacterial powder served as a blank control.

[0088] After the reaction was complete, 200 μL of the reaction solution was centrifuged at 12000 rpm for 3 min. 100 μL of the supernatant was then extracted with 1 mL of ethyl acetate for 30 min. After extraction, the mixture was centrifuged at 12000 rpm for 1 min, and 200 μL of the upper organic phase was collected. The content of each component in the sample was determined using gas chromatography. Three parallel experiments were performed each time, and the mean and standard error were calculated.

[0089] The gas chromatography conditions were as follows: Gas chromatograph, Agilent 6890N; Chiral column, BGB-174 (column length 30m, column inner diameter 250μm, stationary phase coating thickness 0.25μm); Detector, FID, 250℃; Carrier gas, N2; Carrier gas flow rate, 1mL / min; Split ratio: 1:20; Injection volume: 1.0μL; Injector temperature: 250℃. The analysis of furfural, furfuryl alcohol, and possible byproducts furoic acid and tetrahydrofurfuryl alcohol employed a specific temperature program: 80℃ for 3 min, increased to 190℃ at 10℃ / min, held at 190℃ for 6 min, for a total of 20 min. Figure 4 As shown, the retention times of furfuryl alcohol and furfural were 8.4 min and 9.0 min, respectively.

[0090] The results are as follows Figure 5 As shown, the conversion rate of furfural in the blank control was 7.9%; the conversion rate of furfural by alcohol dehydrogenase YahK was 68.8%; the conversion rate of furfural by alcohol dehydrogenase YsADH was 60.0%; the conversion rate of furfural by alcohol dehydrogenase AdhP was 53.6%; and the conversion rate of furfural by alcohol dehydrogenase YjgB was 26.8%. Therefore, alcohol dehydrogenase YahK is preferred.

[0091] 2. Comparison of furfuryl alcohol synthesis catalyzed by different glucose dehydrogenases and coenzymes.

[0092] The alcohol dehydrogenase YahK lyophilized bacterial powder prepared in Example 3 was compared with the glucose dehydrogenase lyophilized bacterial powder (glucose dehydrogenase BmGDH). M6 Glucose dehydrogenase (BsGDH or EsGDH) was mixed in a 1:1 mass ratio as a catalyst, and 50 mM Tris-HCl buffer at pH 7.0 was used as the reaction medium. The coenzyme NADP was added. + or NAD + Other operations and reaction conditions are the same as in step 1, forming a total reaction system of 10 mL. Under the same conditions, uninduced glucose dehydrogenase lyophilized bacterial powder is used as a blank control.

[0093] The results are as follows Figure 6 As shown, when NADP is selected as the coenzyme + At that time, glucose dehydrogenase BmGDH M6 The conversion rate of furfural was 68.75%, the conversion rate of glucose dehydrogenase BsGDH was 61.92%, the conversion rate of glucose dehydrogenase EsGDH was 39.72%, and the conversion rate of furfural in the blank control was 21.33%. When NAD+ was selected as the coenzyme... + At that time, glucose dehydrogenase BmGDH M6The conversion rate of furfural was 54.96%, while that of glucose dehydrogenase BsGDH was 45.76%, and that of glucose dehydrogenase EsGDH was 35.03%. The conversion rate of furfural in the blank control was 17.27%. Therefore, glucose dehydrogenase BmGDH is the preferred enzyme. M6 Preferred coenzyme NADP + .

[0094] Example 6: Optimal temperature for inducing expression of alcohol dehydrogenase YahK

[0095] The engineered bacteria expressing the alcohol dehydrogenase YahK were prepared into lyophilized bacterial powder according to the method in Example 3, except that the induction temperature was set to 12–36℃ (12℃, 16℃, 20℃, 24℃, 28℃, 32℃, and 36℃ were selected), and lyophilized bacterial powders of the alcohol dehydrogenase YahK were prepared at different induction temperatures. The catalytic reaction was carried out according to the operation and reaction system in step 1 of Example 5. Three parallel experiments were performed each time, and the average value and standard error were calculated. The results are as follows: Figure 7 As shown, the optimal induction temperature is 24℃.

[0096] Example 7: Optimal IPTG dosage for inducing YahK alcohol dehydrogenase expression

[0097] The engineered bacteria expressing alcohol dehydrogenase YahK were prepared into lyophilized bacterial powder according to the method in Example 3, except that the amount of IPTG added to induce expression was set to 0–0.6 mM (0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, and 0.6 mM), and lyophilized bacterial powders of alcohol dehydrogenase YahK with different amounts of inducer were prepared. The catalytic reaction was carried out according to the operation and reaction system in step 1 of Example 5. Three parallel experiments were performed each time, and the average value and standard error were calculated. The results are as follows: Figure 8 As shown, the optimal amount of IPTG as the inducing agent is 0.2 mM.

[0098] Example 8: Optimal temperature for the synthesis of furfuryl alcohol via dual-enzyme coupling catalysis using furfural as a substrate

[0099] The alcohol dehydrogenase YahK lyophilized bacterial powder and glucose dehydrogenase BmGDH prepared by the method in Example 3 were selected. M6 The freeze-dried bacterial powder was used as a catalyst, and the reaction system of step 1 in Example 5 was used for the catalytic reaction. The difference was that the temperature of the reaction system was set to 20–50°C (selected as 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C), while other operations were the same as in Example 5. Three sets of parallel experiments were performed each time, and the average value and standard error were calculated. The results are as follows: Figure 9 As shown, the optimal reaction temperature is 30℃.

[0100] Example 9: Optimal pH of the reaction system for the synthesis of furfural alcohol by dual-enzyme coupling catalysis using furfural as a substrate

[0101] The alcohol dehydrogenase YahK lyophilized bacterial powder and glucose dehydrogenase BmGDH prepared by the method in Example 3 were selected. M6 The lyophilized bacterial powder was used as a catalyst, and the reaction system of step 1 in Example 5 was used for the catalytic reaction. The difference was that the pH of the reaction system was set to 4–9 (4, 5, 6, 7, 8, and 9) in a 50 mM Tris-HCl buffer solution. Other operations and the reaction system were the same as in Example 5. Three parallel experiments were performed each time, and the average value and standard error were calculated. The results are as follows: Figure 10 As shown, the conversion rate is higher when the pH is neutral or weakly acidic, and reaches as high as 68.75% when the pH is 7. The optimal reaction pH is 7 and the buffer solution is 50 mM Tris-HCl.

[0102] Example 10: The optimal coenzyme NADP for the two-enzyme coupled catalytic synthesis of furfural alcohol using furfural as a substrate. + Added amount

[0103] The alcohol dehydrogenase YahK lyophilized bacterial powder and glucose dehydrogenase BmGDH prepared by the method in Example 3 were selected. M6 The lyophilized bacterial powder was used as a catalyst, and the reaction system of step 1 in Example 5 was used for the catalytic reaction, the difference being the use of the coenzyme NADP. + The final concentration was set to 0–0.5 mM (selecting 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mM), and other operations and reaction systems were the same as in Example 5. Three parallel experiments were performed each time, and the average value and standard error were calculated. The results are as follows: Figure 11 As shown, when NADP + Without addition, the conversion rate of furfural is 36.26%, NADP + When the concentration is between 0-0.5 mM, NADP + Increasing the concentration of NADP promotes the reaction; at a concentration of 0.2 mM, the conversion rate is 68.75%. + When the concentration of NADP is greater than 0.2 mM, the increase in conversion rate is not significant. Therefore, 0.2 mM NADP is used in the reaction. + Relatively economical.

[0104] Example 11: Optimal furfural to glucose concentration ratio in the reaction system for the synthesis of furfuryl alcohol using furfural as a substrate via dual-enzyme coupling catalysis.

[0105] The alcohol dehydrogenase YahK lyophilized bacterial powder and glucose dehydrogenase BmGDH prepared by the method in Example 3 were selected. M6The freeze-dried bacterial powder was used as a catalyst, and the reaction system of step 1 in Example 5 was used for the catalytic reaction. The difference was that the final concentration ratio of furfural to glucose was set to 1:0.5–2.5 (selecting 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5), where the final concentration of furfural was 500 mM. Other operations and the reaction system were the same as in Example 5. Three sets of parallel experiments were performed each time, and the average value and standard error were calculated. The results are as follows: Figure 12 As shown, increasing the amount of glucose as a co-substrate improves the conversion rate of furfural. When the final concentration ratio of furfural to glucose is 1:1, the conversion rate of furfural is 61.66%. Further increases in glucose concentration do not significantly increase the conversion rate. Therefore, using a 1:1 final concentration ratio of furfural to glucose is economical in the reaction.

[0106] Example 12: Alcohol dehydrogenase YahK and glucose dehydrogenase BmGDH in the dual-enzyme coupled catalytic synthesis of furfural as a substrate for furfuryl alcohol. M6 Optimal mass ratio

[0107] The alcohol dehydrogenase YahK lyophilized bacterial powder and glucose dehydrogenase BmGDH prepared by the method in Example 3 were selected. M6 The lyophilized bacterial powder was used as a catalyst, and the reaction system of step 1 in Example 5 was used for the catalytic reaction. The difference was that the alcohol dehydrogenase YahK and glucose dehydrogenase BmGDH were added to the reaction system. M6 The mass ratio of the freeze-dried bacterial powder was set to 0.2–5:1 (selecting 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, and 5:1), and other operations and reaction systems were the same as in Example 5. Three parallel experiments were performed each time, and the average value and standard error were calculated. The results are as follows: Figure 13 As shown, the optimal alcohol dehydrogenase YahK and glucose dehydrogenase BmGDH for the catalytic system M6 The mass ratio of the lyophilized bacterial powder was 1:1, meaning that in a 10 mL system, the ratio of alcohol dehydrogenase YahK to glucose dehydrogenase BmGDH was 1:1. M6 Add the ingredients in a ratio of 0.15g:0.15g.

[0108] Example 13: Reaction process at different substrate concentrations during the synthesis of furfuryl alcohol via dual-enzyme coupling catalysis

[0109] The alcohol dehydrogenase YahK lyophilized bacterial powder and glucose dehydrogenase BmGDH prepared by the method in Example 3 were selected. M6 The freeze-dried bacterial powder was used as a catalyst, and the reaction system of step 1 in Example 5 was used for the catalytic reaction, except that the final substrate concentrations were 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, and 1000 mM, and the final furfural to glucose concentration ratio was 1:1. Gas phase analysis was performed at regular intervals, and the reaction time was 24 hours. All other operations were the same as in Example 5. Figure 14As shown, at a substrate concentration of 500 mM, furfural can be completely converted to furfuryl alcohol in 10 h; at a substrate concentration of 600 mM, the conversion rate of furfural is 88.05% in 24 h. With the increase of furfural concentration, high concentrations of aldehyde substrates inhibit the catalyst, and the conversion rate decreases. At a substrate concentration of 1000 mM, furfural is hardly converted.

[0110] Example 14: Synthesis of high-concentration furfuryl alcohol by a dual-enzyme coupled catalytic process combined with a substrate feed process

[0111] The lyophilized bacterial powder expressing alcohol dehydrogenase YahK and the glucose dehydrogenase BmGDH expressed were prepared using the method in Example 3. M6 The lyophilized bacterial powder was used as a catalyst, and the reaction system of step 1 in Example 5 was used for catalytic reaction. The difference was that furfural and glucose were added continuously at a constant rate using a micro-injection pump (Baoding Qili Constant Flow Pump Co., Ltd.). The substrate added was furfural stock solution, and the co-substrate added was 1.8M glucose aqueous solution. The constant flow rate of furfural and glucose was 16.7 μmol / min, the final concentration ratio was 1:1, and the addition was stopped after 15 h. The reaction continued for 30 h, and samples were taken periodically for gas phase detection. The results are as follows: Figure 15 As shown, the conversion rate of furfural was >99% from 1 to 10 hours, indicating complete conversion of the added furfural, during which time furfuryl alcohol steadily increased. With continued furfural addition, from 11 to 15 hours, furfural substrate accumulation gradually began, reducing the biocatalyst's conversion efficiency and inhibiting its activity. At 15 hours, the overall furfural conversion rate was 74.65%. After 15 hours, the addition was stopped, but the reaction continued. The accumulated furfural was gradually consumed, indicating that the biocatalyst remained active; at this point, the furfural added in the first 15 hours was completely converted to furfuryl alcohol. Gas chromatography analysis confirmed the results. Figure 16 As shown, the final reaction solution contained no substrate furfural or byproducts furoic acid and tetrahydrofurfuryl alcohol, indicating that furfural was completely converted to furfuryl alcohol and that the established process has excellent chemoselectivity. Calculations showed that 0.015 mol of furfural was completely converted to furfuryl alcohol in the 10 mL reaction system, with a cumulative product concentration of 1500 mM.

[0112] Example 15: Scale-up and product identification of high-concentration furfuryl alcohol synthesis using a dual-enzyme coupled catalytic process combined with substrate feed.

[0113] The alcohol dehydrogenase YahK lyophilized bacterial powder and glucose dehydrogenase BmGDH prepared by the method in Example 3 were selected. M6 4.5g of freeze-dried bacterial powder was mixed at a mass ratio of 1:1 to serve as a catalyst, with a final catalyst concentration of 30g / L. The final concentration ratio of furfural to glucose was 1:1. NADP coenzyme was also added. +A 300 mL reaction system was prepared using 50 mM Tris-HCl buffer solution (0.2 mM, pH 7.0). The reaction temperature was 30 °C, pH 7.0, and the rotation speed was 400 rpm. Furfural and glucose were added continuously at a constant rate using a micro-injection pump (Baoding Qili Constant Flow Pump Co., Ltd.). The substrate added was stock furfural, and the co-substrate was 1.2 M glucose aqueous solution. The constant flow rate for both furfural and glucose was 501.0 μmol / min. The reaction was stopped after 10 h of addition, and samples were taken periodically for gas chromatography analysis. The results are as follows: Figure 17 As shown, the furfural conversion rate was >99% from 1 to 10 h. It was calculated that a total of 0.3 mol of furfural (28.82 g) was converted into furfuryl alcohol (29.43 g) in the 300 mL reaction system, and the cumulative concentration of the product reached 1000 mM. At this time, the space-time yield of furfuryl alcohol was about 235 g / (L·d).

[0114] After the scale-up reaction was completed, the reaction solution was centrifuged at 12,000 rpm for 10 min, the supernatant was collected, and 4 times the volume of ethyl acetate was added. Extraction was carried out at 200 rpm and 30 °C for 1 h. After extraction, the solution was centrifuged at 12,000 rpm for 10 min, and the upper organic phase was collected. Since the reaction conversion rate was >99%, there were almost no substrate and intermediate product residues. The upper organic phase was then subjected to vacuum rotary evaporation to remove the ethyl acetate, directly yielding the product.

[0115] Gas chromatography-mass spectrometry analysis confirmed that the substrate and product in the reaction solution were furfural and furfuryl alcohol, respectively. Figure 18 As shown in Figures a and b. The gas chromatograph-mass spectrometer was an Agilent 7890A / 5975C. The gas chromatographic conditions were as in Example 5 (except for the FID detector, which was not required). The mass spectrometry detection conditions were as follows: auxiliary heater temperature, 250°C; MS quadrupole temperature, 150°C; ion source temperature, 230°C; mass spectrometry scan range, 30-500 amu; emission current, 200 μA; electron energy, 70 eV.

Claims

1. A process for the synthesis of furfuryl alcohol by a two-enzyme cascade, characterized in that, The method involves: inducing the expression of engineered alcohol dehydrogenase and engineered glucose dehydrogenase bacteria separately, and then freeze-drying the wet bacterial cells. The resulting freeze-dried alcohol dehydrogenase and glucose dehydrogenase bacterial powders are mixed at a mass ratio of 1:1 and used as a catalyst. Furfural is used as the substrate, glucose as the co-substrate, and NADP is used as the catalyst. ＋ Using a coenzyme, a reaction system was constructed with a buffer solution of pH 6-7 as the reaction medium. After the reaction was completed at 30-35℃ and 400 rpm, the reaction solution was separated and purified to obtain furfuryl alcohol. The engineered alcohol dehydrogenase strain was constructed by introducing the alcohol dehydrogenase gene into Escherichia coli; the alcohol dehydrogenase gene is the alcohol dehydrogenase YahK gene with the nucleotide sequence shown in SEQ ID NO.5; The glucose dehydrogenase genetically engineered bacterium is constructed by introducing a glucose dehydrogenase gene into Escherichia coli; the glucose dehydrogenase gene is a glucose dehydrogenase BmGDH whose nucleotide sequence is shown in SEQ ID NO. 9 M6 ; In the reaction system, the catalyst is added at a concentration of 20-40 g / L; the final concentration of the substrate furfural is added at 500-900 mM; the furfural to glucose concentration ratio is 1:1; and the final concentration of the coenzyme is added at 0.2 mM.

2. The method of claim 1, wherein, The reaction medium is a 50 mM Tris-HCl buffer solution with a pH of 7.

0.

3. The method of claim 1, wherein, The substrate was added as a furfural stock solution, and the co-substrate was added as a 1.2-1.8 M glucose aqueous solution.

4. The method of claim 1, wherein, The lyophilized bacterial powders of alcohol dehydrogenase and glucose dehydrogenase were prepared as follows: The engineered bacteria of alcohol dehydrogenase and glucose dehydrogenase were inoculated into LB liquid medium containing a final concentration of 100 μg / mL kanamycin, and cultured overnight at 37°C and 200 rpm. Then, they were transferred at a volume concentration of 2% to LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm until the bacterial cell concentration reached OD500. 600 To obtain an induction culture solution, add IPTG to the culture at a final concentration of 0-0.6 mM to a final concentration of 0-0.6 mM, and induce culture at 12-26℃ for 12 h. Then, centrifuge the induction culture solution at 4℃ and 8000 rpm for 10 min and discard the supernatant. Resuspend the bacterial cells in 50 mM Tris-HCl buffer at pH 8.0, centrifuge at 4℃ and 8000 rpm for 10 min, discard the supernatant, and collect the wet bacterial cells. Pre-freeze the obtained wet bacterial cells at -20℃ for two days, and then freeze-dry them at -40℃ for 48 h in a freeze dryer to obtain lyophilized alcohol dehydrogenase and lyophilized glucose dehydrogenase bacterial powders, respectively.

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