CN115992190A - Method for synthesizing furfuryl alcohol by double-enzyme coupling - Google Patents

Abstract

The invention discloses a method for synthesizing high-concentration furfuryl alcohol by double enzyme coupling, which uses alcohol dehydrogenase freeze-dried bacterial powder and glucose dehydrogenase freeze-dried bacterial powder as a catalyst after being mixed, uses furfural as a substrate, uses glucose as an auxiliary substrate and uses NADP ⁺ Or NAD ⁺ The reaction system is formed by taking buffer solution with pH value of 4-9 as a reaction medium, and furfuryl alcohol is obtained after the reaction is completed under the conditions of 20-50 ℃ and 400rpm, and the reaction solution is separated and purified. The biocatalysis system is applicable to furfural with concentration higher than 500mM,500mM achieves 100% conversion within 10 h. The inhibition of aldehyde substrates to the biocatalyst is further removed by adopting a substrate continuous flow processing technology, the concentration of the product can reach 1500mM, and the space-time yield can reach 235 g/(L.d) at the highest.

Description

Method for synthesizing furfuryl alcohol by double-enzyme coupling

Field of the art

The invention belongs to the field of biocatalysis, and relates to a method for synthesizing furfuryl alcohol by double-enzyme coupling catalysis with furfural as a substrate.

(II) background art

Furfuryl alcohol is an important fine chemical organic synthetic raw material, and the furfuryl alcohol has the most wide application in the production of casting resin. Furfuryl alcohol is also widely used in the synthesis of cold-resistant plasticizers, additives, rocket fuels, synthetic fibers, rubber, and the like. Furfuryl alcohol is also an intermediate in the production of fragrances, perfumes, medicines, pesticides, such as chemical intermediates for the synthesis of lysine, vitamin C and levulinic acid. Furfuryl alcohol may be converted from furfural, which is primarily derived from biomass material, including: corncob, bagasse, corn cob, sunflower hull, oat hull, cotton seed hull, rice hull, etc. At present, the industrial production process of furfural is mature, the source is rich, and the price is low.

The process of producing furfuryl alcohol with furfural has multiple chemical processes, however, the catalyst of the chemical process is complex, the chemical reaction needs harsh conditions such as high temperature, high pressure and the like, and involves organic solvents and toxic chemicals, and extremely toxic and corrosive waste residues are difficult to treat, so that serious environmental problems are caused. In addition, c=c of furfural is more easily reduced than c=o, how to effectively perform selective hydrogenation, reducing the production of byproducts, is very challenging for chemical processes, which biological processes can effectively cope with based on their excellent chemical selectivity. Compared with chemical synthesis methods, the biological enzyme method has the advantages of high selectivity, mild reaction and high efficiency. In general, biological catalysis of furfural to furfuryl alcohol has become a research hotspot, but the research on synthesis of furfuryl alcohol by biological enzyme method has not yet been completed. The existing biocatalyst often cannot realize industrialized application due to low catalytic efficiency, low concentration of applicable substrate and low recycling efficiency of coenzyme.

In order to improve the atom economy of biocatalysis synthesis, the reaction design fuses a coenzyme circulation system: alcohol dehydrogenase YahK catalyzes furfural hydrogenation to furfuryl alcohol and NADP using NADPH ⁺ While glucose dehydrogenase BmGDH _M6 Then act to drive the circulation of the coenzyme, utilizing glucose and NADP ⁺ Gluconic acid and NADPH are produced. In order to increase the synthesis efficiency of furfuryl alcohol, various alcohol dehydrogenases (YsADH, adhP, yahK, yjgB) and various glucose dehydrogenases (BmGDH) _M6 BsGDH, esGDH) are preferred. The biocatalysis system can tolerate high-concentration furfural (more than or equal to 500 mM) and realize 100 percent conversion rate within 10 hours. In order to further remove the inhibition of the high-concentration aldehyde substrate to the biocatalyst, a continuous constant-speed fed-batch process is adopted, so that higher space-time yield is obtained.

(III) summary of the invention

The invention aims to provide a method for synthesizing furfuryl alcohol by double enzyme coupling, which takes furfural as a substrate and glucose as an auxiliary substrate, and an alcohol dehydrogenase YahK which is optimized catalyzes furfuryl alcohol hydrogenation to generate furfuryl alcohol and NADP by utilizing NADPH ⁺ And preferably glucose dehydrogenase BmGDH _M6 Then act to drive the circulation of the coenzyme, utilizing glucose and NADP ⁺ Gluconic acid and NADPH are produced. The biocatalysis system can tolerate high-concentration furfural (more than or equal to 500 mM) and realize 100 percent conversion rate. The inhibition of the high-concentration aldehyde substrate to the biocatalyst is further relieved by adopting a constant-speed continuous flow processing technology, the concentration of the product reaches 1500mM, and the space-time yield can reach 235 g/(L.d) at the highest. The method has high atom economy and catalytic effectHigh rate, high concentration of applicable substrate, high coenzyme circulation efficiency and the like.

The technical scheme adopted by the invention is as follows:

the invention provides a method for synthesizing furfuryl alcohol by reducing furfuraldehyde under the catalysis of double enzyme coupling, which comprises the following steps: respectively carrying out induced expression on alcohol dehydrogenase genetic engineering bacteria and glucose dehydrogenase genetic engineering bacteria, freeze-drying wet bacteria, mixing the obtained alcohol dehydrogenase freeze-dried bacteria powder and glucose dehydrogenase freeze-dried bacteria powder, taking furfural as a substrate, glucose as an auxiliary substrate and NADP as a catalyst ⁺ Or NAD ⁺ The reaction system is formed by taking buffer solution with pH value of 4-9 as a reaction medium, and furfuryl alcohol is obtained after the reaction is completed under the conditions of 20-50 ℃ and 400rpm, and the reaction solution is separated and purified. The biocatalysis system can tolerate high-concentration furfural (more than or equal to 500 mM) and realize 100 percent conversion rate. In the reaction process of the invention, an automatic titration system is utilized to maintain the pH constant, and the alkali liquor used for titration is 1M NaOH aqueous solution.

Further, the alcohol dehydrogenase gene engineering bacteria are constructed by introducing alcohol dehydrogenase genes into escherichia coli, wherein the alcohol dehydrogenase genes comprise, but are not limited to, alcohol dehydrogenase YsADH genes (nucleotide sequences and amino acid sequences are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2), alcohol dehydrogenase AdhP genes (nucleotide sequences and amino acid sequences are respectively shown in SEQ ID NO.3 and SEQ ID NO. 4), alcohol dehydrogenase YahK genes (nucleotide sequences and amino acid sequences are respectively shown in SEQ ID NO.5 and SEQ ID NO. 6), and alcohol dehydrogenase YjgB genes (nucleotide sequences and amino acid sequences are respectively shown in SEQ ID NO.7 and SEQ ID NO. 8).

Further, it is preferable that the alcohol dehydrogenase gene is an alcohol dehydrogenase YahK gene; the alcohol dehydrogenase YahK gene is derived from escherichia coli, the GenBank accession number is WP_128491393.1, the escherichia coli genome is obtained by using a small purification kit of TaKaLa genome DNA, and the primer YahK-F, yahK-R is used for obtaining the alcohol dehydrogenase YahK coding gene from the escherichia coli genome. The construction method of the alcohol dehydrogenase genetic engineering bacteria comprises the following steps: inserting an alcohol dehydrogenase YahK gene shown in SEQ ID NO.5 into BamH I and Xho I restriction enzyme cutting sites of the pET28a vector to obtain a recombinant vector pET28a-YahK; and (3) introducing the recombinant vector pET28a-YahK into a host cell E.coli BL21 (DE 3) to obtain recombinant genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-YahK.

Further, the glucose dehydrogenase genetically engineered bacterium is constructed by introducing a glucose dehydrogenase gene into escherichia coli; the glucose dehydrogenase gene comprises, but is not limited to, glucose dehydrogenase BmGDH with a nucleotide sequence shown as SEQ ID NO.9 _M6 Glucose dehydrogenase BsGDH with nucleotide sequence shown as SEQ ID NO.11, and glucose dehydrogenase EsGDH with nucleotide sequence shown as SEQ ID NO. 13.

Further, it is preferable that the glucose dehydrogenase gene is glucose dehydrogenase BmGDH _M6 A gene; the glucose dehydrogenase BmGDH _M6 Mutant with BmGDH gene from bacillus megatherium (Bacillus megaterium), genBank accession number of AAA22475, bmGDH _M6 The nucleotide sequence and the amino acid sequence of the polypeptide are respectively shown as SEQ ID NO.9 and SEQ ID NO.10, and the gene synthesis service is provided by the Hangzhou qing department biotechnology Co.

Further, the glucose dehydrogenase-containing BmGDH _M6 The engineering bacteria of the gene are glucose dehydrogenase BmGDH _M6 And (3) constructing and obtaining the gene-introduced escherichia coli. The method comprises the following steps: the glucose dehydrogenase BmGDH shown in SEQ ID NO.9 _M6 The encoding gene is inserted into BamH I and Xho I restriction enzyme cutting sites of pET28a vector to obtain recombinant vector pET28a-BmGDH _M6 The method comprises the steps of carrying out a first treatment on the surface of the Recombinant vector pET28a-BmGDH _M6 Introducing into host cell E.coli BL21 (DE 3) to obtain recombinant genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-BmGDH _M6 。

Further, the alcohol dehydrogenase freeze-dried bacterial powder and the glucose dehydrogenase freeze-dried bacterial powder are mixed according to a mass ratio of 0.2-5:1, preferably 1:1.

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

Further, the reaction time is 6 to 30 hours, preferably the reaction temperature is 30 ℃, and the reaction medium is preferably 50mM Tris-HCl buffer solution with pH 7.0.

When the addition amount of the substrate is not more than 900mM (preferably 500 to 900 mM), the substrate and the cosubstrate are added to the reaction system at one time; when the substrate addition amount is higher than 900mM (preferably 1000-1500 mM), in order to further remove the inhibition of high-concentration aldehyde substrates on the biocatalyst and obtain higher space-time yield, the substrates and the auxiliary substrates are respectively added in a constant-speed continuous flow adding mode, the fed-batch substrate is furfural stock solution, and the fed-batch auxiliary substrate is a glucose aqueous solution with the concentration of 1.2-1.8M; the feeding time is 1-15 h, and the reaction is continued for 0-15 h after the feeding is finished; the substrates are fed at the same rate so that the concentration ratio of the substrates to the cosubstrate added to the reaction system is 1:0.5-2.5 (preferably 1:1), and the cumulative substrate addition concentration is 500-1500mM. Preferably, the substrate is fed at a rate of 16.7-501.0. Mu. Mol/min.

Further, in a 10mL reaction system, the constant flow acceleration of the furfural and the glucose is 16.7 mu mol/min, the feeding time is 15h, and the reaction is continued for 15h after the feeding is finished; in a 300mL reaction system, constant flow acceleration of furfural and glucose is 501.0 mu mol/min, and the reaction is stopped after 10h of feeding.

Further, the alcohol dehydrogenase freeze-dried bacterial powder and the glucose dehydrogenase freeze-dried bacterial powder are prepared according to the following method: alcohol dehydrogenase genetically engineered bacteria (preferably E.coli BL21 (DE 3)/pET 28 a-YahK) and glucose dehydrogenase genetically engineered bacteria (preferably E.coli BL21 (DE 3)/pET 28 a-BmGDH) are respectively prepared _M6 ) Inoculating into LB liquid medium containing 100 μg/mL kanamycin, culturing at 37deg.C and 200rpm for overnight, transferring into LB liquid medium containing 100 μg/mL kanamycin at 2% by volume, and culturing at 37deg.C and 200rpm to cell concentration OD ₆₀₀ Adding IPTG with a final concentration of 0-0.6 mM (preferably 0.2 mM) to the culture to 0.6-0.8, and performing induction culture at 12-26 ℃ (preferably 24 ℃) for 12h to obtain an induction culture solution; centrifuging the induced culture solution at 4deg.C and 8000rpm for 10min, and discarding supernatant; then, the cells were resuspended in 50mM Tris-HCl buffer, pH 8.0, centrifuged at 8000rpm at 4℃for 10min, the supernatant was discarded, and the wet cells were collected; to be obtainedPre-freezing wet thallus for two days at-20deg.C, lyophilizing at-40deg.C for 48 hr to obtain alcohol dehydrogenase lyophilized powder and glucose dehydrogenase lyophilized powder (preferably alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH) _M6 Freeze-dried bacterial powder).

Further, the reaction liquid separation and purification method comprises the following steps: centrifuging the reaction solution at 12000rpm for 10min, collecting supernatant, adding ethyl acetate with volume 4 times of that of the reaction solution, extracting at 200rpm and 30deg.C for 1 hr, centrifuging at 12000rpm for 10min after extraction, and collecting upper organic phase; because the reaction conversion rate is more than 99%, almost no substrate remains, and the ethyl acetate is removed from the upper organic phase by vacuum rotary evaporation, so that the product furfuryl alcohol is directly obtained.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for synthesizing furfuryl alcohol by double enzyme coupling catalysis, which takes furfural as a substrate, glucose as an auxiliary substrate, and alcohol dehydrogenase YahK is utilized to catalyze furfuryl alcohol and NADP by utilizing NADPH to catalyze furfuryl alcohol hydrogenation ⁺ While glucose dehydrogenase BmGDH _M6 Then act to drive the circulation of the coenzyme, utilizing glucose and NADP ⁺ Gluconic acid and NADPH were produced (FIG. 1). When the substrate is added at one time, the reaction system is suitable for furfural concentration higher than 500mM, and 100% conversion is realized within 10h for 500mM substrate. The inhibition of aldehyde substrates to the biocatalyst is further removed by adopting a constant-speed continuous flow processing technology, the accumulation concentration of the product can reach 1500mM, and the space-time yield can reach 235 g/(L.d) at the highest. No by-product was detected in the reaction system, indicating that the established reaction system has excellent chemoselectivity.

(IV) description of the drawings

FIG. 1 is a schematic diagram of a method for synthesizing furfuryl alcohol by double enzyme coupling catalysis with furfural as a substrate and glucose as an auxiliary substrate.

FIG. 2 is a SDS-PAGE gel of the culture medium of the genetically engineered bacterium of example 3 before and after induction; left to right, lanes M, blue plus II protein marker; lane 1, induced genetically engineered bacterium e.coli BL21 (DE 3)/pET 28a-AdhP, the bolded band corresponds to AdhP, the molecular weight of which is 35kDa; lane 2, the induced genetically engineered E.coli BL21 (DE 3)/pET 28a-YahK, bolded band corresponds toIs YahK, and has a molecular weight of 38kDa; lane 3, the induced genetically engineered bacterium e.coli BL21 (DE 3)/pET 28 a-ysaldh, the bolded band corresponds to ysaldh, with a molecular weight of 36kDa; lane 4, induced genetically engineered bacterium e.coli BL21 (DE 3)/pET 28a-YjgB, bolded bands corresponding to YjgB, having a molecular weight of 37kDa; lane 5, uninduced genetically engineered bacterium e.coli BL21 (DE 3)/pET 28a-YahK; lane 6, induced genetically engineered bacterium e.coli BL21 (DE 3)/pET 28a-BsGDH, bolded bands corresponding to BsGDH, with a molecular weight of 28kDa; lane 7, the induced genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-EsGDH, the bolded band corresponds to EsGDH, and the molecular weight is 28kDa; lane 8, induced genetically engineered E.coli BL21 (DE 3)/pET 28a-BmGDH _M6 The thickened bands correspond to BmGDH _M6 The molecular weight is 28kDa.

FIG. 3 is a standard curve of protein concentration by BCA method in example 4.

FIG. 4 is a gas chromatogram of example 5, step 1; standard furfuryl alcohol (8.4 min) and furfural (9.0 min).

FIG. 5 is a bar graph showing the conversion of furfuryl alcohol by the catalytic synthesis of different alcohol dehydrogenases in step 1 of example 5.

FIG. 6 is a bar graph showing the conversion of furfuryl alcohol by catalytic synthesis under different glucose dehydrogenases and different coenzymes in step 2 of example 5.

FIG. 7 is a graph showing the conversion rate of alcohol dehydrogenase YahK at different induction temperatures in example 6.

FIG. 8 is a graph showing the conversion of alcohol dehydrogenase YahK at various inducer additions in example 7.

FIG. 9 is a graph showing the conversion rate of furfuryl alcohol synthesized by double enzyme-coupled catalysis using furfuraldehyde as a substrate at different temperatures in example 8.

FIG. 10 is a graph showing the conversion rate of furfuryl alcohol synthesized by self-circulation using furfuraldehyde as a substrate based on coenzyme at different pH values in example 9.

FIG. 11 shows the different coenzymes NADP of example 10 ⁺ And under the addition amount, furfuraldehyde is used as a substrate to synthesize the conversion rate curve of furfuryl alcohol by double enzyme coupling catalysis.

FIG. 12 is a graph showing the conversion rate of furfuryl alcohol synthesized by double enzyme-coupled catalysis using furfural as a substrate in different concentration ratios of furfural to glucose in example 11.

FIG. 13 shows various alcohol dehydrogenases YahK and glucose dehydrogenase BmGDH of example 12 _M6 And the conversion rate curve of furfuryl alcohol is synthesized by using furfural as a substrate through double-enzyme coupling catalysis under the mass ratio.

FIG. 14 is a graph showing the progress of the reaction at various substrate concentrations for the dual enzyme-coupled catalytic synthesis of furfuryl alcohol of example 13.

FIG. 15 is a flow chart showing the process of synthesizing furfuryl alcohol by double enzyme-coupled catalysis in example 14.

FIG. 16 shows the gas phase results of the reaction solution and possible by-product standards of example 14; (a) a reaction solution; (b) a furoic acid standard; (c) tetrahydrofurfuryl alcohol standard.

FIG. 17 shows the progress of product accumulation in the amplification reaction of example 15.

FIG. 18 is a gas-mass spectrometry (GC-MS) spectrum of the substrate furfural (a) and the product furfuryl alcohol (b) of example 15.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

LB liquid medium composition: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl and water as solvent, and regulating the pH value to 7.0-7.5 by using 1M NaOH. Autoclaving at 121deg.C for 20min and storing at 4deg.C.

Example 1: acquisition of alcohol dehydrogenase encoding Gene

1. Acquisition of alcohol dehydrogenase YsADH coding Gene

The published gene encoding alcohol dehydrogenase YsADH from Joker's bacteria (Yokenella sp. WZY 002) has been synthesized artificially, genBank accession No. KF887947.1, and has been published in patent application CN201310188883.9, and the nucleotide sequence and amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. Acquisition of alcohol dehydrogenase AdhP coding Gene

E.coli BL21 (DE 3) genome was obtained using a small purification kit of TaKaLa genomic DNA. The primer AdhP-F, adhP-R is used to obtain alcohol dehydrogenase AdhP encoding gene from coligenome, genBank accession No. EFJ66826.1, and the nucleotide sequence and amino acid sequence 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'.

3. Acquisition of alcohol dehydrogenase YahK encoding Gene

E.coli BL21 (DE 3) genome was obtained using a small purification kit of TaKaLa genomic DNA. The primer YahK-F, yahK-R is utilized to obtain an alcohol dehydrogenase YahK coding gene from the escherichia coli genome, the GenBank accession number is WP_128491393.1, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6. The primers were as follows: yahK-F:5'-CAAATGGGTCGCGGATCCATGAAGATCAAAGCTGTTGGTGC-3'; yahK-R:5'-GGTGGTGGTGGTGCTCGAGTCAGTCTGTTAGTGTGCGATTATCG-3'.

SEQ ID NO.6

MKIKAVGAYSAKQPLEPMDITRREPGPNDVKIEIAYCGVCHSDLHQVRSEWAGTVYPCVPGHEIVGRVVAVGDQVEKYAPGDLVGVGCIVDSCKHCEECEDGLENYCDHMTGTYNSPTPDEPGHTLGGYSQQIVVHERYVLRIRHPQEQLAAVAPLLCAGITTYSPLRHWQAGPGKKVGVVGIGGLGHMGIKLAHAMGAHVVAFTTSEAKREAAKALGADEVVNSRNADEMAAHLKSFDFILNTVAAPHNLDDFTTLLKRDGTMTLVGAPATPHKSPEVFNLIMKRRAIAGSMIGGIPETQEMLDFCAEHGIVADIEMIRADQINEAYERMLRGDVKYRFVIDNRTLTD&。

4. Acquisition of alcohol dehydrogenase YjgB encoding Gene

E.coli BL21 (DE 3) genome was obtained using a small purification kit of TaKaLa genomic DNA. The primer YjgB-F, yjgB-R is utilized to obtain an alcohol dehydrogenase YjgB coding gene from the escherichia coli genome, the GenBank accession number is AAA97166.1, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8. The primers were as follows: yjgB-F:5'-CAAATGGGTCGCGGATCCATGTCGATGATAAAAAGCTACGCC-3'; yjgB-R:5'-GGTGGTGGTGGTGCTCGAGTCAGAAATCGGCTTTCAGCAC-3'.

Example 2: acquisition of glucose dehydrogenase encoding Gene

1. Glucose dehydrogenase BmGDH _M6 Acquisition of coding Gene

GenBank accession of glucose dehydrogenase BmGDH from Bacillus megaterium (Bacillus megaterium)The accession number is AAA22475, and the mutant BmGDH is obtained by carrying out Q252L/E170K/S100P/K166R/V72I/K137R multi-site substitution on the glucose dehydrogenase BmGDH amino acid sequence _M6 (disclosed in patent application 2020103075429). For glucose dehydrogenase mutant BmGDH _M6 The coding gene is synthesized artificially after codon optimization (the Hangzhou qingke biotechnology Co., ltd. Provides gene synthesis service), and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10.

SEQ ID NO.10

MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDIINLVQSAIKEFGKLDVMINNAGLENPVPSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIRGTVINMSSVHEKIPWPLFVHYAASKGGMRLMTKTLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG&。

2. Acquisition of Gene encoding glucose dehydrogenase BsGDH

The gene encoding glucose dehydrogenase BsGDH from bacillus subtilis (Bacillus subtilis) has the GenBank accession number of AFQ56330.1, and is artificially synthesized after codon optimization (the gene synthesis service is provided by the Qingzhou department biotechnology Co., ltd.) and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12.

3. Acquisition of the Gene encoding glucose dehydrogenase EsGDH

The coding gene of glucose dehydrogenase EsGDH from escherichia coli (Exiguobacterium sibiricum), genBank accession number is KM817194.1, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID NO.13 and SEQ ID NO.14 after codon optimization and artificial synthesis (the gene synthesis service is provided by Qingzhou department biotechnology Co., ltd.).

Example 3: preparation of engineering bacteria for expressing alcohol dehydrogenase gene and wet thallus and freeze-dried bacterial powder for expressing glucose dehydrogenase gene engineering bacteria

1. Construction of engineering bacteria for expressing alcohol dehydrogenase and engineering bacteria for expressing glucose dehydrogenase

Expression of alcohol dehydrogenase genetically engineered bacteria: the alcohol dehydrogenase YsADH encoding gene, the alcohol dehydrogenase AdhP encoding gene, the alcohol dehydrogenase YahK encoding gene and the alcohol dehydrogenase YjgB encoding gene are respectively inserted between BamH I and Xho I sites on the plasmid pET28a to obtain recombinant plasmids pET28a-YsADH, pET28a-AdhP, pET28a-YahK and pET28a-YjgB. The recombinant plasmids are respectively introduced into competent cells E.coli BL21 (DE 3) to obtain engineering bacteria E.coli BL21 (DE 3)/pET 28a-YsADH, E.coli BL21 (DE 3)/pET 28a-AdhP, E.coli BL21 (DE 3)/pET 28a-YahK and E.coli BL21 (DE 3)/pET 28a-YjgB.

Expressing glucose dehydrogenase genetically engineered bacteria: glucose dehydrogenase BmGDH _M6 The coding gene, glucose dehydrogenase BsGDH coding gene and glucose dehydrogenase EsGDH coding gene are respectively inserted between BamH I and Xho I sites on plasmid pET28a to obtain recombinant plasmid pET28a-BmGDH _M6 pET28a-BsGDH, pET28a-EsGDH. The recombinant plasmids are respectively introduced into competent cells E.coli BL21 (DE 3) to obtain engineering bacteria E.coli BL21 (DE 3)/pET 28a-BmGDH _M6 、E.coli BL21(DE3)/pET28a-BsGDH、E.coli BL21(DE3)/pET28a-EsGDH。

The sequencing of the extracted plasmid of each engineering bacterium shows that each alcohol dehydrogenase and glucose dehydrogenase gene are inserted without error.

2. Preparation of genetically engineered bacterium for expressing alcohol dehydrogenase and wet bacterium for expressing glucose dehydrogenase

The wet cell is prepared as follows: the genetically engineered bacteria E.coli BL21 (DE 3)/pET 28a-YsADH, E.coli BL21 (DE 3)/pET 28a-AdhP, E.coli BL21 (DE 3)/pET 28a-YahK, E.coli BL21 (DE 3)/pET 28a-YjgB and E.coli BL21 (DE 3)/pET 28a-BmGDH are prepared _M6 E.coli BL21 (DE 3)/pET 28a-BsGDH and E.coli BL21 (DE 3)/pET 28a-EsGDH were inoculated into LB liquid medium containing kanamycin at a final concentration of 100. Mu.g/mL, cultured overnight at 37℃at 200rpm, then transferred into LB liquid medium containing kanamycin at 100. Mu.g/mL at an inoculum size of 2% by volume, and cultured at 37℃at 200rpm to a cell concentration OD ₆₀₀ To 0.6-0.8, IPTG with a final concentration of 0.2mM was added to the culture, and the culture was induced at 24℃for 12 hours to obtain an induction culture solution. Under the same conditions, the uninduced control culture medium was a culture medium without addition of IPTG. Centrifuging the induced culture solution at 4deg.C and 8000rpm for 10min, and discarding supernatantA liquid; then, the cells were resuspended in 50mM Tris-HCl buffer, pH 8.0, centrifuged at 8000rpm at 4℃for 10min, the supernatant was discarded, and the wet cells were collected as biocatalysts and stored at-20℃for further use.

Preparation of SDS-PAGE detection samples: taking 1mL of each of the uninduced control culture solution and the induced culture solution, centrifuging at 12000rpm for 1min, discarding the supernatant, and reserving the thalli. The cells were then resuspended to a bacterial suspension by adding 100. Mu.L of ultrapure water to each cell. Then, 20. Mu.L of each bacterial suspension was added and mixed with 4. Mu.L of 6x Protein Loading Buffer, and the mixture was boiled for 10 minutes. After boiling was completed, the mixture was centrifuged at 12000rpm for 1min, and 15. Mu.L of each supernatant was used for SDS-PAGE detection, and the protein Marker was BluePlus Protein Marker (14-120 kDa). As shown in FIG. 2, SDS-PAGE shows that alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK, alcohol dehydrogenase YjgB and glucose dehydrogenase BmGDH _M6 Glucose dehydrogenase BsGDH and glucose dehydrogenase EsGDH were successfully expressed in E.coli.

3. Preparation of freeze-dried bacterial powder for expressing alcohol dehydrogenase gene engineering bacteria and glucose dehydrogenase gene engineering bacteria

Pre-freezing the wet thallus obtained in step 2 at-20deg.C for two days, lyophilizing at-40deg.C for 48 hr to obtain lyophilized bacterial powder (specifically alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK, alcohol dehydrogenase YjgB) expressing alcohol dehydrogenase and lyophilized bacterial powder (specifically glucose dehydrogenase BmGDH) expressing glucose dehydrogenase _M6 Glucose dehydrogenase BsGDH, glucose dehydrogenase EsGDH).

Example 4: specific enzyme activity determination of alcohol dehydrogenase

1. Preparation of crude enzyme solution of alcohol dehydrogenase

10mL of 50mM Tris-HCl buffer solution with pH of 7.0 is added into each 0.06g of the freeze-dried bacterial powder prepared in the embodiment 3 for expressing alcohol dehydrogenase, the mixture is stirred into bacterial suspension by a glass rod, the bacterial suspension is crushed by ultrasonic waves for 15min under the condition of ice bath (0 ℃), the ultrasonic waves work for 2s, the interval time is 4s, and the ultrasonic power is 210W. Centrifuging the bacterial suspension after ultrasonic disruption at 8000rpm and 4 ℃ for 10min, obtaining supernatant which is crude enzyme liquid, and storing at 4 ℃ for standby.

2. Volumetric specific activity determination of alcohol dehydrogenase

The enzyme activity of the alcohol dehydrogenase was calculated by measuring the change of absorbance at 340nm of NADPH by a single-factor kinetic method of an enzyme-labeled instrument. The enzyme activity detection system added in the ELISA plate comprises: 10mM furaldehyde, 0.1mM NADPH, 15. Mu.L crude enzyme solution, 300. Mu.L supplemented with 50mM Tris-HCl buffer pH 7.0, and after incubation of the system at 30℃for 5min, the absorbance change at 340nm was measured using an enzyme-labeled instrument. The enzyme activity unit U is defined as converting 1. Mu. Mol of NADPH to NADP per minute at 30℃and ⁺ The amount of enzyme required. Three replicates were run each time, and the mean and standard error were calculated. Volumetric enzyme activity of alcohol dehydrogenase was calculated as in equation 1.

Equation 1:

d: dilution factor 1; a is that ₁ : absorbance of the sample; a is that ₂ : blank control absorbance; v (V) _t : the total reaction system was 300. Mu.L.

e: molar absorption coefficient, constant 6220; v (V) _s : the volume of the enzyme solution is 15 mu L; d: the optical path length was 1cm.

3. Protein concentration determination of alcohol dehydrogenase

Drawing a protein concentration standard curve according to the protein concentration determination kit by the BCA method, drawing the standard curve by taking the protein content as an abscissa and the light absorption value as an ordinate, and as shown in figure 3, wherein the measured linear relation formula is y=0.0011x+0.1648, y is the light absorption value at 562nm, x is the protein concentration (mg/mL) of BSA solution, and the standard deviation is R ² ＝0.999。

When protein concentration of the alcohol dehydrogenase crude enzyme solution was measured using the BCA method protein concentration measurement kit, three sets of parallel experiments were performed each time, and the average value and standard error were calculated. The protein concentration of the alcohol dehydrogenase YahK crude enzyme solution is 5.90mg/mL; the protein concentration of the alcohol dehydrogenase YsADH crude enzyme solution is 3.84mg/mL; the protein concentration of the alcohol dehydrogenase AdhP crude enzyme solution is 4.83mg/mL; the protein concentration of the crude enzyme solution of alcohol dehydrogenase YjgB was 5.33mg/mL.

4. Specific enzyme activity

The ratio of the volume enzyme activity to the protein concentration can obtain the corresponding specific enzyme activity. The specific enzyme activity of the alcohol dehydrogenase YsADH crude enzyme solution is 433.45U/g, the specific enzyme activity of the alcohol dehydrogenase AdhP crude enzyme solution is 399.61U/g, the specific enzyme activity of the alcohol dehydrogenase YahK crude enzyme solution is 526.92U/g, and the specific enzyme activity of the alcohol dehydrogenase YjgB crude enzyme solution is 297.83U/g.

Example 5: construction of initial reaction system for synthesizing furfuryl alcohol by double enzyme coupling catalysis with furfural as substrate

1. Comparison of furfuryl alcohol catalyzed by different alcohol dehydrogenases

The lyophilized powder of alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK, and alcohol dehydrogenase YjgB prepared in example 3 were respectively used with glucose dehydrogenase BmGDH _M6 The freeze-dried bacterial powder is mixed according to the mass ratio of 1:1 to be used as a catalyst, added into 50mM Tris-HCl buffer solution with pH of 7.0, furfural is used as a substrate, glucose is used as an auxiliary substrate, and coenzyme NADP is added ⁺ 10mL of the total reaction system was constituted. In the reaction process, an automatic titration system is utilized to maintain the pH constant, and the alkali liquor used for titration is 1M NaOH aqueous solution.

The 10mL reaction system, which was not initially condition optimized, was as follows: substrate furfural final concentration 500mM, co-substrate glucose final concentration 1250mM, coenzyme NADP ⁺ Final concentration 0.2mM, alcohol dehydrogenase lyophilized powder (alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK or alcohol dehydrogenase YjgB) and glucose dehydrogenase BmGDH _M6 The addition amount of the dry bacterial powder is 15g/L, wherein the ethanol dehydrogenase freeze-dried bacterial powder and the glucose dehydrogenase freeze-dried bacterial powder are mixed according to the mass ratio of 1:1 (0.15 g:0.15 g), added into 50mM Tris-HCl buffer solution with pH of 7.0, and reacted for 6 hours at the pH of 7.0, 400rpm and 30 ℃. Under the same conditions, the non-induced alcohol dehydrogenase freeze-dried bacterial powder is used as a blank control.

After the completion of the reaction, 200. Mu.L of the reaction mixture was centrifuged at 12000rpm for 3min, and 100. Mu.L of the supernatant was extracted with 1mL of ethyl acetate for 30min. After the extraction was completed, the mixture was centrifuged at 12000rpm for 1min, 200. Mu.L of the upper organic phase was collected, and the contents of the respective components in the sample were measured by gas chromatography. Three replicates were run each time, and the mean and standard error were calculated.

Gas chromatographyThe conditions were as follows: gas chromatograph, agilent 6890N; chiral chromatographic column, BGB-174 (column length 30m, column inner diameter 250 μm, fixing solution coating thickness 0.25 μm); detector, FID,250 ℃; carrier gas, N ₂ The method comprises the steps of carrying out a first treatment on the surface of the Carrier gas flow, 1mL/min; split ratio: 1:20; sample injection amount: 1.0. Mu.L; sample inlet temperature: 250 ℃. Analysis of furfural, furfuryl alcohol, and possible byproducts furoic acid and tetrahydrofurfuryl alcohol uses a specific temperature program: the temperature is maintained at 80 ℃ for 3min, and the temperature is increased to 190 ℃ at 10 ℃ per minute, and the temperature is maintained at 190 ℃ for 6min for 20min. As shown in fig. 4, the retention times of furfuryl alcohol and furfuryl aldehyde were 8.4min and 9.0min, respectively.

The results are shown in fig. 5, with a conversion of 7.9% for the blank to furfural; the conversion rate of alcohol dehydrogenase YahK to furfural is 68.8%; the conversion rate of alcohol dehydrogenase YsADH to furfural is 60.0%; the conversion rate of alcohol dehydrogenase AdhP to furfural is 53.6%; the conversion of furfural by alcohol dehydrogenase, alcohol dehydrogenase YjgB, was 26.8%. As can be seen, the alcohol dehydrogenase YahK is preferred.

2. Comparison of catalytic Synthesis of furfuryl alcohol under different glucose dehydrogenases and different coenzymes

The lyophilized powder of alcohol dehydrogenase YahK prepared in example 3 was mixed with lyophilized powder of glucose dehydrogenase (glucose dehydrogenase BmGDH) _M6 Glucose dehydrogenase BsGDH or glucose dehydrogenase EsGDH) in a mass ratio of 1:1, 50mM Tris-HCl buffer solution with pH of 7.0 as reaction medium, adding coenzyme NADP ⁺ Or NAD ⁺ Other operations and reaction conditions are the same as in the step 1, so that a 10mL reaction total system is formed. Under the same conditions, the non-induced glucose dehydrogenase freeze-dried bacterial powder is used as a blank control.

As a result, as shown in FIG. 6, when NADP was selected as the coenzyme ⁺ Glucose dehydrogenase BmGDH when used _M6 The conversion rate of the glucose dehydrogenase BsGDH to the furfural is 68.75%, the conversion rate of the glucose dehydrogenase BsGDH to the furfural is 61.92%, the conversion rate of the glucose dehydrogenase EsGDH to the furfural is 39.72%, and the conversion rate of the blank control to the furfural is 21.33%. When the coenzyme is NAD ⁺ Glucose dehydrogenase BmGDH when used _M6 The conversion rate of the glucose dehydrogenase BsGDH to the furfural is 54.96 percent, the conversion rate of the glucose dehydrogenase BsGDH to the furfural is 45.76 percent, and the conversion rate of the glucose dehydrogenase EsGDH to the furfural is35.03% and 17.27% conversion of furfural to blank. As can be seen, glucose dehydrogenase BmGDH is preferred _M6 Preferably coenzyme NADP ⁺ 。

Example 6: optimum temperature for alcohol dehydrogenase YahK-induced expression

The alcohol dehydrogenase YahK gene engineering bacteria are prepared into freeze-dried bacterial powder according to the method of the embodiment 3, wherein the temperature of the induced expression condition is set to be 12-36 ℃ (12 ℃, 16 ℃,20 ℃,24 ℃, 28 ℃, 32 ℃ and 36 ℃), and the alcohol dehydrogenase YahK freeze-dried bacterial powder under different induction temperatures is respectively prepared. Catalytic reactions were carried out as in example 5, step 1. Three replicates were run each time, and the mean and standard error were calculated. As a result, the optimum induction temperature was 24℃as shown in FIG. 7.

Example 7: optimum inducer IPTG addition amount for alcohol dehydrogenase YahK induced expression

The alcohol dehydrogenase YahK gene engineering bacteria expressing alcohol dehydrogenase are prepared into freeze-dried bacterial powder according to the method of the example 3, wherein the addition amount of IPTG for inducing expression is set to be 0-0.6 mM (0 mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM and 0.6mM are selected), and the alcohol dehydrogenase YahK freeze-dried bacterial powder with different addition amounts of inducers is respectively prepared. Catalytic reactions were carried out as in example 5, step 1. Three replicates were run each time, and the mean and standard error were calculated. As a result, as shown in FIG. 8, the optimum inducer IPTG was added at an amount of 0.2mM.

Example 8: double-enzyme coupling catalytic synthesis furfuryl alcohol reaction system with furfuraldehyde as substrate

The alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 The freeze-dried bacterial powder is used as a catalyst, and the reaction system in the step 1 of the example 5 is adopted for catalytic reaction, except that the temperature of the reaction system is set to 20-50 ℃ (20 ℃,25 ℃,30 ℃, 35 ℃,40 ℃, 45 ℃ and 50 ℃), and other operations are the same as the example 5. Three replicates were run each time, and the mean and standard error were calculated. As a result, as shown in FIG. 9, the optimum reaction temperature was 30 ℃.

Example 9: optimum pH of furfuryl alcohol synthesizing reaction system by double enzyme coupling catalysis with furfuraldehyde as substrate

The alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 The lyophilized powder was used as a catalyst and the reaction system of step 1 of example 5 was used for the catalytic reaction, except that the pH of the reaction system was set to 4 to 9 (4, 5, 6, 7, 8 and 9 were selected) in 50mM Tris-HCl buffer, and the other operations and reaction system were the same as in example 5. Three replicates were run each time, and the mean and standard error were calculated. As a result, as shown in FIG. 10, the conversion was high when the pH was neutral or weakly acidic, and as high as 68.75% when the pH was 7, the optimal reaction pH was 7, and the buffer was 50mM Tris-HCl.

Example 10: double-enzyme coupling catalytic synthesis of furfuryl alcohol reaction system optimal coenzyme NADP by using furfural as substrate ⁺ Additive amount

The alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 The lyophilized powder is used as a catalyst, and the reaction system of the step 1 of the example 5 is adopted for catalytic reaction, except that coenzyme NADP ⁺ The final concentration was set at 0 to 0.5mM (0, 0.1, 0.2, 0.3, 0.4 and 0.5mM were selected), and the other operations and reaction systems were the same as in example 5. Three replicates were run each time, and the mean and standard error were calculated. The results are shown in FIG. 11 when NADP ⁺ When not added, the conversion of furfural was 36.26%, NADP ⁺ NADP at a concentration between 0 and 0.5mM ⁺ The increase in (2) favors the reaction, and the conversion is 68.75% at a concentration of 0.2mM. When NADP ⁺ At a concentration of more than 0.2mM, the conversion rate is not increased significantly. Thus, 0.2mM NADP was used in the reaction ⁺ Is economical.

Example 11: furfurol and glucose optimal concentration ratio of furfurol reaction system synthesized by double enzyme coupling catalysis with furfurol as substrate

The alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 The freeze-dried bacterial powder is used as a catalyst, and the reaction system in the step 1 of the embodiment 5 is adopted for catalytic reaction, wherein the difference is that the final concentration ratio of furfural to glucose is set to be 1:0.5-2.5 (1:0.5, 1:1 and 1:1 are selected5, 1:2, 1:2.5) wherein the final concentration of furfural was 500mM, the other procedures and reaction systems were as in example 5. Three replicates were run each time, and the mean and standard error were calculated. As shown in fig. 12, the increase of the co-substrate glucose increased the conversion of furfural, which was 61.66% when the final concentration ratio of furfural to glucose was 1:1, and did not increase significantly when the glucose concentration continued to increase. Therefore, the final concentration ratio of furfural to glucose used in the reaction is economical.

Example 12: alcohol dehydrogenase YahK and glucose dehydrogenase BmGDH of furfuryl alcohol reaction system synthesized by double enzyme coupling catalysis with furfural as substrate _M6 Optimum mass ratio

The alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 The lyophilized powder is used as a catalyst, and the reaction system of the step 1 of the example 5 is adopted for catalytic reaction, except that alcohol dehydrogenase YahK and glucose dehydrogenase BmGDH of the reaction system _M6 The mass ratio of the freeze-dried bacterial powder is set to be 0.2-5:1 (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 are the same as in example 5. Three replicates were run each time, and the mean and standard error were calculated. As a result, FIG. 13 shows that the catalytic system was optimized for alcohol dehydrogenase YahK and glucose dehydrogenase BmGDH _M6 The mass ratio of the freeze-dried bacterial powder of (1:1), namely in a 10mL system, alcohol dehydrogenase YahK and glucose dehydrogenase BmGDH _M6 Added in a mixed ratio of 0.15g to 0.15 g.

Example 13: reaction progress under different substrate concentrations during double-enzyme coupling catalytic synthesis of furfuryl alcohol

The alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 The lyophilized powder is used as a catalyst, and the reaction system in the step 1 of the example 5 is adopted for catalytic reaction, wherein the final concentration of the substrate is 500mM, 600mM, 700mM, 800mM, 900mM and 1000mM respectively, and the final concentration ratio of furfural to glucose is 1:1. The gas phase was detected by sampling at regular time, the reaction time was 24 hours, and the rest was the same as in example 5. As shown in FIG. 14, at a substrate concentration of 500mM, 10 hours can completely convert furfural to furfuryl alcohol; substrate(s)At a concentration of 600mM, the 24h furfural conversion was 88.05%. With the increase of the concentration of the furfural, the catalyst is inhibited by the high-concentration aldehyde substrate, the conversion rate is reduced, and the furfural is hardly converted when the concentration of the substrate is 1000 mM.

Example 14: synthesis of high-concentration furfuryl alcohol by combining double enzyme coupling catalytic process with substrate fed-batch process

The lyophilized powder for expressing alcohol dehydrogenase YahK and the lyophilized powder for expressing glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 The freeze-dried bacterial powder in the step 1 of the embodiment 5 is used as a catalyst for catalytic reaction, and the difference is that furfural and glucose are added by a microinjection pump (a constant flow pump Co., ltd.) in a constant-speed continuous flow adding mode, a fed-batch substrate is furfural stock solution, a fed-batch auxiliary substrate is 1.8M glucose aqueous solution, the constant flow acceleration of the furfural and glucose is 16.7 mu mol/min, the final concentration ratio is 1:1, the reaction is stopped after 15h of feeding, the reaction is continued until 30h, and gas phase detection is carried out by taking samples regularly. As shown in FIG. 15, the conversion rate of furfural in the process of 1-10 h is all>99% of the furfurol fed in was completely converted, at which time the furfuryl alcohol was continuously and steadily increased. Furfural is continuously added, accumulation of substrate furfural is gradually started in 11-15 hours, the conversion efficiency of the biocatalyst to the furfural is reduced, and the activity is inhibited. When the reaction time is 15h, the conversion rate of the whole furfural in the system is 74.65%. Stopping feeding after 15 hours, continuing the reaction, and gradually consuming accumulated furfural, which indicates that the biocatalyst still has activity, and completely converting the fed-batch furfural into furfuryl alcohol in the first 15 hours. The results of gas phase detection verification show that the final reaction liquid has no substrate furfural, side products furoic acid and tetrahydrofurfuryl alcohol, which show that the furfuraldehyde is completely converted into furfuryl alcohol, and also show that the established process has excellent chemical selectivity. It was calculated that 0.015mol of furfural in the 10mL reaction system was completely converted to furfuryl alcohol and the cumulative concentration of product reached 1500mM.

Example 15: technological amplification and product identification of synthesizing high-concentration furfuryl alcohol by combining double enzyme coupling catalytic process with substrate fed-batch process

The alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH prepared in the method of example 3 are selected _M6 4.5g of freeze-dried bacterial powder is mixed according to the mass ratio of 1:1 to be used as a catalyst, the final concentration of the catalyst added is 30g/L, the final concentration ratio of furfural to glucose is 1:1, and the coenzyme NADP is the same as that of the catalyst ⁺ 50mM Tris-HCl buffer, 0.2mM concentration, pH 7.0, constituted a 300mL reaction system at 30℃pH 7.0 and a rotation speed of 400rpm. Furfural and glucose are added by a microinjection pump (a constant-pressure constant-flow pump Co., ltd.) in a constant-speed continuous flow adding mode, a fed-batch substrate is furfural stock solution, a fed-batch auxiliary substrate is a 1.2M glucose water solution, constant flow acceleration of the furfural and the glucose is 501.0 mu mol/min, the reaction is stopped after 10 hours of fed-batch, and gas phase detection is performed by timing sampling. The results are shown in figure 17, and the conversion rate of the furfural is 1-10 h>99% of the total 0.3mol of furfurol (28.82 g) was converted to furfuryl alcohol (29.43 g) in the 300mL reaction system, and the cumulative concentration of the product reached 1000mM, at which time the space-time yield of furfuryl alcohol was about 235 g/(L.d).

After the amplification reaction was completed, the reaction mixture was centrifuged at 12000rpm for 10 minutes, the supernatant was collected, ethyl acetate was added in an amount of 4 times the volume of the reaction mixture, the mixture was extracted at 200rpm and 30℃for 1 hour, and after the completion of the extraction, the mixture was centrifuged at 12000rpm for 10 minutes, and the upper organic phase was collected. Because the reaction conversion rate is more than 99%, almost no substrate and intermediate product remain, and the ethyl acetate is removed from the upper organic phase by vacuum rotary evaporation, so that the product is directly obtained.

The substrates and products in the reaction solution were confirmed to be furfural and furfuryl alcohol, respectively, by gas chromatography-mass spectrometry analysis, as shown in fig. 18 a and b. The gas chromatograph-mass spectrometer model was Agilent 7890A/5975C, and the gas chromatograph conditions were as in example 5 (without detector FID, except for the relevant conditions), and the mass spectrometric detection conditions were as follows: auxiliary heater temperature, 250 ℃; MS quadrupole temperature, 150 ℃; ion source temperature, 230 ℃; mass spectrum scanning range, 30-500amu; emission current, 200 μa; electron energy, 70eV.

Claims (10)

1. A method for synthesizing furfuryl alcohol by double enzyme coupling, which is characterized by comprising the following steps: inducing expression of alcohol dehydrogenase gene engineering bacteria and glucose dehydrogenase gene engineering bacteria, lyophilizing wet thallus, and obtaining alcohol dehydrogenaseThe freeze-dried bacterial powder and glucose dehydrogenase freeze-dried bacterial powder are mixed and then used as a catalyst, furfural is used as a substrate, glucose is used as an auxiliary substrate, and NADP is used ⁺ Or NAD ⁺ The reaction system is formed by taking buffer solution with pH value of 4-9 as a reaction medium, and furfuryl alcohol is obtained after the reaction is completed under the conditions of 20-50 ℃ and 400rpm, and the reaction solution is separated and purified.

2. The method according to claim 1, wherein the alcohol dehydrogenase genetically engineered bacterium is constructed by introducing an alcohol dehydrogenase gene into escherichia coli; the alcohol dehydrogenase genes include, but are not limited to, an alcohol dehydrogenase YsADH gene with a nucleotide sequence shown as SEQ ID NO.1, an alcohol dehydrogenase AdhP gene with a nucleotide sequence shown as SEQ ID NO.3, an alcohol dehydrogenase YahK gene with a nucleotide sequence shown as SEQ ID NO.5, and an alcohol dehydrogenase YjgB gene with a nucleotide sequence shown as SEQ ID NO. 7.

3. The method according to claim 1, wherein the glucose dehydrogenase genetically engineered bacterium is constructed by introducing a glucose dehydrogenase gene into E.coli; the glucose dehydrogenase gene comprises, but is not limited to, glucose dehydrogenase BmGDH with a nucleotide sequence shown as SEQ ID NO.9 _M6 Glucose dehydrogenase BsGDH with nucleotide sequence shown as SEQ ID NO.11, and glucose dehydrogenase EsGDH with nucleotide sequence shown as SEQ ID NO. 13.

4. The method according to claim 1, wherein the alcohol dehydrogenase lyophilized powder and the glucose dehydrogenase lyophilized powder are mixed in a mass ratio of 0.2-5:1.

5. The method according to claim 1, wherein the catalyst is added in an amount of 20 to 40g/L to the reaction system; the final concentration of the substrate furfural is 500-1500mM, and the concentration ratio of the furfural to glucose is 1:0.5-2.5; the final concentration of the coenzyme is 0-0.5 mM.

6. The method of claim 1, wherein the reaction medium is 50mM Tris-HCl buffer at pH 7.0.

7. The method according to claim 5, wherein the substrate and the cosubstrate are added to the reaction system at one time when the substrate addition amount is not more than 900 mM; when the substrate addition amount is higher than 900mM, the substrate and the cosubstrate are respectively added in a constant-speed continuous flow manner.

8. The method of claim 7, wherein the substrate is fed as a stock solution of furfural and the co-substrate is fed as an aqueous solution of 1.2-1.8M glucose; feeding at the same speed for 1-15 h, and continuously reacting for 0-15 h after the feeding is finished, so that the concentration ratio of the substrate to the auxiliary substrate in the fed reaction system is 1:0.5-2.5; the substrate is fed at a rate of 16.7-501.0. Mu. Mol/min.

9. The method of claim 8, wherein in a 10mL reaction system, the constant flow acceleration of the furfural and the glucose is 16.7 mu mol/min, the feeding time is 15h, and the reaction is continued for 15h after the feeding is finished; in a 300mL reaction system, constant flow acceleration of furfural and glucose is 501.0 mu mol/min, and the reaction is stopped after 10h of feeding.

10. The method of claim 1, wherein the alcohol dehydrogenase lyophilized powder and the glucose dehydrogenase lyophilized powder are each prepared as follows: inoculating alcohol dehydrogenase genetically engineered bacteria and glucose dehydrogenase genetically engineered bacteria into LB liquid medium containing kanamycin with final concentration of 100 mug/mL, culturing overnight at 37 ℃ and 200rpm, transferring into LB liquid medium containing kanamycin with volume concentration of 2%, and culturing at 37 ℃ and 200rpm to cell concentration OD ₆₀₀ Adding IPTG with the final concentration of 0-0.6 mM to the culture at 0.6-0.8, and carrying out induction culture at 12-26 ℃ for 12h to obtain an induction culture solution; centrifuging the induced culture solution at 4deg.C and 8000rpm for 10min, and discarding supernatant; then with 50mM Tris-HCl buffer pH 8.0Re-suspending the thallus, centrifuging at 4deg.C and 8000rpm for 10min, discarding supernatant, and collecting wet thallus; and (3) pre-freezing the obtained wet thalli for two days at the temperature of minus 20 ℃, and freeze-drying the thalli for 48 hours at the temperature of minus 40 ℃ in a freeze dryer to obtain freeze-dried alcohol dehydrogenase powder and freeze-dried glucose dehydrogenase powder respectively.

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