Alcohol dehydrogenase mutant and application thereof in synthesis of furfuryl alcohol

Abstract

The application discloses an alcohol dehydrogenase mutant and application thereof in synthesis of furfuryl alcohol, and the amino acid sequence of the alcohol dehydrogenase mutant is shown in SEQ ID NO. 3. The mutant YahK-Y114W improves the preference for coenzyme NADH, so that the catalytic efficiency of the mutant for furfural can reach the level of a wild type using coenzyme NADPH. Wet mycelium expressing the mutant YahK-Y114W is used as a biocatalyst, 500 mM furfural is used as a substrate, 500 mM glucose is used as a co-substrate, glucose dehydrogenase is used as an auxiliary enzyme, and NADH is used as a coenzyme to construct a reaction system, and the reaction is carried out at 30 DEG C and 600 rpm for 6 h; the yield of furfuryl alcohol can reach 76.04%, compared with the wild type enzyme, the yield is increased by 12.59%, the preference of the alcohol dehydrogenase for the coenzyme is successfully changed, and the catalytic activity is greatly improved.

Description

(I) Technical Field

[0001] This invention belongs to the field of biocatalysis and relates to an alcohol dehydrogenase mutant YahK-Y114W and its application in the catalytic reduction of furfural to furfuryl alcohol. (II) Background Technology

[0002] Nicotinamide coenzymes are widely present in redox reactions and play an important role in life activities. Most redox reactions require the coenzyme NAD(P)H to proceed smoothly. Redox enzymes show a preference for either NAD(H) or NADP(H). Compared to NAD(H), NADP(H) is approximately five times more expensive and less stable. Due to the needs of synthetic biology research and the cost requirements of industrial production, the modification of redox enzymes to achieve coenzyme specificity has attracted considerable attention from researchers.

[0003] Furfural, containing a furan ring, serves as a bridge connecting biomass resources and high-value-added chemicals. Among the reduction products of furfural, furfuryl alcohol is one of the most important downstream products, widely applicable in the chemical industry and possessing significant economic value and practical importance. Alcohol dehydrogenases catalyze the reduction of furfural to furfuryl alcohol, a reaction requiring the participation of nicotinamide coenzyme. In the bioreduction of furfural, highly active alcohol dehydrogenases tend to favor NADP(H). Therefore, modifying NADP(H)-biased alcohol dehydrogenases using protein engineering techniques to convert them from coenzyme-biased to NAD(H)-biased can help reduce economic costs in actual production processes. Furthermore, constructing a coenzyme regeneration system can effectively address the coenzyme demand problem in oxidoreductase systems, ensuring a continuous supply of coenzymes. (III) Summary of the Invention

[0004] The purpose of this invention is to provide an alcohol dehydrogenase mutant and its application in the catalytic reduction of furfural to furfuryl alcohol. This invention selects the alcohol dehydrogenase YahK gene from *Escherichia coli* as the research object. Through molecular docking and site-directed mutagenesis, a YahK-Y114W alcohol dehydrogenase mutant that prefers the coenzyme NAD(H) was successfully obtained. The yield of furfuryl alcohol synthesized by alcohol dehydrogenase YahK using NADP(H) is 63.45%, while the modified YahK-Y114W alcohol dehydrogenase mutant achieves a yield of 76.04% when using the coenzyme NAD(H) to catalyze the reduction of furfural to furfuryl alcohol.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides an alcohol dehydrogenase mutant (denoted as YahK-Y114W), which is obtained by mutating tyrosine at position 114 of the amino acid sequence shown in SEQ ID NO.1 to tryptophan. The amino acid sequence of the alcohol dehydrogenase mutant YahK-Y114W is shown in SEQ ID NO.3.

[0007] The present invention also provides a gene encoding the alcohol dehydrogenase mutant YahK-Y114W, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0008] This invention also relates to a recombinant vector containing the encoding gene of the alcohol dehydrogenase mutant YahK-Y114W and a genetically engineered bacterium constructed from the vector. The recombinant vector is based on pET28a, and the genetically engineered bacterium uses E. coli BL21(DE3) as the host bacterium.

[0009] The genetically engineered bacteria were obtained by the following method: using plasmid pET28a-YahK as a template, the full plasmid was amplified by reverse PCR using primers with mutant bases. The PCR product was digested with DpnI enzyme to methylate the template, and the enzyme digestion product was transformed into E. coli BL21(DE3) to obtain the engineered bacteria E. coli BL21(DE3) / pET28a-YahK-Y114W containing the alcohol dehydrogenase mutant YahK-Y114W gene. The plasmid containing the recombinant alcohol dehydrogenase mutant encoding gene was named pET28a-YahK-Y114W.

[0010] Furthermore, this invention also provides an application of the alcohol dehydrogenase mutant YahK-Y114W in the catalytic synthesis of furfuryl alcohol from furfural. The application involves using wet cells of the engineered bacterium *E. coli* BL21(DE3) / pET28a-YahK-Y114W, containing the alcohol dehydrogenase mutant encoding gene, as a catalyst. Furfural is used as a substrate, and NADH, a coenzyme, and a co-substrate are added. A pH 4-9 buffer solution is used as the reaction medium to construct the reaction system. The reaction is carried out at 20-50°C and 500 °C. After the reaction is completed at -800 rpm (preferably at 30℃ and 600 rpm for 6 hours), a furfuryl alcohol-containing reaction solution is obtained. The reaction solution is then separated and purified to obtain furfuryl alcohol. The coenzymes include glucose dehydrogenase, isopropanol dehydrogenase, and formate dehydrogenase. The cosubstrate includes glucose, isopropanol, and sodium formate. The coenzymes and cosubstrate constitute a coenzyme system: glucose dehydrogenase / glucose system, isopropanol dehydrogenase / isopropanol system, and formate dehydrogenase / sodium formate system.

[0011] Furthermore, in the reaction system, the catalyst dosage is 20-100 g / L based on wet bacterial cells, preferably 80 g / L; the amount of furfural added is 300-500 mM, preferably 500 mM; the amount of NADH added is 0.1-0.5 mM, preferably 0.2 mM; and the ratio of co-substrate to substrate concentration is 1:0.5-2.5, preferably 1:1.

[0012] Furthermore, the glucose dehydrogenase, isopropanol dehydrogenase, and formate dehydrogenase are each added in the form of wet cells obtained by fermentation culture of engineered bacteria containing the corresponding coding genes. The amount of wet cells added is 20-100 g / L (the mass ratio of wet cell to catalyst is 1:0.2-5), preferably 40 g / L; the amino acid sequences of the glucose dehydrogenase, isopropanol dehydrogenase, and formate dehydrogenase are SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, respectively.

[0013] Further, the wet bacterial cells were prepared as follows: The engineered strain *E. coli* BL21(DE3) / pET28a-YahK-Y114W, containing the gene encoding the alcohol dehydrogenase mutant *YahK-Y114W*, was inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 8-12 h to obtain a seed culture; the seed culture was then inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% and cultured at 37°C and 200 rpm until OD... 600 The concentration was 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM. The mixture was induced overnight at 24°C and 200 rpm. The induction culture was centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was discarded and the wet cells were collected.

[0014] The glucose dehydrogenase, isopropanol dehydrogenase, and formate dehydrogenase were each prepared by fermentation culture of engineered bacteria containing the corresponding coding gene to obtain wet cells, using the same method as engineered bacteria containing the coding gene of alcohol dehydrogenase mutant.

[0015] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following: the alcohol dehydrogenase mutant YahK-Y114W, compared with wild-type YahK, prefers to utilize the coenzyme NADH, and its catalytic ability is superior to that of YahK utilizing the coenzyme NADPH. Using the wet cell E. coli BL21(DE3) / pET28a-YahK-Y114W expressing the mutant YahK-Y114W as a biocatalyst, with furfural 500mM as the substrate and NADH 0.2mM as the coenzyme, and selecting a glucose dehydrogenase / glucose cycling system, the reaction was carried out at 30℃ and 600rpm for 6h, and the yield of furfuryl alcohol reached 76.04%, which is higher than the product yield of 63.04% when YahK utilizes the coenzyme NADPH. (iv) Description of the attached drawings

[0016] Figure 1 This is a schematic diagram of furfural synthesized into furfuryl alcohol by alcohol dehydrogenase catalysis.

[0017] Figure 2 Agarose gel electrophoresis patterns of PCR products of YahK and its mutants; lane M, marker;

[0018] Lanes 1-14, YahK, V39L, K176E, C107I, Y114W, A159D, T162Q, K177R, T205N, S207K, E208A, S225Q, M293F, A209R.

[0019] Figure 3 SDS-PAGE analysis of wet cells induced to express YahK and its mutants; Lane M, Marker; Lanes 1-14, YahK, V39L, K176E, C107I, Y114W, A159D, T162Q, K177R, T205N, S207K, E208A, S225Q, M293F, A209R.

[0020] Figure 4 Agarose gel electrophoresis image of the PCR product encoding the alcohol dehydrogenase mutant YahK-Y114W; lane M is the marker; lane 1 is the gene encoding the alcohol dehydrogenase YahK; lane 2 is the gene encoding the alcohol dehydrogenase mutant YahK-Y114W.

[0021] Figure 5 SDS-PAGE image of the alcohol dehydrogenase mutant YahK-Y114W; lane M is the marker; lane 1 is the uninduced E. coli BL21(DE3) / pET28a-YahK bacterial culture; lane 2 is the induced E. coli BL21(DE3) / pET28a-YahK wet bacterial culture; lane 3 is the purified enzyme solution obtained by isolating and purifying the alcohol dehydrogenase YahK after induction; lane 4 is the purified enzyme solution obtained by isolating and purifying the alcohol dehydrogenase mutant YahK-Y114W.

[0022] Figure 6 A standard curve for protein concentration determination using the BCA method.

[0023] Figure 7 GC chromatograms of furfuryl alcohol and furfural standards.

[0024] Figure 8SDS-PAGE images of wet cells containing three helper enzymes; lane M is the marker; lane 1, E. coli BL21(DE3) / pET28a-ADH; lane 2, E. coli BL21(DE3) / pET28a-CtFDH; lane 3, E. coli BL21(DE3) / pET28a-BmGDH M6 .

[0025] Figure 9 The effect of reaction temperature on the conversion rate of furfural catalyzed by YahK-Y114W.

[0026] Figure 10 The effect of pH on the conversion rate of furfural catalyzed by YahK-Y114W.

[0027] Figure 11 For NAD + Effect of YahK-Y114W addition on furfural conversion rate.

[0028] Figure 12 The effect of the furfural to glucose concentration ratio on the furfural conversion rate catalyzed by YahK-Y114W.

[0029] Figure 13 The effect of the mass ratio of alcohol dehydrogenase to coenzyme on the furfural conversion rate catalyzed by YahK-Y114W.

[0030] Figure 14 This is a graph showing the substrate conversion rate of the alcohol dehydrogenase YahK and its mutant YahK-Y114W during the furfural reaction. (V) Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0032] Example 1: Construction and culture of genetically engineered E. coli BL21(DE3) / pET28a-YahK

[0033] The genome of *E. coli* BL21(DE3) was obtained using the TaKaLa genomic DNA extraction kit as a template. PCR amplification was then performed using primers YahK-F and YahK-R to obtain the gene encoding the alcohol dehydrogenase YahK (nucleotide sequence shown in SEQ ID NO.2, amino acid sequence shown in SEQ ID NO.1). Using a one-step cloning kit, the obtained alcohol dehydrogenase YahK gene was inserted into the BamHI and XhoI sites of the pET28a linearized vector to obtain the recombinant plasmid pET28a-YahK. The recombinant plasmid pET28a-YahK was transformed into competent *E. coli* BL21(DE3) cells to obtain the recombinant strain *E. coli* BL21(DE3) / pET28a-YahK. Verification of the constructed genetically engineered bacteria by plasmid extraction and sequencing confirmed the correct insertion of the alcohol dehydrogenase gene.

[0034] The recombinant bacteria were streaked onto LB solid medium containing 100 μg / mL kanamycin and incubated overnight at 37°C. A single colony was picked and inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin and incubated overnight at 37°C and 200 rpm to obtain the seed culture. Fresh seed culture was then diluted with 40% glycerol at a 1:1 volume ratio and stored at -80°C.

[0035] The preservation solution of strain E. coli BL21(DE3) / pET28a-YahK was removed from the -80℃ freezer and thawed on ice. 5 μL of the preserved bacterial solution was streaked onto an LB agar plate containing 100 μg / mL kanamycin resistance and incubated at 37℃ for 12 h for activation. A single colony was picked and transferred to 50 mL of LB liquid medium containing 100 μg / mL kanamycin and incubated at 37℃ in a 200 rpm shaking incubator for 12 hours. The bacterial solution was then sequenced. After successful sequencing, the obtained seed culture was confirmed as the genetically engineered E. coli BL21(DE3) / pET28a-YahK. The plasmid pET28a-YahK was extracted from the E. coli BL21(DE3) / pET28a-YahK bacterial solution.

[0036] The LB liquid medium consisted of 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and distilled water as the solvent, with a pH of 7.0–7.5.

[0037] LB solid medium is LB liquid medium with 20 g / L agar added.

[0038] YahK-F:

[0039] 5'-CAAATGGGTCGC GGATCC ATGAAGATCAAAGCTGTTGGTGC-3';

[0040] YahK-R:

[0041] 5'-GGTGGTGGTGGTG CTCGAG TCAGTCTGTTAGTGTGCGATTATCG-3',

[0042] Example 2: Rational design and key site identification of the YahK mutant of alcohol dehydrogenase

[0043] Alcohol dehydrogenase YahK is derived from *E. coli*, with a nucleotide sequence (SEQ ID NO.2) of 1050 bp and a corresponding amino acid sequence (SEQ ID NO.1) of 350 aa. Its molecular weight is approximately 37.98 kDa. A crystal structure of alcohol dehydrogenase YahK is available, and this structure was used as the basis for modeling. The YahK model was docked with furfural and NADPH, respectively, and the model was visualized and analyzed in Pymol to determine the coenzyme binding sites, which are 13 amino acid residues: V39, C107, Y114, A159, T162, K176, K177, T205, S207, E208, A209, S225, and M293.

[0044] The primers used to mutate each coenzyme binding site and replace amino acid residues are shown in Table 1.

[0045] Table 1. Primers for whole-plasmid PCR to construct mutant libraries

[0046]

[0047]

[0048] Using plasmid pET28a-YahK from Example 1 as a template, and employing the upstream and downstream primers listed in Table 1, the corresponding coding gene was amplified using a specific PCR program within the PCR system shown in Table 2. The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min; followed by a cycle of denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and holding at 72℃ for 80 s, repeated 30 times; finally, holding at 72℃ for 5 min.

[0049] Table 2 PCR system for full plasmid mutation

[0050]

[0051] After amplification, the PCR products were detected by 0.8% agarose gel electrophoresis. A bright band was visible at 8000 bp under ultraviolet light, which matched the theoretical value of the plasmid. The results are as follows. Figure 2 As shown.

[0052] The target fragment was directly transformed into the host bacterium *Escherichia coli* BL21(D3), and 100 μL of competent cells were thawed on ice. A final concentration of 30 ng / μL of the mutant PCR product was added to the competent cells. The mixture was then plated on LB agar containing 100 μg / mL kan resistance and incubated overnight upside down at 37°C. This yielded the mutant engineered bacterium *E. coli* BL21(DE3) / pET28a-YahK-M, where M represents the mutant.

[0053] The above-mentioned E. coli BL21(DE3) / pET28a-YahK-M mutant was induced to express using the method described in Example 4. The expression effect was verified by SDS-PAGE electrophoresis of wet bacterial cells, and the results are as follows: Figure 3 As shown, the molecular weight of YahK protein is approximately 37.98 kDa. The protein bands expressed by each mutant strain are significantly thicker and located at around 40 kDa, which is the correct size.

[0054] To verify the preference of different mutants for two coenzymes, crude enzyme solutions were prepared from the original YahK strain and the mutant strain according to the method in Example 4, and the crude enzyme activities were determined using NADPH and NADH respectively according to the method in Example 5. Among these mutants, only the mutant Y114W significantly altered the coenzyme preference. The crude enzyme activity of YahK using NADH was 0.54 U / mg, and the specific activity using NADPH was 0.75 U / mg, with a coenzyme NADH / NADPH crude enzyme activity ratio of 0.72; the crude enzyme activity of mutant Y114W using NADH was 1.25 U / mg, and the specific activity using NADPH was 0.25 U / mg, with a coenzyme NADH / NADPH crude enzyme activity ratio of 5; the coenzyme NADH / NADPH crude enzyme activity ratio increased from 0.72 to 5. The enzyme activity assay preliminarily screened out the YahK-Y114W mutant, which will be further verified using catalytic reaction results.

[0055] Example 3: Construction of recombinant expression plasmid pET28a-YahK-Y114W and genetically engineered bacteria E.coli BL21(DE3) / pET28a-YahK-Y114W.

[0056] 1. Recombinant expression plasmid pET28a-YahK-Y114W

[0057] Using the plasmid pET28a-YahK prepared in Example 1 as a template, a single-point mutation of the key amino acid (Y114W) was performed using the Y114W-F / R primers. Reverse PCR amplification was performed using the reaction system in Table 3, and an appropriate amount of PCR product was verified by agarose gel electrophoresis. Figure 4 As shown, after electrophoresis, a bright band can be seen at 8000bp when observed under ultraviolet light, which is consistent with the theoretical value of the plasmid.

[0058] The complete plasmid was cloned using reverse PCR and transformed into E. coli BL21(DE3). The plasmid was extracted and sequenced, and the sequencing results were analyzed using software. The mutant plasmid pET28a-YahK-Y114W was successfully obtained. The nucleotide sequence of the mutant YahK-Y114W is shown in SEQ ID NO.4, and its amino acid sequence is shown in SEQ ID NO.3.

[0059] The primers are as follows:

[0060] Y114W-F:5'-ACCGGCACC TGG AACTCGCCGACGCC-3';

[0061] Y114W-R: 5'-CGGCGAGTT CCA GGTGCCGGTCATGTGAT-3'.

[0062] The reverse PCR amplification system is shown in Table 3.

[0063] Table 3 PCR amplification reaction system

[0064]

[0065] The PCR reaction process was as follows: pre-denaturation: 95℃, 5 min; complete denaturation: 95℃, 15 s; annealing: 62℃, 15 s; extension: 72℃, 90 s; 30 cycles; second extension: 72℃, 5 min; then incubate at 4℃.

[0066] 2. Genetically engineered E. coli BL21(DE3) / pET28a-YahK-Y114W

[0067] The PCR product was digested at 37°C for 1 hour to remove the methylated template. The digestion system is shown in Table 4.

[0068] Table 4. Digestion system for methylated template in PCR products

[0069]

[0070]

[0071] The PCR product digested with DpnI (the nucleotide sequence of the mutant gene YahK-Y114W carried by the plasmid is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.3) was directly transformed into the host bacterium *E. coli* BL21(DE3) to obtain the genetically engineered *E. coli* strain *E. coli* BL21(DE3) / pET28a-YahK-Y114W. After colony PCR verification, the transformants were inoculated into LB broth containing 100 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. The cells were collected by centrifugation, the plasmid was extracted, and sequenced. Sequencing analysis revealed that tyrosine at position 114 was successfully converted to tryptophan.

[0072] Example 4: Induced expression and purification of alcohol dehydrogenase YahK and its mutant YahK-Y114W

[0073] Genetically engineered bacteria E. coli BL21(DE3) / pET28a-YahK and E. coli BL21(DE3) / pET28a-YahK-Y114W were inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 8–12 h to obtain seed culture. The seed culture was then inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% and cultured at 37°C and 200 rpm until OD (dose-to-volume) reached. 600 The concentration was 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM. The mixture was induced overnight at 24°C and 200 rpm to obtain the induction culture medium. The induction culture medium was then centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was discarded, and the wet bacterial cells were collected.

[0074] Add an appropriate amount of Tris-HCl buffer (pH 7.0, 50mM) to the above wet bacterial cells at a ratio of 1g wet bacterial cells to 20mL Tris-HCl buffer (pH 7.0, 50mM). Sonicate the mixture at 400W for 10min (1s working, 3s rest). Centrifuge the mixture at 4℃ and 8000rpm for 10min. Repeat the centrifugation three times to obtain the supernatant, which is the crude enzyme solution.

[0075] According to Ni-NTA metal chelate affinity chromatography (purchased from Bio-Rad, abbreviated as Ni), 2+ (Column, inner diameter 1.6 cm, height 15 cm) Instructions for use: Take 15 mL of crude enzyme solution and load it into the pre-equilibrated Ni... 2+In the column, the target protein and other contaminating proteins were eluted sequentially with elution buffers containing 5 mM imidazole, 50 mM imidazole, 100 mM imidazole, 200 mM imidazole, and 500 mM imidazole (elution composition: imidazole of the corresponding concentration, 300 mM sodium chloride, solvent: 50 mM Tris-HCl buffer, pH 6.0). The elution rate was 2.0 mL / min for each concentration of elution, and 3 column volumes were eluted with each concentration of elution buffer. The eluent corresponding to the 200 mM imidazole eluent was collected and desalted and concentrated by centrifugation at 4 °C and 5000 rpm for 30 min using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The retentate was used as the pure enzyme solution of YahK and YahK-Y114W and stored at -20 °C for later use.

[0076] The purified enzyme solutions of alcohol dehydrogenase YahK and its mutant YahK-Y114W were verified by SDS-PAGE gel electrophoresis, and the results are as follows: Figure 5 As shown, the alcohol dehydrogenase mutant YahK-Y114W appears as a single band after SDS-PAGE electrophoresis. The theoretical subunit size of the alcohol dehydrogenase mutant YahK-Y114W is approximately 37.98 kDa, and its apparent size on SDS-PAGE electrophoresis matches the theoretical molecular weight.

[0077] Example 5: Determination of the activity and kinetic parameters of alcohol dehydrogenase YahK and its mutant YahK-Y114W

[0078] 1. Determine the protein concentration standard curve

[0079] The protein concentration of the crude enzyme solution was determined using a BCA (bicinchoninic acid) protein concentration assay kit. This method primarily utilizes the reaction of proteins with Cu under alkaline conditions. 2+ Reduced to Cu + This ion can form a purple complex with solution A in the BCA kit. This complex has absorbance at 562 nm and exhibits good linearity.

[0080] BCA Standard Curve: Following the BCA kit instructions, BCA Reagent A to BCA Reagent B were mixed at a ratio of 50:1 (volume ratio) to prepare the BCA working solution. 200 μL of the BCA working solution was added to each well of the microplate, followed by 20 μL of appropriately diluted BCA protein. Each sample was tested in triplicate. After vortexing, the microplate was incubated at 37°C for 30 min. The standard curve was then measured using the microplate reader. 562 The absorbance was determined by testing. A standard curve was plotted with protein concentration on the x-axis and absorbance on the y-axis. The linear relationship was expressed by the formula y = 0.0011x + 0.1648, and the standard deviation was R. 2 =0.999, such as Figure 6As shown.

[0081] The method for determining the protein concentration in the sample is as follows: The pure enzyme solution of alcohol dehydrogenase YahK and its mutant YahK-Y114W prepared by the method in Example 4 was diluted with ultrapure water to the linear range that meets the standard curve. 20 μL of diluted sample was added to 200 μL of BCA working solution; the mixture was shaken and placed at 37°C for 30 min; the absorbance value at 562 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The protein content of the sample was calculated based on the absorbance value and the protein standard curve.

[0082] 2. Enzyme activity assay method

[0083] The enzyme activity of alcohol dehydrogenase was calculated by measuring the decrease in absorbance of coenzyme (NADPH or NADH) at 340 nm using a single-factor kinetic method on an ELISA reader. Enzyme activity detection system: 10 mM furfural substrate, 0.1 mM coenzyme (NADPH or NADH), 1 μL of purified alcohol dehydrogenase YahK and its mutant YahK-Y114W prepared according to the method in Example 4, were added to a final volume of 300 μL with 50 mM Tris-HCl buffer (pH 7.0). The mixture was added to an ELISA plate and incubated at 30°C for 5 min. The absorbance at 340 nm was then measured. Three parallel experiments were performed each time. Enzyme activity was calculated according to formula (1), and specific enzyme activity was calculated according to formula (2). The results are shown in Table 5. The concentration of purified YahK protein was 10.2 mg / mL, and the specific enzyme activity measured using coenzyme NADPH was 5.28 U / mg. The concentration of YahK-Y114W protein was 9.1 mg / mL, and the specific enzyme activity measured using coenzyme NADH was 5.95 U / mg, which was 0.67 U / mg higher than that of YahK before modification. After purification, the ratio of coenzyme NADH / NADPH activity increased from 0.54 to 1.81, confirming that the preference of YahK for coenzyme was successfully changed.

[0084] Enzyme activity unit U is defined as the ability of an enzyme to convert 1 μmol of coenzyme (NADPH or NADH) into coenzyme (NADP) within 1 minute at 30°C. + or NAD + The amount of enzyme required.

[0085] The formulas for calculating the volumetric enzyme activity and specific enzyme activity of alcohol dehydrogenase are as follows:

[0086]

[0087]

[0088] △A represents the change in absorbance; t represents the reaction time in min; L represents the optical path length in cm; V1 represents the system volume in mL; V2 represents the enzyme solution volume in mL.

[0089] Table 5. Determination of pure enzyme activity

[0090]

[0091] 3. Kinetic parameters of YahK and YahK-Y114W were determined.

[0092] The enzyme activity assay in step 2 was performed by adding different concentrations of furfural (0.3mM, 0.5mM, 1mM, 2mM, 10mM, 12mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, 100mM) to measure the activity of alcohol dehydrogenase and its mutants. The furfural concentration was plotted on the x-axis, and the enzyme activity on the y-axis, and the results were calculated using PrismDemo software. Since the activities of YahK and YahK-Y114W decreased at high furfural concentrations, substrate inhibition kinetics were used for fitting, and the results are shown in Table 6. For wild-type YahK, k... cat / K m It is 27.32s -1 ·mM -1 For the mutant YahK-Y114W, when NADH is used as a cofactor, k cat / K m It is 29.33s -1 ·mM -1 It is 2.59 times that of the form with NADPH as a cofactor, and 2.01 s higher than the wild type. -1 ·mM -1 This change further indicates that the mutant YahK-Y114W prefers NADH as a cofactor.

[0093] Table 6. Kinetic parameters of YahK and YahK-Y114W

[0094]

[0095] Example 6: GC analysis of the enzymatic catalytic process for the production of furfural from furfural to furfuryl alcohol

[0096] The catalytic effect of furfural on the conversion to furfuryl alcohol was detected by gas chromatography. The gas chromatograph was a SHMADZUGC-2014, the gas column was a BGB-174 (30m × 250μm × 0.25μm); the FID detector was set to 250℃; N2 was used as the carrier gas at a flow rate of 1mL / min; the split ratio was 1:20; the injection volume was 1.0μL; and the injection port temperature was 250℃.

[0097] The temperature program for furfural and furfuryl alcohol was as follows: hold at 80℃ for 9 min, then increase to 190℃ at a rate of 10℃ / min for a total of 20 min. The retention times for furfuryl alcohol and furfural were 8.4 min and 9.0 min, respectively. A suitable amount of the reaction solution was centrifuged to remove bacterial cells, and the supernatant was extracted with 800 μL of ethyl acetate. After extraction, 500 μL of the upper organic phase was collected and an appropriate amount of anhydrous sodium sulfate was added to remove water. 100 μL of the supernatant was transferred to a clean gas chromatography bottle, and the contents of furfural and furfuryl alcohol in the sample were determined by gas chromatography.

[0098] Gas chromatograms of the substrate furfural and the product furfuryl alcohol standards are shown below. Figure 7 As shown.

[0099] Substrate conversion rate = Amount of substrate participating in the reaction / Total amount of substrate.

[0100] Example 7: Three Coenzyme Cycling Systems and Their Applications

[0101] The coenzyme glucose dehydrogenase BmGDH was separately added. M6 The coding genes for isopropanol dehydrogenase ADH (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6), isopropanol dehydrogenase ADH (amino acid sequence as shown in SEQ ID NO. 7, nucleotide sequence as shown in SEQ ID NO. 8), and formate dehydrogenase CtFDH (amino acid sequence as shown in SEQ ID NO. 9, nucleotide sequence as shown in SEQ ID NO. 10) were inserted into the BamHI and XhoI sites of the pET28a vector to obtain the corresponding recombinant plasmids. The recombinant plasmids were then transformed into competent E. coli BL21(DE3) cells to obtain the corresponding recombinant strain E. coli BL21(DE3) / pET28a-BmGDH. M6 E. coli BL21(DE3) / pET28a-ADH and E. coli BL21(DE3) / pET28a-CtFDH were constructed. After plasmid extraction and sequencing verification, the gene insertion was confirmed to be correct. The engineered bacteria were streaked onto LB agar containing 100 μg / mL Kan and incubated overnight at 37°C. Single colonies were picked and incubated overnight in 50 mL LB broth containing 100 μg / mL Kan at 37°C and 200 rpm to obtain seed culture. Fresh seed culture was then mixed with 40% glycerol at a 1:1 volume ratio and stored at -80°C.

[0102] E. coli BL21(DE3) / pET28a-BmGDH to be stored at -80℃ M6Glycerol tubes containing E. coli BL21(DE3) / pET28a-ADH and E. coli BL21(DE3) / pET28a-CtFDH strains were thawed, and 10 μL of the bacterial suspension was streaked onto LB agar plates containing 100 μg / mL Kan, and incubated upside down at 37°C for 12–16 h. Single colonies were then selected and cultured overnight at 37°C and 200 rpm in 50 mL LB liquid medium containing 100 μg / mL Kan to obtain seed culture. Induction of expression and cell collection were the same as in Example 4. SDS-PAGE analysis of the induced wet cells is shown below. Figure 8 As shown, glucose dehydrogenase BmGDH was obtained. M6 Wet bacterial cells, isopropanol dehydrogenase ADH wet bacterial cells, and formate dehydrogenase CtFDH wet bacterial cells should be stored at -20°C for later use.

[0103] Glucose dehydrogenase / glucose coenzyme cycle system: furfural final concentration 500mM, alcohol dehydrogenase YahK or alcohol dehydrogenase mutant YahK-Y114W wet cell final concentration 60g / L prepared by the method in Example 3, glucose dehydrogenase BmGDH M6 Final wet cell concentration: 60 g / L; final glucose concentration: 500 mM; coenzyme NAD... + The final concentration was 0.2 mM. A 10 mL reaction system was then formed by adding Tris-HCl buffer (pH 7.0, 50 mM). The pH was maintained at 7.0 by adding 1 M NaOH dropwise. The reaction was carried out at 600 rpm and a 30°C water bath for 6 hours. A 200 μL sample of the reaction solution was taken, and the contents of furfural and furfuryl alcohol in the sample were determined using gas chromatography as described in Example 6. The substrate conversion rate was calculated, and the results are shown in Table 7.

[0104] Isopropanol dehydrogenase / isopropanol coenzyme cycling system: furfural final concentration 500 mM, alcohol dehydrogenase YahK or alcohol dehydrogenase mutant YahK-Y114W wet cell final concentration 60 g / L prepared by the method in Example 3, isopropanol dehydrogenase ADH wet cell final concentration 60 g / L, isopropanol final concentration 500 mM, coenzyme NAD + The final concentration was 0.2 mM. A 10 mL reaction mixture was then added to Tris-HCl buffer (pH 7.0, 50 mM), and the pH was maintained at 7.0 by adding 0.5 M HCl dropwise. The reaction was carried out at 600 rpm and a 30°C water bath for 6 h. A 200 μL sample of the reaction solution was taken, and the contents of furfural and furfuryl alcohol in the sample were determined using gas chromatography as described in Example 6. The substrate conversion rate was calculated, and the results are shown in Table 7.

[0105] Formate dehydrogenase / sodium formate coenzyme cycle system: furfural final concentration 500 mM, alcohol dehydrogenase YahK or alcohol dehydrogenase mutant YahK-Y114W prepared by the method in Example 3 final concentration of wet cells 60 g / L, formate dehydrogenase CtFDH final concentration of wet cells 60 g / L, sodium formate final concentration 500 mM, coenzyme NAD + The final concentration was 0.2 mM. A 10 mL reaction mixture was then added to Tris-HCl buffer (pH 7.0, 50 mM), and the pH was maintained at 7.0 by adding 0.5 M HCl dropwise. The reaction was carried out at 600 rpm and a 30°C water bath for 6 hours. A 200 μL sample of the reaction solution was taken, and the contents of furfural and furfuryl alcohol in the sample were determined using gas chromatography as described in Example 6. The substrate conversion rate was calculated, and the results are shown in Table 7.

[0106] As shown in Table 7, the mutant YahK-Y114W exhibited the highest catalytic conversion rate with the glucose dehydrogenase cycle, followed by isopropanol dehydrogenase, while formate dehydrogenase showed the worst catalytic effect. Considering factors such as conversion efficiency, co-substrate price, regenerated enzyme stability, and byproduct separation, the glucose dehydrogenase cycle was selected.

[0107] Table 7 Comparison of conversion rates of different coenzyme cycling systems

[0108]

[0109] Example 8: Investigating the optimal reaction temperature for the alcohol dehydrogenase mutant YahK-Y114W to catalyze the reduction of furfural to furfuryl alcohol at different temperatures.

[0110] Reaction system: furfural final concentration 500mM, wet cells of alcohol dehydrogenase mutant YahK-Y114W, and glucose dehydrogenase BmGDH M6 The final concentration of wet bacterial cells was 60 g / L, the final glucose concentration was 500 mM (furfural to glucose ratio was 1:1), and the coenzyme NAD... + The final concentration was 0.2 mM. Tris-HCl buffer (pH 7.0, 50 mM) was added to bring the volume to 10 mL. The pH was maintained at 7.0 by adding 1 M NaOH dropwise. The reaction was carried out at 600 rpm in a water bath at 20-50°C (selected temperatures: 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C) for 6 hours. A 200 μL sample of the reaction solution was taken, centrifuged to remove bacterial cells, and the supernatant was extracted with 800 μL of ethyl acetate. After extraction, 500 μL of the upper organic phase was taken and an appropriate amount of anhydrous sodium sulfate was added to remove water. 100 μL of the supernatant was then used for gas chromatography analysis to determine the content of furfural and furfuryl alcohol in the sample, as described in Example 6.

[0111] The results are as follows Figure 9As shown, the highest conversion rate, reaching 75%, was achieved at a reaction temperature of 30℃. Within the temperature range of 30-35℃, the mutant maintained high activity; however, the conversion rate of furfural decreased sharply when the catalytic reaction temperature reached 45℃. Therefore, the temperature for this reaction system was determined to be 30℃.

[0112] Example 9: Investigating the optimal pH for the reduction of furfural to furfuryl alcohol catalyzed by the alcohol dehydrogenase mutant YahK-Y114W.

[0113] The reaction temperature in Example 8 was set to 30°C, and the reaction pH range was 4.0-9.0. Buffer solutions with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 were prepared. During the reaction, an automatic pH control system was used to maintain a constant pH. The alkali solution used for titration was 1M NaOH solution. Other operations were the same as in Example 8, and the results were as follows. Figure 10 As shown, the conversion rate increases with increasing pH from 4.0 to 7.0. When the pH is 7.0, the conversion rate of furfural by YahK-Y114W is 74.9% after 6 hours of reaction. When the pH exceeds 7.0, the enzyme activity decreases significantly, and the conversion rate is almost zero when the pH reaches 9.0. Therefore, the pH of the reaction system was selected as 7.0.

[0114] Example 10: Investigating the optimal amount of coenzyme added for the reduction of furfural to furfuryl alcohol catalyzed by the alcohol dehydrogenase mutant YahK-Y114W.

[0115] The reaction temperature in Example 8 was set to 30°C, and coenzyme NAD... + The final concentration was set to 0-0.5 mM (selecting 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mM). Other operations were the same as in Example 8, and the results were as follows: Figure 11 As shown, when NAD is not added + At that time, the conversion rate of furfural was 15.26%, verifying the fact that *E. coli* cells contain coenzymes and can spontaneously achieve coenzyme cycling. Figure 11 It is known that adding coenzymes increases the catalytic conversion rate, and this rate increases with increasing coenzyme concentration. When the coenzyme NAD... + At a concentration of 0.2 mM, the transformation efficiency of the mutant reached a relatively high level. However, when the coenzyme NAD... + When the concentration of the coenzyme exceeds 0.2 mM and continues to increase, the conversion rate only improves slightly. Therefore, considering the economics of industrial applications, the optimal coenzyme addition for this catalytic system is determined to be 0.2 mM.

[0116] Example 11: Investigating the optimal glucose addition for the reduction of furfural to furfural alcohol catalyzed by the alcohol dehydrogenase mutant YahK-Y114W

[0117] The reaction temperature in Example 8 was set to 30°C, and the final concentration ratio of furfural to glucose was set to 1:0.5-2.5 (selecting 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5). Other operations were the same as in Example 8. The results are as follows: Figure 12 As shown, increasing the amount of glucose as a co-substrate improves the conversion rate of furfural to some extent. When the final concentration ratio of furfural to glucose is 1:1, the conversion rate of furfural reaches a relatively high level of 73%. As the glucose concentration continues to increase, the increase in conversion rate is not significant. Therefore, a final concentration of furfural to glucose of 1:1 is used in the reaction.

[0118] Example 12: Optimal mass ratio of alcohol dehydrogenase to coenzyme in the reaction system of furfural reduction to furfuryl alcohol catalyzed by the alcohol dehydrogenase mutant YahK-Y114W.

[0119] In Example 8, the reaction temperature was set to 30°C. With the total amount of biocatalyst added (120 g / L) remaining constant, the alcohol dehydrogenase YahK-Y114W and glucose dehydrogenase BmGDH in the reaction system were... M6 The wet cell mass ratio was set to 0.2-5:1 (1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, and 5:1 were selected for the reaction, and other operations were the same as in Example 8. The results are as follows. Figure 13 As shown, the optimal catalytic system consists of alcohol dehydrogenase YahK-Y114W and glucose dehydrogenase BmGDH. M6 The wet cell mass ratio was 2:1, meaning that in a 10 mL system, the ratio of alcohol dehydrogenase YahK-Y114W to glucose dehydrogenase BmGDH was... M6 Add the ingredients in a ratio of 0.8g:0.4g.

[0120] Example 13: The reaction process of furfural reduction catalyzed by alcohol dehydrogenase YahK and its mutant YahK-Y114W

[0121] Wet cells of the genetically engineered E. coli BL21(DE3) / pET28a-YahK-Y114W, obtained through induced expression, were used as a catalyst at a concentration of 80 g / L. A final concentration of 500 mM furfural and 0.2 mM coenzyme NAD were added. + Glucose dehydrogenase BmGDH at a final concentration of 40 g / L M6 Wet bacterial cells and glucose (final concentration 500 mM) were used as the co-substrate to form a 10 mL reaction system, with Tris-HCl buffer (pH 7.0, 50 mM) as the reaction medium. The reaction was carried out at 30°C and 600 rpm. At regular intervals, 200 μL of the reaction solution was taken and the substrate conversion rate was analyzed by GC as described in Example 6. The results are as follows: Figure 14 As shown, the yield of furfuryl alcohol can reach 76.04% in 6 hours.

[0122] In the control group, the catalyst was replaced with wet cells of E. coli BL21(DE3) / pET28a-YahK obtained through induced expression, and the coenzyme was replaced with NADPH. Under the same reaction conditions, the yield of furfuryl alcohol was 63.45% after 6 hours. The results indicate that the molecularly modified YahK-Y114W can utilize the more inexpensive NADH as a coenzyme in catalytic reactions.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The alcohol dehydrogenase mutant was obtained by mutating tyrosine at position 114 of the amino acid sequence shown in SEQ ID NO.1 to tryptophan.

2. The encoding gene of the alcohol dehydrogenase mutant of claim 1.

3. A recombinant vector containing the gene encoded by claim 2.

4. A recombinant genetically engineered bacterium constructed from the recombinant vector of claim 3.

5. The application of the alcohol dehydrogenase mutant of claim 1 in the catalytic synthesis of furfural from furfural.

6. The application as described in claim 5, characterized in that, The application is as follows: using wet bacterial cells obtained by inducing expression of engineered bacteria containing an alcohol dehydrogenase mutant gene as a catalyst, furfural as a substrate, NADH, coenzyme, and co-substrate are added, and a pH 4-9 buffer solution is used as the reaction medium to form a reaction system. After the reaction is completed at 20-50 ℃ and 500-800 rpm, a furfural-containing reaction solution is obtained. The reaction solution is then separated and purified to obtain furfural alcohol. The coenzyme and co-substrate are one of the following combinations: glucose dehydrogenase and glucose, isopropanol dehydrogenase and isopropanol, formate dehydrogenase and sodium formate.

7. The application as described in claim 6, characterized in that, In the reaction system, the catalyst dosage is 20-100 g / L based on wet bacterial cells, the furfural dosage is 300-500 mM, and the NADH dosage is 0.1-0.5 mM; the co-substrate to substrate concentration ratio is 1:

1.

8. The application as described in claim 6, characterized in that, The glucose dehydrogenase, isopropanol dehydrogenase, and formate dehydrogenase are each added in the form of wet cells obtained by fermentation culture of engineered bacteria containing the corresponding coding genes, and the amount of wet cells added is 20-100 g / L.

9. The application as described in claim 8, characterized in that, The amino acid sequences of the glucose dehydrogenase, isopropanol dehydrogenase, and formate dehydrogenase are SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, respectively.

10. The application as described in claim 6, characterized in that, Engineered bacteria containing the alcohol dehydrogenase mutant encoding gene were inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured at 37 ℃ and 200 rpm for 8–12 h to obtain seed culture. The seed culture was then inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% and cultured at 37 ℃ and 200 rpm until OD reached. 600 The concentration was 0.6–0.8, and then IPTG was added to a final concentration of 0.2 mM. The mixture was induced overnight at 24 °C and 200 rpm. The induction culture was centrifuged at 4 °C and 8000 rpm for 10 min, the supernatant was discarded, and the wet cells were collected.

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