CN115992191A - Method for synthesizing 2, 5-furandimethanol by reducing 5-hydroxymethylfurfural under double enzyme coupling catalysis - Google Patents

Abstract

The invention discloses a method for synthesizing 2, 5-furandimethanol by double enzyme coupling catalysis of 5-hydroxymethylfurfural reduction, which comprises the steps of freeze-drying thalli obtained by respective induced expression of alcohol dehydrogenase genetic engineering bacteria and glucose dehydrogenase genetic engineering bacteria, mixing the obtained alcohol dehydrogenase freeze-dried bacterial powder with glucose dehydrogenase freeze-dried bacterial powder to be used as a catalyst, taking 5-hydroxymethylfurfural as a substrate, glucose as an auxiliary substrate and NADP (NADP) ⁺ The reaction system is formed by taking buffer solution with pH value of 4-9 as a reaction medium, and the 2, 5-furandimethanol is obtained after the reaction is completed under the conditions of 20-50 ℃ and 400 rpm. The biocatalysis system can tolerate high-concentration 5-hydroxymethylfurfural. The inhibition of aldehyde substrates to the biocatalyst is further removed by adopting a constant-speed continuous flow processing technology, the concentration of the product can reach 1300mM, and the space-time yield can reach 224 g/(L.d) at the highest.

Description

Method for synthesizing 2, 5-furandimethanol by reducing 5-hydroxymethylfurfural under double enzyme coupling catalysis

Field of the art

The invention belongs to the field of biocatalysis, and relates to a method for synthesizing 2, 5-furandimethanol by using 5-hydroxymethylfurfural as a substrate and performing double-enzyme coupling catalysis on the 5-hydroxymethylfurfural.

(II) background art

2, 5-furandimethanol is used as a diol with high added value and has important application in the preparation research of synthetic resin and polyheterocyclic compound of medicines. Along with the gradual reduction of fossil resources, the catalytic preparation of 2, 5-furandimethanol by using renewable biomass-based platform molecules of 5-hydroxymethylfurfural is widely focused by people, and is one of the important research points for realizing the high-value utilization of biomass resources.

At present, the hydrogenation of 5-hydroxymethylfurfural to prepare 2, 5-furandimethanol is still dominant by using a chemical catalyst, however, the chemical method requires harsh reaction conditions such as high temperature, high pressure and the like, relates to organic solvents and toxic chemicals, and is difficult to treat highly toxic and corrosive waste residues, so that serious environmental problems are caused. In addition, in the process of preparing 2, 5-furandimethanol by hydrogenating 5-hydroxymethylfurfural, selecting a proper catalyst and reaction conditions, the selective hydrogenation and conversion of 5-hydroxymethylfurfural into 2, 5-furandimethanol and the obtaining of higher yield remain a great challenge. The biocatalysis reduction of 5-hydroxymethylfurfural to produce 2, 5-furandimethanol becomes an important alternative method for chemical catalysis due to the advantages of high catalytic efficiency, good selectivity, mild reaction conditions and the like. In general, the biological method for catalyzing the production of 2, 5-furandimethanol by 5-hydroxymethylfurfural has become a research hot spot, but the research on synthesizing 2, 5-furandimethanol by a biological enzyme method has not been mature. 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 the biocatalytic synthesis, the reaction design incorporates a reaction system based on glucose dehydrogenase (BmGDH _M6 ) Is a coenzyme circulation system: alcohol dehydrogenase YahK catalyzes the hydrogenation of 5-hydroxymethylfurfural to 2, 5-furandimethanol 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 2, 5-furandimethanol, various alcohol dehydrogenases (YsADH, adhP, yahK, yjgB, preferably YahK) are used as biocatalysts. The biocatalytic system is tolerant of high concentrations of 5-hydroxymethylfurfural (500 mM). In order to further remove the inhibition of the high-concentration aldehyde substrate to the biocatalyst, a constant-speed continuous flow 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 2, 5-furandimethanol by reducing 5-hydroxymethylfurfural through double enzyme coupling catalysis, which takes 5-hydroxymethylfurfural as a substrate, takes glucose as an auxiliary substrate, and uses alcohol dehydrogenase YahK which is optimized to catalyze 5-hydroxymethylfurfural to hydrogenate to generate 2, 5-furandimethanol and NADP by using 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 biocatalytic system is tolerant of high concentrations of 5-hydroxymethylfurfural (500 mM). 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 can reach 1300mM, and the space-time yield can reach 224 g/(L.d) at the highest. The method has the advantages of high atom economy, high catalytic efficiency, high concentration of applicable substrates, high coenzyme circulation efficiency and the like.

The technical scheme adopted by the invention is as follows:

the invention provides a method for synthesizing 2, 5-furandimethanol by reducing 5-hydroxymethylfurfural through double enzyme coupling catalysis, which comprises the following steps: respectively carrying out induced expression on alcohol dehydrogenase genetic engineering bacteria and glucose dehydrogenase genetic engineering bacteria to obtain wet bacterial freeze-drying, mixing the obtained alcohol dehydrogenase freeze-dried bacterial powder with glucose dehydrogenase freeze-dried bacterial powder to obtain a catalyst, taking 5-hydroxymethylfurfural as a substrate, glucose as an auxiliary substrate and NADP (sodium phosphate) ⁺ The reaction system is formed by taking buffer solution with pH value of 4-9 as a reaction medium, and the 2, 5-furandimethanol is obtained after the reaction is completed under the conditions of 20-50 ℃ and 400 rpm. The biocatalysis system can tolerate high concentration 5-hydroxymethylfurfural (500 mM) to achieve 100% conversion. 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 an alcohol dehydrogenase gene (preferably YahK) into escherichia coli; the alcohol dehydrogenase genes include, but are not limited to, an alcohol dehydrogenase YsADH encoding gene (SEQ ID NO. 1), an alcohol dehydrogenase AdhP encoding gene (SEQ ID NO. 3), an alcohol dehydrogenase YahK encoding gene (SEQ ID NO. 5), and an alcohol dehydrogenase YjgB encoding gene (SEQ ID NO. 7); preferably, the alcohol dehydrogenase gene is an alcohol dehydrogenase YahK gene, and is derived from escherichia coli, the GenBank accession number is WP_128491393.1, and a small purification kit of TaKaLa genome DNA is utilized to obtain escherichia coli genome; the primer is designed to clone the alcohol dehydrogenase YahK coding gene from the escherichia coli genome, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6. The construction method of the alcohol dehydrogenase YahK genetic engineering bacteria comprises the following steps: inserting an alcohol dehydrogenase YahK encoding 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 obtained by introducing glucose dehydrogenase (preferably BmGDH _M6 ) And (3) constructing and obtaining the gene-introduced escherichia coli. The glucose dehydrogenase gene is preferablyGlucose dehydrogenase BmGDH _M6 The nucleotide sequence and the amino acid sequence of the gene are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10. Glucose dehydrogenase BmGDH _M6 The construction method of the genetically engineered bacteria 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 is 500-1500 mM (preferably 500 mM), and the concentration ratio of the substrate 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).

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.

Further, when the substrate addition amount is 700mM or less, the substrate and the cosubstrate are added to the reaction system at once; when the substrate addition amount is higher than 700mM, in order to further remove the inhibition of the high-concentration aldehyde substrate on the biocatalyst and obtain higher space-time yield, the substrate and the auxiliary substrate are added in a constant-speed continuous flow adding mode, wherein the fed-batch substrate is 5-hydroxymethylfurfural stock solution, and the fed-batch auxiliary substrate is 1.2-1.8M glucose aqueous solution; the substrate and the auxiliary substrate are fed at the speed of 10-360 mu mol/min for 1-18 h respectively, and the reaction is continued for 0-7 h after the fed-batch is finished; the substrate and the co-substrate flow have the same acceleration, and the cumulative addition amount of the substrate is 500-1300mM.

Further, in a 10mL reaction system, the constant flow acceleration of the furfural and the glucose is 11.9 mu mol/min, the feeding time is 18h, and the reaction is continued for 7h after the feeding is finished; in a 300mL reaction system, the constant flow acceleration of furfural and glucose is 357.1 mu mol/min, and the reaction is stopped after 11h 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 gene engineering bacteria and glucose dehydrogenase gene engineering bacteria (preferably E.coli BL21 (DE 3)/pET 28a-YahK and E.coli BL21 (DE 3)/pET 28 a-BmGDH) _M6 ) Inoculated into LB liquid medium containing 100. Mu.g/mL kanamycin at a final concentration, cultured overnight at 37℃and 200rpm, then transferred into LB liquid medium containing 100. Mu.g/mL kanamycin at an inoculum size of 2% by volume, and cultured to a cell concentration OD at 37℃and 200rpm 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 carrying out induction culture for 12h at 12-36 ℃ (preferably 24 ℃) 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; and (3) pre-freezing the obtained wet thalli for two days at the temperature of minus 20 ℃, and freeze-drying the thalli in a freeze dryer at the temperature of minus 40 ℃ for 48 hours to obtain each freeze-dried bacterial powder.

Further, the reaction liquid separation and purification method comprises the following steps: the reaction solution was centrifuged at 12000rpm for 10min, the supernatant was taken, ethyl acetate was added in an amount of 4 times the volume of the reaction solution, extraction was performed at 200rpm and 30℃for 1h, and after the completion of the extraction, the mixture was centrifuged at 12000rpm for 10min, and the upper organic phase was taken. 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 is directly obtained.

Compared with the prior art, the invention has the beneficial effects that: the method for synthesizing 2, 5-furandimethanol by double enzyme coupling catalysis uses 5-hydroxymethylfurfural as a substrate and glucose as an auxiliary substrate, and the preferential alcohol dehydrogenase YahK catalyzes the hydrogenation of 5-hydroxymethylfurfural to generate 2, 5-furandimethanol and NADP by using NADPH ⁺ And preferably 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). The biocatalytic system is tolerant of high concentrations of 5-hydroxymethylfurfural (500 mM). Using a constant-speed continuous flowThe processing technology further removes the inhibition of the high-concentration aldehyde substrate on the biocatalyst, the accumulation concentration of the product can reach 1300mM, and the space-time yield can reach 224 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 2, 5-furandimethanol by double enzyme coupling catalysis with 5-hydroxymethylfurfural as a substrate and glucose as an auxiliary substrate.

FIG. 2 is a SDS-PAGE gel of the culture medium of the alcohol dehydrogenase genetically engineered bacteria 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, induced genetically engineered bacterium e.coli BL21 (DE 3)/pET 28a-YahK, with a thicker band corresponding to YahK, having 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.

FIG. 3 is a SDS-PAGE gel of the culture medium of the glucose dehydrogenase genetically engineered bacteria of example 3 before and after induction; from left to right, lane 1, induced genetically engineered E.coli BL21 (DE 3)/pET 28a-BmGDH _M6 The thickened bands correspond to BmGDH _M6 Molecular weight of 28kDa; lanes M, blue plus II protein marker.

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

FIG. 5 is a gas chromatogram of example 5; standard 5-hydroxymethyl furfuryl alcohol (14.4 min) and 2, 5-furandicarboxaldehyde (15.7 min).

FIG. 6 is a bar graph of the conversion of 5-hydroxymethylfurfural by catalytic synthesis with different alcohol dehydrogenases of example 5.

FIG. 7 is a flow chart showing the process of synthesizing 2, 5-furandimethanol by double enzyme coupling catalysis in example 6.

FIG. 8 shows the gas phase results of the reaction solution of example 6 and possible by-product standards, (a) the reaction solution; (b) a2, 5-diformylfuran standard; (c) 2, 5-dimethyloltetrahydrofuran standard.

FIG. 9 shows the progress of product accumulation in the amplification reaction of example 7.

FIG. 10 is a gas-mass spectrometry (GC-MS) spectrum of the substrate 5-hydroxymethylfurfural (a) and the product 2, 5-furandimethanol (b) of example 7.

(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

The GenBank accession number of glucose dehydrogenase BmGDH from bacillus megatherium (Bacillus megaterium) is AAA22475, and mutant BmGDH is obtained by performing Q252L/E170K/S100P/K166R/V72I/K137R multi-site substitution on the amino acid sequence of glucose dehydrogenase BmGDH _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&。

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 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 is inserted between BamH I and Xho I sites on plasmid pET28a to obtain recombinant plasmid pET28a-BmGDH _M6 . The recombinant plasmid is led into competent cells E.coli BL21 (DE 3) to obtain engineering bacteria E.coli BL21 (DE 3)/pET 28a-BmGDH _M6 。

The engineering bacteria show that the alcohol dehydrogenase and glucose dehydrogenase genes are inserted into each other without error through the sequencing of the extracted plasmid.

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 respectively used for preparing the recombinant bacteria _M6 Inoculating to the final productCulturing overnight at 37deg.C and 200rpm in LB liquid medium containing 100 μg/mL kanamycin, transferring to LB liquid medium containing 100 μg/mL kanamycin at an inoculum size of 2% by volume, and culturing at 37deg.C and 200rpm to 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 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 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, 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 and FIG. 3, SDS-PAGE shows that alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK, alcohol dehydrogenase YjgB and glucose dehydrogenase BmGDH _M6 All expressed successfully 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 at-20deg.C for two days, lyophilizing at-40deg.C for 48 hr to obtain lyophilized powder of alcohol dehydrogenase (alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK, alcohol dehydrogenase YjgB) and lyophilized powder of glucose dehydrogenase (glucose dehydrogenase BmGDH) _M6 )。

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 example 3, the mixture is stirred into bacterial suspension by a glass rod, the bacterial suspension is crushed by ultrasound for 15min under the ice bath (0 ℃) condition, the ultrasound is operated for 2s, the interval is 4s, and the ultrasound 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 5-hydroxymethylfurfural, 0.1mM NADPH, 15. Mu.L crude enzyme solution, 300. Mu.L of 50mM Tris-HCl buffer solution with pH 7.0 were used for supplementing, and after the system was incubated at 30℃for 5min, the absorbance change at 340nm was detected by 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 a standard curve by taking the protein content as an abscissa and the light absorption value as an ordinate, and as shown in fig. 4, the measured linear relation formula is y=0.0011x+0.1648, wherein 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 365.01U/g, the specific enzyme activity of the alcohol dehydrogenase AdhP crude enzyme solution is 292.59U/g, the specific enzyme activity of the alcohol dehydrogenase YahK crude enzyme solution is 393.41U/g, and the specific enzyme activity of the alcohol dehydrogenase YjgB crude enzyme solution is 256.01U/g.

Example 5: construction of initial reaction system for synthesizing 2, 5-furandimethanol by double enzyme coupling catalysis of 5-hydroxymethylfurfural

1. Comparison of catalytic Synthesis of 5-hydroxymethylfurfural by different alcohol dehydrogenases

The alcohol dehydrogenase YsADH lyophilized powder, the alcohol dehydrogenase AdhP lyophilized powder, the alcohol dehydrogenase YahK lyophilized powder and the alcohol dehydrogenase YjgB lyophilized powder prepared in example 3 were respectively mixed with glucose dehydrogenase BmGDH _M6 The freeze-dried bacterial powder is mixed according to the mass ratio of 1:1 as a catalyst, added into 50mM Tris-HCl buffer solution with pH of 7.0, and added with coenzyme NADP by taking 5-hydroxymethylfurfural as a substrate and glucose as an auxiliary substrate ⁺ 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 reaction system, which was not initially subjected to condition optimization, was as follows: substrate 5-hydroxymethylfurfural final concentration 500mM, cosubstrate glucose final concentration 1250mM, coenzyme NADP ⁺ Final concentration 0.2mM, alcohol dehydrogenase YsADH, alcohol dehydrogenase AdhP, alcohol dehydrogenase YahK, alcohol dehydrogenase YjgB and glucose dehydrogenase BmGDH _M6 The addition amount of the freeze-dried bacterial powder is 15g/L, wherein the dry bacterial powder of alcohol dehydrogenase and the dry bacterial powder of glucose dehydrogenase 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 powderIs 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.

The gas chromatography 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 5-hydroxymethylfurfural, 2, 5-furandimethanol and possible byproducts 2, 5-diformylfuran and 2, 5-dimethyloltetrahydrofuran employed specific temperature ramp program: maintaining at 90deg.C for 3min, heating to 190deg.C at 10deg.C/min, and maintaining at 190deg.C for 7min for 20min. As shown in fig. 5, the retention times of 2, 5-furandimethanol and 5-hydroxymethylfurfuryl alcohol were 14.4min and 15.7min, respectively.

The results are shown in FIG. 6, where the conversion of 5-hydroxymethylfurfural by the blank is 7.1%; the conversion rate of YahK to 5-hydroxymethylfurfural is 41.0%; the conversion of 5-hydroxymethylfurfural by alcohol dehydrogenase YsADH was 34.1%; the conversion rate of alcohol dehydrogenase AdhP to 5-hydroxymethylfurfural is 30.1%; the conversion of 5-hydroxymethylfurfural by alcohol dehydrogenase YjgB was 16.6%. As can be seen, the alcohol dehydrogenase YahK is preferred.

Example 6: synthesis of high-concentration 2, 5-furandimethanol by combining double enzyme coupling catalytic process with substrate fed-batch process

Adopts the reaction system and the reaction conditions of the example 5, selects alcohol dehydrogenase YahK freeze-dried bacterial powder and glucose dehydrogenase BmGDH _M6 The mass ratio of the freeze-dried bacterial powder is 1:1 (0.15 g:0.15 g) as a catalyst, and the final concentration of the catalyst is 30g/L. The difference is that 5-hydroxymethyl furfural and glucose are added by a microinjection pump of a constant flow pump limited company with a constant-speed continuous flow feeding mode, the fed substrate is 5-hydroxymethyl furfural stock solution, the fed auxiliary substrate is 1.8M glucose aqueous solution, and the constant flow acceleration of the 5-hydroxymethyl furfural and the glucose aqueous solution is11.9 mu mol/min, 5-hydroxymethylfurfural is added in a cumulative final concentration of 1300mM, the ratio of 5-hydroxymethylfurfural to glucose is 1:1, the reaction is stopped after 18h of feeding, the reaction is continued until 25h, and gas phase detection is carried out by sampling at fixed time.

As shown in FIG. 7, the conversion rate of 5-hydroxymethylfurfural in the process of 1-11 h is more than 99%, the fed-batch of 5-hydroxymethylfurfural is completely converted, and the 2, 5-furandimethanol product is continuously and steadily increased. And 5-hydroxymethylfurfural is continuously added, accumulation of 5-hydroxymethylfurfural is gradually started in 12-18 hours, the conversion efficiency of the biocatalyst on the 5-hydroxymethylfurfural is reduced, and the activity is inhibited. When the reaction time is 18h, the overall conversion rate of the 5-hydroxymethylfurfural in the system is 77.84%. Stopping feeding after 18 hours, continuing the reaction, gradually consuming the accumulated 5-hydroxymethylfurfural, indicating that the biocatalyst still has activity, and completely converting the 5-hydroxymethylfurfural fed in the previous 18 hours into 2, 5-furandimethanol. The gas phase detection shows that the final reaction liquid has no substrate 5-hydroxymethyl furfural and byproducts 2, 5-diformylfuran and 2, 5-dihydroxymethyltetrahydrofuran, which show that the 5-hydroxymethyl furfural is completely converted into 2, 5-furandimethanol, and the established process has excellent chemical selectivity. The total 0.013mol of 5-hydroxymethylfurfural in the 10mL reaction system is completely converted into 2, 5-furandimethanol, and the concentration of the 2, 5-furandimethanol reaches 1300mM.

Example 7: process amplification and product identification for synthesizing high concentration 2, 5-furandimethanol by combining double enzyme coupling catalytic process with substrate fed-batch process

The reaction system of example 5 was expanded to 300mL, and alcohol dehydrogenase YahK lyophilized powder and glucose dehydrogenase BmGDH _M6 The method comprises the steps of adding a 5-hydroxymethylfurfural stock solution as a fed-batch substrate by using a microinjection pump of a constant-pressure constant-flow pump company, wherein the fed-batch substrate is a glucose aqueous solution of 1.2M, constant flow acceleration of the 5-hydroxymethylfurfural and the glucose aqueous solution is 357.1 mu mol/min, stopping feeding after feeding for 11 hours, and carrying out gas phase detection by taking samples at fixed time, wherein the cumulative addition concentration of the 5-hydroxymethylfurfural is 800mM, and the addition concentration ratio of the 5-hydroxymethylfurfural to the glucose is 1:1.

As a result, as shown in FIG. 9, the conversion of 5-hydroxymethylfurfural was >99% in 1 to 11 hours, and it was calculated that 0.24mol of 5-hydroxymethylfurfural (30.27 g) was converted into 2, 5-furandimethanol (30.75 g) in the 300mL reaction system, and the cumulative concentration of the product was 800mM, whereby the space-time yield of 2, 5-furandimethanol was 224 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, the ethyl acetate is removed from the upper organic phase by vacuum rotary evaporation, and the product 2, 5-furandimethanol is directly obtained.

The substrates and products in the reaction solution were confirmed to be 5-hydroxymethylfurfural and 2, 5-furandimethanol, respectively, by gas chromatography-mass spectrometry analysis, as shown in fig. 10 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 2, 5-furandimethanol by reducing 5-hydroxymethylfurfural through double enzyme coupling catalysis is characterized by comprising the following steps: freeze-drying thalli obtained by respective induced expression of alcohol dehydrogenase gene engineering bacteria and glucose dehydrogenase gene engineering bacteria, mixing the obtained alcohol dehydrogenase freeze-dried bacterial powder with glucose dehydrogenase freeze-dried bacterial powder to be used as a catalyst, taking 5-hydroxymethylfurfural as a substrate, glucose as an auxiliary substrate and NADP (sodium phosphate) ⁺ The reaction system is formed by taking buffer solution with pH value of 4-9 as a reaction medium, and the 2, 5-furandimethanol is obtained after the reaction is completed under the conditions of 20-50 ℃ and 400 rpm.

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 comprise, but are not limited to, alcohol dehydrogenase YsADH with a nucleotide sequence shown as SEQ ID NO.1, alcohol dehydrogenase AdhP with a nucleotide sequence shown as SEQ ID NO.3, alcohol dehydrogenase YahK with a nucleotide sequence shown as SEQ ID NO.5 and alcohol dehydrogenase YjgB 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 genes include, but are not limited to, glucose dehydrogenase BmGDH _M6 The nucleotide sequence is shown as SEQ ID NO. 9.

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 4, wherein the catalyst is added in an amount of 20 to 40g/L to the reaction system; the final concentration of the substrate is 500-1300mM, and the concentration ratio of the substrate to the 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 temperature is 30 ℃ and the reaction medium is 50mM Tris-HCl buffer at pH 7.0.

7. The method of claim 5, wherein the substrate and co-substrate are added to the reaction system in one portion when the substrate addition is less than or equal to 700 mM; when the substrate addition amount is higher than 700mM, the substrate and the cosubstrate are respectively added by adopting a constant-speed continuous flow method.

8. The method of claim 7, wherein the fed-batch substrate is a stock solution of 5-hydroxymethylfurfural and the fed-batch co-substrate is an aqueous solution of glucose in the range of 1.2 to 1.8M; the substrate and the auxiliary substrate are fed at the speed of 10-360 mu mol/min for 1-18 h respectively, and the reaction is continued for 0-7 h after the fed-batch is finished; the substrate and the co-substrate flow have the same acceleration, and the cumulative addition amount of the substrate is 500-1300mM.

9. The method of claim 8, wherein in a 10mL reaction system, the constant flow acceleration of the furfural and the glucose is 11.9 mu mol/min, the feeding time is 18h, and the reaction is continued for 7h after the feeding is finished; in a 300mL reaction system, the constant flow acceleration of furfural and glucose is 357.1 mu mol/min, and the reaction is stopped after 11h of feeding.

10. The method of claim 1, wherein the alcohol dehydrogenase lyophilized powder and the glucose dehydrogenase lyophilized powder are 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%, 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 for 12h at 12-36 ℃ 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; and (3) pre-freezing the obtained wet thalli for two days at the temperature of minus 20 ℃, and freeze-drying the thalli in a freeze dryer at the temperature of minus 40 ℃ for 48 hours to obtain each freeze-dried bacterial powder.

Images