Nadhph-dependent alcohol dehydrogenase ecyjgb mutant and application thereof

Abstract

This invention discloses an NADPH-dependent alcohol dehydrogenase EcYjgB mutant and its applications. The mutant is based on the wild-type alcohol dehydrogenase EcYjgB with mutations occurring at one or more of the following sites: S46, E51, W52, F54, T92, I108, A114, P116, I118, N240, G262, V264, L265, and A286. The amino acid sequence of the wild-type alcohol dehydrogenase EcYjgB is shown in SEQ ID. 1. This mutant is used to catalyze the selective reduction of high concentrations of 5-hydroxymethylfurfural or furfural to synthesize 2,5-furandiethanol or furfuryl alcohol, respectively. Compared to the wild-type enzyme, this mutant exhibits significantly improved catalytic efficiency and significantly reduced substrate inhibition at high substrate concentrations. It is green and efficient, requires no organic reagents, shortens reaction time, and significantly reduces the difficulty and cost of subsequent separation and purification.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and green biomanufacturing, and relates to a mutant of NADPH-dependent alcohol dehydrogenase EcYjgB and its application in the synthesis of 2,5-furandiethanol and furfuryl alcohol. Background Technology

[0002] 2,5-Furfural (BHMF) is a structurally symmetrical and stable furan glycol, an important precursor for the production of bio-based polymers (such as polyesters, polyurethanes, and polyethers), biofuels, and pharmaceutical intermediates. Furfuryl alcohol is mainly used in the production of furan resins, tetrahydrofurfuryl alcohol, and pharmaceutical intermediates; it is also a good solvent for varnishes and pigments, and a rocket fuel. Currently, there are many methods for the chemical catalytic synthesis of BHMF and furfuryl alcohol, typically using hydrogen as a hydrogen donor and a metal catalyst to catalyze the reduction of 5-hydroxymethylfurfural (HMF) or furfural. In contrast, biocatalytic synthesis of BHMF and furfuryl alcohol offers advantages such as mild reaction conditions, high reaction selectivity, and a more environmentally friendly process. Selecting suitable enzymes / cells as biocatalysts for the large-scale production of BHMF or furfuryl alcohol has significant economic and practical value.

[0003] The NADPH-dependent alcohol dehydrogenase EcYjgB, derived from *Escherichia coli*, belongs to the cinnamyl alcohol dehydrogenase (CAD) subfamily within the medium-chain dehydrogenase / reductase (MDR) superfamily. This enzyme exhibits high catalytic activity towards aldehyde substrates with aromatic rings (such as HMF), making it a highly efficient catalyst for the enzymatic synthesis of BHMF. However, HMF severely inhibits EcYjgB's substrate activity; its catalytic efficiency decreases significantly with increasing substrate concentration, leading to low reaction efficiency and high product separation costs, thus limiting the industrial and large-scale application of this biocatalytic process. Therefore, there is an urgent need to develop an alcohol dehydrogenase with reduced substrate inhibition that maintains good catalytic performance even at high substrate concentrations. In view of this, the present invention is proposed. Summary of the Invention

[0004] The main objective of this invention is to provide an alcohol dehydrogenase EcYjgB mutant to solve the problems of strong substrate inhibition and poor catalytic activity in the catalytic reduction of HMF to BHMF by wild-type alcohol dehydrogenase EcYjgB in the prior art.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] According to a first aspect of the present invention, a mutant of NADPH-dependent alcohol dehydrogenase EcYjgB is provided, comprising a protein mutated based on the wild-type alcohol dehydrogenase EcYjgB shown in SEQ ID NO.1, wherein the mutation includes mutations occurring at any one or more of the following sites: S46, E51, W52, F54, T92, I108, A114, P116, I118, N240, G262, V264, L265, A286.

[0007] Furthermore, each of the above mutations is independently selected from the following: S46A, E51D, W52F, F54G or F54I or F54L, T92S or T92Y, I108F, A114C or A114G or A114I or A114M or A114S or A114V or A114Y, P116S, I118R or I118S, N240I or N240L or N240S or N240T, G262A or G262S, V264G, L265E or L265S, A286G, where the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid.

[0008] Further, the mutations include at least one of the following: S46A; E51D; W52F; F54G; F54I; F54L; T92S; T92Y; I108F; A114C; A114G; A114I; A114M; A114S; A114V; A114Y; P116S; I118R; I118S; N240I; N240L; N240S; N240T; G262A; G262S; V 264G; L265E; L265S; A286G; A114C+I118S; A114C+P116S; A114I+P116S; G262A+A286G; G262S+L265 E; G262S+V264G; N240I+A286G; N240T+V264G; P116S+I118S; S46A+G262A; S46A+N240T; S46A+T92S; S46A+T92Y; S46A+V264G; S46A+W52F; T92S+A286G; T92S+N240T; T92Y+A286G; T92Y+G262A; T92Y+N240I; T92Y+N240T; V264G+A286G; W52F+A286G; W52F+F54G; W52F+G262A; W52F+N240I; W52F+N240T; W52F+T92Y; W52F+V264G; S46A+W52F+N240T; S46A+W52F+F54G; A114C+P116S+I118S; G262A+V264G+L265E; G262S+V264G+L265E; T92Y+N240T+A 286G; E51D+W52F+F54G; W52F+F54G+N240T; W52F+F54G+A286G; S46A+W52F+L265 E; E51D+W52F+F54G+N240T; E51D+W52F+F54G+L265E; E51D+W52F+F54G+I118S; E 51D+W52F+F54G+A286G; E51D+W52F+F54G+T92Y; E51D+W52F+F54G+V264G+L265E.

[0009] A recombinant plasmid, said recombinant plasmid being a pET-28a(+) plasmid carrying the gene encoding the EcYjgB mutant alcohol dehydrogenase of claim 3 or 4.

[0010] Preferably, the recombinant plasmid also carries the glucose dehydrogenase gene.

[0011] Preferably, the glucose dehydrogenase gene is as shown in SEQ ID. 3; the recombinant plasmid is a pET-Duet-1 plasmid carrying the above two genes.

[0012] A recombinant bacterium, wherein the recombinant bacterium is obtained by introducing the recombinant plasmid into Escherichia coli BL21(DE3).

[0013] Application of the recombinant bacteria in the synthesis of 2,5-furandiethanol or furfuryl alcohol.

[0014] The alcohol dehydrogenase mutant is used in the form of crude enzyme solution, lyophilized crude enzyme solution, pure enzyme, or whole cells. Preferably, wet bacterial cells obtained after induction of recombinant bacteria are used as catalysts, 5-hydroxymethylfurfural (HMF) or furfural is used as substrate, glucose is used as co-substrate, sodium bicarbonate or calcium carbonate is used to neutralize the acid produced in the reaction, phosphate buffer is used as the reaction medium, and 2,5-furandiethanol (BHMF) or furfuryl alcohol is obtained after the reaction.

[0015] The structural formulas of HMF, BHMF, furfural, and furfuryl alcohol are as follows:

[0016]

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The alcohol dehydrogenase EcYjgB mutant provided by the present invention has significantly improved catalytic efficiency and reduced substrate inhibition under higher concentrations of 5-hydroxymethylfurfural or furfural conditions compared with the wild-type enzyme. This is beneficial for the reaction system to operate under higher substrate loading conditions. It is green and efficient, and does not require the addition of organic reagents such as ethyl acetate or vinyl acetate to shorten the reaction time. This makes the process easier to achieve continuous and stable operation, and significantly reduces the difficulty and cost of subsequent separation and purification.

[0019] (2) Compared with free enzyme catalysis, using whole-cell catalysts can not only reduce the cost of catalyst preparation, but also because enzyme proteins have better stability under the protection of cell membranes; in addition, compared with enzyme catalysis, intracellular coenzyme cycling is easier to achieve and does not require the addition of expensive coenzymes. Attached Figure Description

[0020] Figure 1 The plasmid map of the co-expression vector pETDuet-YjgB-GDH constructed in Example 4.

[0021] Figure 2 The reaction process curve for the expanded synthesis of BHMF catalyzed by the co-expressed recombinant bacteria in Example 12 is shown.

[0022] Figure 3The nuclear magnetic resonance hydrogen spectrum of the BHMF prepared in Example 12 was expanded. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. The following embodiments are used to illustrate the present invention, but should not be used to limit the scope of the present invention.

[0024] The pET28a vector, pETDuet-1 plasmid, and E. coli BL21(DE3) used in the examples were all purchased from Novagen.

[0025] Example 1: Obtaining the EcYjgB mutant of endogenous alcohol dehydrogenase in Escherichia coli

[0026] The amino acid sequence of wild-type Escherichia coli alcohol dehydrogenase EcYjgB is shown in SEQ ID NO.1, and its gene sequence is shown in SEQ ID NO.2. The gene sequence of glucose dehydrogenase is shown in SEQ ID NO.3.

[0027] The EcYjgB mutant of endogenous alcohol dehydrogenase in *E. coli* was constructed using whole-plasmid PCR. The recombinant vector pET-28a-EcYjgB, carrying the wild-type *E. coli* endogenous alcohol dehydrogenase EcYjgB, was used as a template. Appropriate primers were designed for PCR amplification. The product was confirmed by agarose gel electrophoresis, and the template was removed by digestion with Dpn I enzyme. Sequencing confirmed the successful acquisition of the recombinant vector expressing the *E. coli* endogenous alcohol dehydrogenase EcYjgB mutant.

[0028] Example 2: Expression of Escherichia coli endogenous alcohol dehydrogenase EcYjgB mutant and preparation of crude enzyme, crude enzyme lyophilized powder, and pure enzyme.

[0029] The recombinant expression vector of the E. coli endogenous alcohol dehydrogenase EcYjgB mutant constructed in Example 1 was transformed into E. coli BL21(DE3) competent cells and plated onto Luria-Bertani (LB) solid medium containing kanamycin sulfate resistance (50 μg / mL). The cells were incubated at 37 °C for 12–16 h until transformants emerged. Single transformants were then inoculated into LB medium containing kanamycin sulfate resistance (50 μg / mL) and incubated overnight at 37 °C and 220 rpm for 12–16 h. Then, the transformed cells were inoculated into LB medium at a ratio of 1% (volume percentage) and incubated at 37 °C and 220 rpm until OD (digestive growth rate) was reached. 600The pH value was approximately 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.3 mM, and the cells were induced and cultured at 20 °C for 20 h. The cells were then collected by centrifugation at 8000 rpm for 5 min at 4 °C. The collected cells were whole cells. The cells were washed with 0.85% (w / v) physiological saline, repeated twice. Afterward, the cells were sonicated and centrifuged at 12000 rpm for 20 min. The supernatant was the crude enzyme solution. The crude enzyme solution was freeze-dried to obtain the crude enzyme lyophilized powder. The crude enzyme solution was purified using a nickel affinity chromatography column to obtain the pure enzyme solution.

[0030] Example 3: Enzyme activity assay of E. coli endogenous alcohol dehydrogenase EcYjgB mutant

[0031] The purified enzyme solution obtained in Example 2 was added to 1 mL of 30°C phosphate buffer (50 mM, pH 7.0) containing HMF (final concentration 20 mM) and NADPH (final concentration 0.2 mM). Enzyme activity was determined by detecting the change in NADPH absorbance at 340 nm during the reaction using a UV-Vis spectrophotometer. One unit of enzyme activity (U) was defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute under the above conditions. The results are shown in Table 1.

[0032] The formula for calculating enzyme activity is: Enzyme activity (U) = E w ×V×10 3 / (6220×L)

[0033] E w V: Change in absorbance at 340 nm per minute; L: Volume of reaction liquid, mL; L: Optical path distance, cm.

[0034] Table 1 Enzyme activity of Escherichia coli endogenous alcohol dehydrogenase EcYjgB mutant

[0035]

[0036] Table 2 Enzyme activity of Escherichia coli endogenous alcohol dehydrogenase EcYjgB mutant

[0037]

[0038] Table 3 Enzyme activity of Escherichia coli endogenous alcohol dehydrogenase EcYjgB mutant

[0039]

[0040] Table 4 Changes before and after the mutation site

[0041]

[0042] Example 4: Construction of a recombinant vector co-expressing the EcYjgB mutant and glucose dehydrogenase

[0043] This embodiment uses the construction of pETDuet-YjgB-GDH, pETDuet-YjgB (S46A+W52F) / GDH, and pETDuet-YjgB (W52F+N240T) / GDH as examples to illustrate the construction of recombinant vectors co-expressing EcYjgB mutants and glucose dehydrogenase (GDH). Using the recombinant vectors pET-28a-YjgB, pET-28a-YjgB (S46A+W52F), and pET-28a-YjgB (W52F+N240T) obtained in Example 1 as templates, the target gene fragment with sticky ends was obtained by PCR fragment amplification. The primers used were YB-F and YB-R (Table 5). Using the empty pET-Duet-1 plasmid as a template, the linearized vector fragment was obtained by PCR fragment amplification. The primers used were DUET1-F and DUET1-R. The target gene fragment with sticky ends and the linearized vector fragment were assembled using a seamless cloning kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). The gene fragment of E. coli endogenous alcohol dehydrogenase EcYjgB or its mutant was inserted into the first multiple cloning site (MCS-1) of the empty pET-Duet-1 plasmid, yielding pETDuet-YjgB, pETDuet-YjgB (S46A+W52F), and pETDuet-YjgB (W52F+N240T).

[0044] Using the GDH-containing plasmid pET-28a-GDH as a template, the target gene fragment with sticky ends was obtained by PCR fragment amplification. The primers used were GDH-F and GDH-R. Using pETDuet-YjgB, pETDuet-YjgB (S46A+W52F), and pETDuet-YjgB (W52F+N240T) as templates, linearized vector fragments were obtained by PCR fragment amplification, using primers DUET2-F and DUET2-R. The target GDH gene fragment with sticky ends and the linearized vector fragment were assembled using a seamless cloning kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). The GDH gene fragment was inserted into the second multiple cloning site (MCS-2) of pETDuet-YjgB, pETDuet-YjgB (S46A+W52F), and pETDuet-YjgB (W52F+N240T), respectively, to obtain pETDuet-YjgB-GDH. Figure 1), pETDuet-YjgB (S46A+W52F) / GDH and pETDuet-YjgB (W52F+N240T) / GDH.

[0045] Table 5 Primer sequences used to construct recombinant vectors

[0046]

[0047] Example 5: Whole-cell catalysis of 500 mM HMF synthesis of BHMF by mutant YjgB(S46A+W52F)

[0048] 500 mM HMF, 1000 mM glucose, and 600 mM NaHCO3 were added to 3 mL of phosphate buffer (200 mM, pH 7.0). Finally, 50 mg / mL (wet weight) of the co-expressing recombinant strain E. coli / pETDuet-YjgB(S46A+W52F) / GDH was added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, small amounts of the reaction solution were periodically taken, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 2.5 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 98%.

[0049] Example 6: Whole-cell catalysis of 500 mM HMF synthesis of BHMF by mutant YjgB(W52F+N240T)

[0050] 500 mM HMF, 1000 mM glucose, and 600 mM NaHCO3 were added to 3 mL of phosphate buffer (200 mM, pH 7.0). Finally, 50 mg / mL (wet weight) of the co-expressing recombinant strain E. coli / pETDuet-YjgB(W52F+N240T) / GDH was added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, small amounts of the reaction solution were periodically taken, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 3 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 92%.

[0051] Example 7: Whole-cell catalysis of 800 mM HMF synthesis of BHMF by mutant YjgB(S46A+W52F)

[0052] 800 mM HMF, 1.6 M glucose, and 960 mM NaHCO3 were added to 3 mL of phosphate buffer (200 mM, pH 7.0). Finally, 50 mg / mL (wet weight) of the co-expressing recombinant strain E. coli / pETDuet-YjgB (S46A+W52F) / GDH was added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, small amounts of the reaction solution were periodically taken, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 6 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 93%.

[0053] Example 8: Whole-cell catalysis of 1 M HMF synthesis of BHMF by mutant YjgB(S46A+W52F)

[0054] 1 M HMF, 2 M glucose, and 1.5 M calcium carbonate were added to 3 mL of phosphate buffer (200 mM, pH 7.0), followed by 50 mg / mL (wet weight) of the co-expressing recombinant E. coli / pETDuet-YjgB (S46A+W52F) / GDH. The reaction was carried out at 30 °C and 150 rpm. Small amounts of the reaction solution were periodically taken during the reaction, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 9 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 97%.

[0055] Example 9: Synthesis of BHMF catalyzed by crude enzyme solution of mutant YjgB(S46A+W52F)

[0056] Add 300 mM HMF, 600 mM glucose, 450 mM calcium carbonate, and 1 mM NADP to 3 mL of phosphate buffer (200 mM, pH 7.0). + Finally, 0.8 mg / mL of the mutant YjgB (S46A+W52F) crude enzyme solution and 1 mg / mL of glucose dehydrogenase (GDH) were added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, small amounts of the reaction solution were periodically taken, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 4 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 96%.

[0057] Example 10: Synthesis of BHMF catalyzed by pure enzyme solution of mutant YjgB(S46A+W52F)

[0058] Add 300 mM HMF, 600 mM glucose, 450 mM calcium carbonate, and 1 mM NADP to 3 mL of phosphate buffer (200 mM, pH 7.0). + Finally, 0.3 mg / mL of purified enzyme solution of the mutant YjgB (S46A+W52F) and 1 mg / mL of glucose dehydrogenase (GDH) were added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, a small amount of reaction solution was taken periodically, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 3 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 97%.

[0059] Example 11: Whole-cell catalytic synthesis of furfuryl alcohol by mutant YjgB(S46A+W52F)

[0060] 300 mM furfural, 600 mM glucose, and 360 mM NaHCO3 were added to 3 mL of phosphate buffer (200 mM, pH 7.0). Finally, 50 mg / mL (wet weight) of the co-expressed recombinant strain E. coli / pETDuet-YjgB(S46A+W52F) / GDH was added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, small amounts of the reaction solution were periodically taken, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of furfuryl alcohol was determined by HPLC. After 2 h of reaction, the furfural conversion rate was >99%, and the furfuryl alcohol yield was 96%.

[0061] Example 12: Whole-cell catalysis of HMF synthesis of BHMF by mutant YjgB(S46A+W52F)

[0062] In a 100 mL serum bottle, add 50 mL of phosphate buffer (200 mM, pH 7.0), then add 500 mM HMF, 1000 mM glucose, 600 mM NaHCO3, and finally add 50 mg / mL (wet weight) of E. coli / pETDuet-YjgB (S46A+W52F) / GDH wet cells. Incubate at 30 °C and 150 rpm. After 4 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 96%. Figure 2 The space-time yield is 15.5 g·L⁻¹. -1·h -1 After the reaction was complete, the reaction solution was collected, placed in a separatory funnel, and extracted five times with an equal volume of ethyl acetate. The organic phase was collected, and anhydrous sodium sulfate was added to remove water. After filtration, ethyl acetate was removed by rotary evaporation to obtain 2.78 g of BHMF crystals, with a separation yield of 90%. Detection by 1H NMR spectroscopy showed that the obtained BHMF crystals had high purity. Figure 3 ).

[0063] Control Example 1: E. coli / pETDuet-YjgB-GDH synthesized BHMF through whole-cell catalysis of 500 mM HMF.

[0064] 500 mM HMF, 1000 mM glucose, and 600 mM NaHCO3 were added to 3 mL of phosphate buffer (200 mM, pH 7.0). Finally, 50 mg / mL (wet weight) of the co-expressing recombinant strain E. coli / pET-Duet-YjgB-GDH was added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, small amounts of the reaction solution were periodically taken, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 7 h of reaction, the HMF conversion rate was >99%, and the BHMF yield was 95%.

[0065] Control Example 2: E. coli / pETDuet-YjgB-GDH synthesized BHMF through whole-cell catalysis of 800 mM HMF.

[0066] 800 mM HMF, 1.6 M glucose, and 960 mM NaHCO3 were added to 3 mL of phosphate buffer (200 mM, pH 7.0). Finally, 50 mg / mL (wet weight) of the co-expressing recombinant strain E. coli / pETDuet-YjgB-GDH was added, and the reaction was carried out at 30 ℃ and 150 rpm. During the reaction, small amounts of the reaction solution were periodically taken, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 10 h of reaction, the HMF conversion rate was 4%, and the BHMF yield was 3%.

[0067] Control Example 3: E. coli / pETDuet-YjgB-GDH synthesized BHMF through whole-cell catalysis of 1000 mM HMF.

[0068] 1 M HMF, 2 M glucose, and 1.2 M NaHCO3 were added to 3 mL of phosphate buffer (200 mM, pH 7.0), followed by 50 mg / mL (wet weight) of the co-expressing recombinant strain E. coli / pETDuet-YjgB-GDH. The reaction was carried out at 30 °C and 150 rpm. Small amounts of the reaction solution were periodically taken during the reaction, inactivated in a boiling water bath, and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic syringe filter, and the yield of BHMF was determined by HPLC. After 10 h of reaction, the HMF conversion rate was 3%, and the BHMF yield was 2%.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0070] SEQ ID NO.1:

[0071] MSMIKSYAAKEAGGELEVYEYDPGELRPQDVEVQVDYCGICHSDLSMIDNEWGFSQYPLVAGHEVIGRVVALGSAAQDKGLQVGQRVGIGWTARSCGHCDACISGNQINCEQGAVPTIMNRGGFAEKLRADWQWVIPLPENIDIESAGPLLCGGITVFKPLLMHHITAT SRVGVIGIGGLGHIAIKLLHAMGCEVTAFSSNPAKEQEVLAMGADKVVNSRDPQALKALAGQFDLIINTVNVSLDWQPYFEALTYGGNFHTVGAVLTPLSVPAFTLIAGDRSVSGSATGTPYELRKLMRFAARSKVAPTTELFPMSKINDAIQHVRDGKARYRVVLKADF

[0072] SEQ ID NO.2:

[0073]

[0074] SEQ ID NO.3:

[0075] 。

Claims

1. A mutant of the NADPH-dependent alcohol dehydrogenase EcYjgB, characterized in that, The mutant is a mutation based on the wild-type alcohol dehydrogenase EcYjgB at any one or more of the following sites: S46, E51, W52, F54, T92, I108, A114, P116, I118, N240, G262, V264, L265, and A286; the amino acid sequence of the wild-type alcohol dehydrogenase EcYjgB is shown in SEQ ID.

1.

2. The mutant according to claim 1, characterized in that, The mutations include at least one of the following: S46A; E51D; W52F; F54G; F54I; F54L; T92S; T92Y; I108F; A114C; A114G; A114I; A114M; A114S; A114V; A114Y; P116S; I118R; I118S; N240I; N240L; N240S; N240T; G262A; G262S; V264G; L265E; L265S; A286G; A114C+I118S; A114C+P116S; A114I+P116S; G262A+A2 86G; G262S+L265E; G262S+V264G; N240I+A286G; N240T+V264G; P116S+I118S; S46A +G262A; S46A+N240T; S46A+T92S; S46A+T92Y; S46A+V264G; S46A+W52F; T92S+A286 G; T92S+N240T; T92Y+A286G; T92Y+G262A; T92Y+N240I; T92Y+N240T; V264G+A286G; W52F+A286G; W52F+F54G; W52F+G262A; W52F+N240I; W52F+N240T; W52F+T92Y; W52F+V264G; S46A+W52F+N240T; S46A+W52F+F54G; A114C+P116S+I118S; G262A+V264G+L265E; G262S+V264G+L265E; T92Y+N240T+A286G; E51D+W52 F+F54G; W52F+F54G+N240T; W52F+F54G+A286G; S46A+W52F+L265E; E51D+W5 2F+F54G+N240T; E51D+W52F+F54G+L265E; E51D+W52F+F54G+I118S; E51D+W 52F+F54G+A286G; E51D+W52F+F54G+T92Y; E51D+W52F+F54G+V264G+L265E.

3. The gene encoding the EcYjgB mutant of the alcohol dehydrogenase as described in claim 1 or 2.

4. A recombinant plasmid, characterized in that, The recombinant plasmid is a pET-28a(+) plasmid carrying the gene encoding the EcYjgB mutant alcohol dehydrogenase as described in claim 3 or 4.

5. The recombinant plasmid according to claim 4, characterized in that, The recombinant plasmid also carries the glucose dehydrogenase gene.

6. The recombinant plasmid according to claim 5, characterized in that, The glucose dehydrogenase gene is shown in SEQ ID. 3; the recombinant plasmid is the pET-Duet-1 plasmid carrying the above two genes.

7. A recombinant bacterium, characterized in that, The recombinant bacteria is obtained by introducing the recombinant plasmid according to any one of claims 4 to 6 into Escherichia coli BL21(DE3).

8. The use of the recombinant bacteria of claim 7 in the synthesis of 2,5-furandiethanol or furfuryl alcohol.

9. The application according to claim 8, characterized in that, The wet cells obtained after induction of recombinant bacteria were used as catalysts, 5-hydroxymethylfurfural or furfural as substrates, glucose as co-substrate, sodium bicarbonate or calcium carbonate to neutralize the acid produced in the reaction, and phosphate buffer as the reaction medium. After the reaction, 2,5-furandiethanol or furfuryl alcohol was obtained.

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