Use of an alcohol dehydrogenase phyadh and mutants thereof

Abstract

This invention discloses an alcohol dehydrogenase, PhyADH, and the applications of its mutants. The PhyADH described in this invention can catalyze reversible hydroxyl oxidation and carbonyl reduction reactions of various alcohols and aldehydes / ketones under mild aqueous conditions, completing the interconversion between alcohols and aldehydes / ketones. At pH 8.0 and 45°C, this enzyme exhibits good conversion ability for substrates such as benzyl alcohol, 2-phenylethanol, 3-phenylpropanol, p-methylbenzyl alcohol, and p-aminobenzyl alcohol. The optimal reaction temperature of PhyADH is approximately 45°C, and the optimal pH is 8.0. It retains high residual activity even after pre-incubation at 40-45°C. The alcohol dehydrogenase PhyADH provided by this invention offers a novel and highly efficient biocatalytic tool for the green and precise synthesis of alcohols and aldehydes / ketones, and has broad application prospects in the enzymatic synthesis of pharmaceutical intermediates and fine chemicals.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of an alcohol dehydrogenase, PhyADH, and its mutants. Background Technology

[0002] Alcohol dehydrogenases (ADHs) are a class of widely distributed oxidoreductases that typically use NAD(H) or NADP(H) as coenzymes to catalyze the oxidation of alcohols and the reduction of corresponding aldehydes and ketones. Since their initial discovery in the liver, numerous ADHs from various sources have been reported. These enzymes not only participate in the metabolism of alcohols such as ethanol in vivo, but also, due to their excellent stereoselectivity, are widely used in the biosynthesis of chiral drugs and fine chemicals, such as in the synthesis of the important chiral drug precursor ethyl 4-chloro-3-hydroxy-butyrate (CHBE).

[0003] Based on amino acid sequence length and metal ion dependence, ADHs are mainly classified into three categories: short-chain ADHs, medium-chain ADHs, and long-chain ADHs. Although the catalytic mechanism of alcohol dehydrogenases has been largely elucidated, different types of ADHs still exhibit significant differences in biochemical properties, such as optimal reaction pH and temperature, organic solvent tolerance, substrate spectrum range, and reaction kinetic parameters. These differences are mainly attributed to the diversity of enzyme proteins in their primary sequence and spatial structure. Meanwhile, currently commonly used industrial alcohol dehydrogenases generally suffer from poor stability, narrow substrate spectrum, and low tolerance to organic solvents, limiting their potential for application in practical production. Therefore, exploring novel, high-performance alcohol dehydrogenase resources and elucidating their spatial structures, especially enzymes derived from thermophilic or extremophile microorganisms, is of great significance for developing efficient and stable biocatalytic processes. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide an alcohol dehydrogenase, PhyADH.

[0005] Another object of the present invention is to provide an alcohol dehydrogenase PhyADH mutant.

[0006] Another object of the present invention is to provide the application of the above-mentioned alcohol dehydrogenase PhyADH and its mutants.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] An alcohol dehydrogenase, PhyADH, is at least one of the following:

[0009] (a) A protein with the amino acid sequence shown in SEQ ID NO.1;

[0010] (b) is a protein obtained by mutating the amino acid sequence shown in (a), with the mutation being one of the following:

[0011] V152D, V152E, V152R, V152S, V152T;

[0012] (c) is a protein obtained by mutation based on the amino acid sequence shown in (b), and the mutation in its sequence is one of the following:

[0013] T145R, T145S, T145E, T145D, T145L;

[0014] (d) is a protein obtained by mutation based on the amino acid sequence shown in (c), and the mutation in its sequence is one of the following:

[0015] N150H, N150T, N150S, N150E, N150D.

[0016] The nucleotide sequence of the alcohol dehydrogenase PhyADH is obtained according to the codon coding rules.

[0017] The gene encoding the alcohol dehydrogenase PhyADH is at least one of the following:

[0018] (a) The nucleotide sequence shown in SEQ ID NO.2;

[0019] (b) is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in (a), wherein the codons 454 to 456 of the nucleotide sequence are mutated to one of GAT, GAA, CGT, AGC, or ACC.

[0020] (c) is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in (b), wherein the codons 433 to 435 of the nucleotide sequence are mutated to one of CGT, AGC, GAA, GAT, or CTG;

[0021] (d) is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in (c), where the codons 478 to 480 of the nucleotide sequence are mutated to one of CAT, ACC, AGC, GAA, or GAT.

[0022] A recombinant expression vector comprising the gene encoding the alcohol dehydrogenase PhyADH.

[0023] The recombinant expression vector has a vector backbone of pET30a.

[0024] An engineered bacterium, comprising the aforementioned recombinant expression vector.

[0025] The starting strain of the engineered bacteria is Escherichia coli.

[0026] A PhyADH alcohol dehydrogenase crystal, formed by crystallizing the PhyADH protein, has a pyramidal morphology and a fine three-dimensional spatial structure containing 387 amino acids. The structure has a unit cell with a = 74.338 Å, b = 74.338 Å, c = 153.653 Å, α = β = γ = 90.0°, and the protein space group of the crystal is P43212.

[0027] The method for preparing the crystal includes the following steps:

[0028] Take the purified alcohol dehydrogenase PhyADH protein solution, adjust the concentration to 12.5 mg / mL, and inoculate it into an equal volume of crystallization buffer. Place the resulting crystallization solution on a plastic coverslip, then drop the crystallization solution upside down onto a crystallization plate, seal the gap in the middle with Vaseline, and place it in a biological incubator at 16℃ for crystal culture. After the crystals grow, collect them.

[0029] The crystal buffer solution is composed of PEG6000 and NaCl, wherein the mass percentage of PEG6000 is 10% and the concentration of NaCl is 2.0 mol / L.

[0030] The above-mentioned alcohol dehydrogenase PhyADH is used in catalyzing the conversion of alcohols into aldehydes and ketones.

[0031] The alcohol compound is benzyl alcohol; preferably at least one of benzyl alcohol, 2-phenylethanol, 1-phenyl-1-propanol, 3-phenylpropanol, p-methylbenzyl alcohol, p-aminobenzyl alcohol, p-hydroxybenzyl alcohol, and p-methoxybenzyl alcohol.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] The PhyADH enzyme and its modified mutant described in this invention can catalyze reversible hydroxyl oxidation and carbonyl reduction reactions of various alcohols and aldehydes / ketones under mild aqueous conditions, completing the interconversion between alcohols and aldehydes / ketones. At pH 8.0 and 45 °C, this enzyme mutant exhibits good conversion ability for substrates such as benzyl alcohol, 2-phenylethanol, 3-phenylpropanol, p-methylbenzyl alcohol, and p-aminobenzyl alcohol. The optimal reaction temperature of PhyADH is approximately 45 °C, and the optimal pH is 8.0. It retains high residual activity even after pre-incubation at 40-45 °C. The alcohol dehydrogenase PhyADH provided by this invention offers a novel and highly efficient biocatalytic tool for the green and precise synthesis of alcohols and aldehydes / ketones, and has broad application prospects in the enzymatic synthesis of pharmaceutical intermediates and fine chemicals. Attached Figure Description

[0034] Figure 1 This is the SDS-PAGE electrophoresis result of the protein after purification in Example 1.

[0035] Figure 2 These are the results of the basic enzymatic properties of PhyADH;

[0036] Figure 3 This is the time curve of the substrate catalyzed by PhyADH;

[0037] Figure 4 This is a protein crystal image of PhyADH;

[0038] Figure 5 This is a three-dimensional protein structure diagram of PhyADH. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0040] Example 1 Expression and purification of alcohol dehydrogenase PhyADH

[0041] 1.1 Construction and Induction Expression of Recombinant Engineered Bacteria

[0042] After digesting the pET30a(+) and the vector containing the alcohol dehydrogenase PhyADH (its nucleotide sequence is shown in SEQ ID No. 2) with EcoRI and SalI respectively, linearized pET30a(+) and PhyADH fragments were obtained. These fragments were then ligated using a seamless cloning method to obtain the recombinant plasmid pET30a(+)-PhyADH, which was transformed into *E. coli* BL21(DE3) competent cells via heat shock. The cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12–16 h. Single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 180 rpm. A 1% inoculum was then transferred to 50 mL of LB liquid medium (containing the same concentration of kanamycin) and cultured at 37°C with shaking at 180 rpm until OD500. 600 ≈0.8. Subsequently, a 5% inoculum was transferred to 400 mL of TB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 200 rpm until OD reached 0.8. 600 =0.6~0.8. Add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mmol / L, and induce culture at 21℃ for 20 h.

[0043] 1.2 Preparation of crude enzyme solution

[0044] After induction, bacterial cells were collected by centrifugation at 4500 rpm for 30 min at 4°C. The supernatant was discarded, and the cells were resuspended in buffer A (50 mmol / L sodium phosphate, 400 mmol / L NaCl, 100 mmol / L KCl, 40 mmol / L imidazole, pH 7.5) at a ratio of 10 mL per gram of wet bacterial cells. The suspension was placed in an ice bath, and cell disruption was performed using an ultrasonic disruptor with the following parameters: 5 s sonication, 5 s interval, total duration 25 min, power 80 W, and 4°C maintained throughout. The disrupted solution was centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was filtered through a 0.45 μm filter membrane to obtain the crude enzyme solution.

[0045] 1.3 Affinity chromatography purification

[0046] The crude enzyme solution was purified using a Ni-NTA affinity chromatography column. The column was first equilibrated with buffer A until the baseline stabilized. The pretreated crude enzyme solution was then loaded at a flow rate of 5 mL / min. After loading, elution with buffer A continued until the UV absorption baseline stabilized. Subsequently, linear or stepwise elution was performed using buffer B (50 mmol / L sodium phosphate, 400 mmol / L NaCl, 100 mmol / L KCl, 500 mmol / L imidazole, pH 7.5), and the elution peaks were collected in separate tubes and stored at 4°C for later use.

[0047] 1.4 Gel size exclusion chromatography purification

[0048] The protein sample collected by affinity chromatography was concentrated using a 10 kDa ultrafiltration tube (5000 rpm, 4℃) to a protein concentration of approximately 10 mg / mL. Further purification was performed using a HiLoad 16 / 60 Superdex 75 pg gel filtration chromatography column. The column was pre-equilibrated with crystallization buffer (20 mmol / L Tris-HCl, 100 mmol / L NaCl, pH 8.0) at a flow rate of 1 mL / min. After baseline stabilization, 1 mL of the concentrate was injected, and eluted with the same buffer and flow rate, collecting the main peak fraction. Protein purity was assessed by 12% SDS-PAGE electrophoresis. The results showed a single target band with a molecular weight of approximately 42 kDa, consistent with the theoretical value. The obtained sample was used for subsequent crystal culture and enzymatic property determination.

[0049] Through a series of heterologous expression operations using engineered bacteria, the alcohol dehydrogenase PhyADH was efficiently expressed in Escherichia coli. SDS-PAGE results showed that the purified sample had a single band at around 42 kDa, indicating that the target protein was successfully expressed and purified.

[0050] Example 2 Enzymatic characterization of alcohol dehydrogenase PhyADH

[0051] 2.1 Enzyme Activity Assay Method

[0052] Based on the principle that NADPH has a characteristic absorption peak at 340 nm, the enzyme activity of PhyADH is determined by monitoring the rate of change of absorbance at 340 nm in the reaction system. Enzyme activity unit (U) is defined as the amount of protein required to oxidize 1 μmol of NADPH per minute under the test conditions. Specific enzyme activity (U / mg) is defined as the enzyme activity units per milligram of enzyme protein. The standard reaction system (total volume 1 mL) is as follows: 950 μL phosphate buffer (50 mmol / L, pH 8.0), 50 μL NADPH solution (final concentration 0.5 mmol / L), 10 μL substrate solution (final concentration 1 mmol / L), and 10 μL purified PhyADH enzyme solution. After mixing the above reaction system at room temperature, it is immediately placed in a spectrophotometer, and the change in absorbance at 340 nm is recorded within 1 minute. The enzyme activity is calculated using the following formula:

[0053] U / mg = (Vt × ΔAbs) 340 ×10 3 ) / (c×Vs×e×L)

[0054] Vt: Total volume of the system (mL);

[0055] ΔAbs 340 Absorption difference at 340 nm;

[0056] c: Enzyme concentration (mg / mL);

[0057] Vs: Enzyme dosage (mL);

[0058] L: Optical path length of the cuvette, 1 cm;

[0059] e: Molar extinction coefficient 6.22 × 10 3 / (mol∙cm).

[0060] 2.2 Effect of temperature on the activity and stability of PhyADH

[0061] 2.2.1 Determination of the optimal reaction temperature

[0062] The oxidation activity of PhyADH against benzyl alcohol was measured at reaction temperatures of 30℃, 35℃, 40℃, 45℃, and 50℃ to investigate the effect of temperature on enzyme activity. The highest measured enzyme activity was set as 100%, and the relative enzyme activities at the other temperatures were calculated.

[0063] The optimal reaction temperature for PhyADH is 45℃. Within the range of 25-45℃, its relative enzyme activity gradually increases with increasing temperature; when the temperature exceeds 45℃, the enzyme activity drops sharply, with a residual activity of about 70% at 50℃ and almost complete inactivation at 60℃.

[0064] 2.2.2 Thermal stability determination

[0065] PhyADH enzyme solutions were incubated in the dark at 40℃, 45℃, and 50℃ for 0, 0.5, 1, 1.5, 2, 3, and 4 hours, respectively. After incubation, the solutions were quickly removed from the ice bath and cooled, and the remaining enzyme activity was measured. The residual enzyme activity under different conditions was calculated with the activity of the unincubated enzyme solution as 100%.

[0066] PhyADH maintained high residual activity after pre-incubation at 40°C; however, the enzyme inactivation rate accelerated significantly at 45°C and above. These results indicate that the PhyADH described in this invention possesses good thermal stability and is suitable for biocatalytic processes under medium- and high-temperature conditions.

[0067] 2.3 Effect of pH on PhyADH activity and stability

[0068] 2.3.1 Determination of the optimal reaction pH

[0069] Reaction systems were prepared using buffer solutions with different pH values ​​(4.0–10.0), and the enzyme activity of PhyADH was determined at 45°C. The buffer systems used included citrate-phosphate buffer (pH 4.0–6.0), phosphate buffer (pH 6.0–8.0), and glycine-NaOH buffer (pH 9.0–10.0). The highest measured enzyme activity was set as 100%, and the relative enzyme activities under the remaining pH conditions were calculated.

[0070] The optimal reaction pH for PhyADH is 8.0. The enzyme exhibits low activity under acidic conditions, but its activity rapidly increases within the pH range of 5.0-8.0, reaching a peak at pH 8.0. When the pH exceeds 9.0, the enzyme activity gradually diminishes.

[0071] 2.3.2 pH stability determination

[0072] PhyADH enzyme solutions were incubated with buffers of different pH values ​​(4.0-10.0) at 4°C in the dark for 0, 1, 2, 3, 4, and 5 hours. After incubation, the residual enzyme activity was measured, with the activity of the unincubated enzyme solution considered as 100%. PhyADH exhibited good stability at pH 8.0, with residual enzyme activity exceeding 80% after 6 hours of incubation.

[0073] 2.4 Substrate Spectral Analysis of PhyADH

[0074] The reaction system was maintained at a constant temperature using a constant-temperature water bath reactor. A 4 mL glass reaction flask was placed on a magnetic stirrer, and the catalytic activity of PhyADH on different aromatic alcohol substrates was determined under optimal reaction conditions (50 mM phosphate buffer, pH 8.0, 45℃). 100 μL of purified PhyADH enzyme solution (enzyme concentration 7.9 mg / mL), 5 mM substrate, 1% (v / v) DMSO, and 10 mM NADP were added. + Alternatively, NADPH and 50 mM phosphate buffer (to a final volume of 1 mL) were reacted at 45°C and 500 rpm for 4 h. After the reaction, the product was extracted with an equal volume of ethyl acetate and then quantitatively analyzed by gas chromatography (Agilent 8890, HP-5 column). The substrate and product were quantified using a standard curve method. (Reference: ACS Catal. 2018, 8, 8680-8684. DOI: 10.1021 / acscatal.8b02355)

[0075] Experimental results are as follows Figure 3As shown in Table 1, the results indicate that PhyADH can efficiently catalyze the oxidation of various aromatic alcohols, exhibiting a broad substrate spectrum, including but not limited to structural analogs such as benzyl alcohol, 2-phenylethanol, 3-phenylpropanol, p-methylbenzyl alcohol, and p-aminobenzyl alcohol. The catalytic products are the corresponding aromatic aldehydes or ketones (such as benzaldehyde). Table 1 shows that PhyADH exhibits the highest catalytic activity for benzyl alcohol, with a conversion rate of 42.13%. The conversion rates for other substrates are: 32.73% for 2-phenylethanol, 22.04% for 3-phenylpropanol, 36.64% for p-methylbenzyl alcohol, and 27.76% for p-aminobenzyl alcohol. (See catalytic time curves...) Figure 3 As shown in the figure, PhyADH typically reaches reaction equilibrium within 8 hours. These results confirm that PhyADH has broad application potential in the stereoselective synthesis of aromatic alcohols.

[0076] Table 1 Catalytic substrate spectrum of wild-type PhyADH

[0077]

[0078] Example 3: Crystal culture and structural analysis of alcohol dehydrogenase PhyADH

[0079] 3.1 Initial screening of crystal growth conditions

[0080] PhyADH crystals were cultured using the hanging drop vapor diffusion method. The purified PhyADH protein solution was replaced with 20 mmol / L Tris-HCl buffer (containing 100 mmol / L NaCl, pH 8.0) and concentrated to 10 mg / mL. Crystallization conditions were screened using Hampton Research's SaltRx series (HR2-110, HR2-112, HR2-144) and Rigaku's Wizard Classic 1–4 kits. The kit name, reagent number, protein concentration, culture temperature, and date were labeled on the side of the 24-well plate. 200 μL of reservoir solution was added to each well in the order of kit numbers. 1 μL of protein solution was added to the center of the coverslip, followed by 1 μL of reservoir solution from the corresponding well, ensuring thorough mixing without air bubbles. The coverslip was then inverted and placed over the wells, compacted, and sealed. The plate was then incubated at 16°C in a crystallizer.

[0081] 3.2 Optimization of Crystallization Conditions

[0082] After 7–14 days of cultivation, crystal growth was observed under an optical microscope. To optimize the growth of microcrystals or crystal clusters, a two-dimensional gradient optimization was performed using a base formulation of 10% PEG6000 and 2.0 mol / L NaCl. The final optimal crystal growth conditions were: protein concentration of 12.5 mg / mL, pool solution composition of 10% (w / v) PEG6000 and 2.0 mol / L NaCl, and growth temperature of 16℃.

[0083] 3.3 Crystal cryoprotection and data collection

[0084] Selected single crystals were placed in corresponding mother liquors and subjected to gradient dehydration treatment sequentially through cryoprotectant solutions containing 5%, 10%, 15%, and 25% (v / v) glycerol, with each step lasting approximately 10–20 seconds. The crystals were then packed into nylon loops and rapidly immersed in liquid nitrogen for cryopreservation.

[0085] Crystal diffraction data were collected at the BL17U1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF). Diffraction images were collected over a 360° range by rotating the crystal at a scan step of 0.1° at a cryogenic temperature of 100 K. The raw data were integrated, scaled, and corrected using the HKL-3000 software package.

[0086] 3.4 Structural Analysis and Refinement

[0087] Molecular replacement was used, with the known Fe²⁺-dependent alcohol dehydrogenase structure (PDB ID: 26GB) as the search model, and the phase was solved using the PHASER program. The initial model was manually corrected using COOT software, and then refined using restricted least squares with the REFMAC5 program. The relevant parameters of the final PhyADH crystal structure model are summarized in Table 2.

[0088] Table 2. Diffraction data and structural correction statistics of PhyADH crystals (an alcohol dehydrogenase).

[0089]

[0090] Example 4: Molecular modification and catalytic application verification of alcohol dehydrogenase PhyADH

[0091] 4.1 Design and Synthesis of Site-Directed Mutation Primers

[0092] To improve the conversion rate of the alcohol dehydrogenase PhyADH to its substrate, a series of mutation sites were designed, and mutant enzymes were obtained using mutant primers. The specific steps are as follows:

[0093] Using recombinant plasmid pET30a(+)-PhyADH as a template, site-directed mutagenesis was performed on selected target sites. Specific primers containing the mutation sites were designed using online primer design software based on the codon bias of *E. coli*. Primer synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0094] 4.2 Construction and Identification of Mutant Expression Vectors

[0095] Mutants were constructed using overlap extension PCR or rapid site-directed mutagenesis. Using pET30a(+)-PhyADH plasmid as a template, PCR amplification was performed using high-fidelity DNA polymerase. The reaction system and procedure are shown in Table 3. PCR products were digested with Dpn I restriction enzyme to remove methylated template plasmids, followed by PCR product purification.

[0096] The purified linearized mutant vector was transformed into *E. coli* Top 10 competent cells using a heat shock method. The transformed cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 12–16 h. Single colonies were picked and inoculated into LB liquid medium containing the same antibiotic and cultured overnight at 37°C with shaking at 220 rpm. Plasmids were extracted and sent to a sequencing company for bidirectional sequencing verification. After confirming the correct mutation site and the absence of unexpected mutations, the mutant expression vector was obtained.

[0097] 4.3 Expression and purification of mutants

[0098] The correctly sequenced mutant plasmid was transformed into the *E. coli* BL21(DE3) expression host. Shake-flask fermentation, induced expression, and protein purification were performed according to the method in Example 1 to obtain high-purity PhyADH mutant protein.

[0099] 4.4 Evaluation of the catalytic performance of mutants

[0100] Under optimal reaction conditions, the catalytic conversion rates of wild-type and a series of mutants for different aromatic alcohol substrates were determined, following the specific methods described in Example 2, section 2.4. The first round of mutations, based on the wild-type, involved mutating V152 to obtain five mutants: V152D, V152E, V152R, V152S, and V152T. The second round of mutations, based on V152R, involved mutating T145 to obtain V152R / T145R, V152R / T145S, V152R / T145E, V152R / T145D, and V152R. The third round of mutations, based on V152R / T145S, mutated N150 to obtain five mutants: V152R / T145S / N150H, V152R / T145S / N150T, V152R / T145S / N150S, V152R / T145S / N150E, and V152R / T145S / N150D.

[0101] The results are shown in Tables 3-5. By introducing multi-point combination mutations, the catalytic efficiency of the enzyme for a series of benzyl alcohol substrates was significantly improved.

[0102] In conversion experiments targeting primary alcohol substrates such as benzyl alcohol, 2-phenylethanol, and 3-phenylpropanol, the mutant PhyADH-V152R achieved conversion rates of 49.90%, 40.70%, and 33.14%, respectively (Table 4), representing increases of approximately 1.18-fold, 1.24-fold, and 1.5-fold compared to the wild-type enzyme (42.13%, 32.73%, and 22.04%, respectively). Furthermore, this mutant also exhibited improved conversion capabilities for other aromatic alcohol substrates. The mutant PhyADH-V152R / T145S (its amino acid sequence is shown in SEQ ID NO.3) achieved conversion rates of 61.53%, 46.38%, and 42.97%, respectively (Table 5), representing increases of approximately 1.46-fold, 1.42-fold, and 1.95-fold compared to the wild-type enzyme.

[0103] Table 3. Mutants of PhyADH mutation site V152 and their catalytic substrate spectra.

[0104]

[0105] Table 4. Mutants at the PhyADH-V152R mutation site T145 and their catalytic substrate profiles.

[0106]

[0107] Table 5. Mutants of PhyADH-V152R / T145S mutation site N150 and their catalytic substrate spectra.

[0108]

[0109] Specifically, in conversion experiments targeting primary alcohol substrates such as benzyl alcohol, 2-phenylethanol, and 3-phenylpropanol, the optimal mutant PhyADH-V152R / T145S / N150E achieved conversion rates of 82.57%, 62.83%, and 57.65%, respectively, representing improvements of approximately 1.96-fold, 1.92-fold, and 3.30-fold compared to the wild-type enzyme (42.13%, 32.73%, and 22.04%, respectively). Furthermore, this mutant also exhibited excellent conversion capabilities for other aromatic alcohol substrates (conversion rates around 80%). These results demonstrate that a mutant alcohol dehydrogenase with significantly enhanced catalytic performance was successfully obtained through structure-guided rational design, greatly expanding the application value of this enzyme in the field of industrial biocatalysis.

[0110] 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.

Claims

1. An alcohol dehydrogenase, PhyADH, characterized in that... It is at least one of the following: (a) A protein with the amino acid sequence shown in SEQ ID NO.1; (b) is a protein obtained by mutating the amino acid sequence shown in (a), with the mutation being one of the following: V152D, V152E, V152R, V152S, V152T; (c) is a protein obtained by mutating the amino acid sequence shown in (b), with the mutation being one of the following: T145R, T145S, T145E, T145D, T145L; (d) is a protein obtained by mutation based on the amino acid sequence shown in (c), and the mutation in its sequence is one of the following: N150H, N150T, N150S, N150E, N150D.

2. The alcohol dehydrogenase PhyADH according to claim 1, characterized in that: The nucleotide sequence of the alcohol dehydrogenase PhyADH is obtained according to the codon coding rules.

3. A gene encoding an alcohol dehydrogenase, PhyADH, characterized in that... It is at least one of the following: (a) The nucleotide sequence shown in SEQ ID NO.2; (b) is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in (a), wherein the codons 454 to 456 of the nucleotide sequence are mutated to one of GAT, GAA, CGT, AGC, or ACC; (c) is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in (b), wherein the codons 433 to 435 of the nucleotide sequence are mutated to one of CGT, AGC, GAA, GAT, or CTG; (d) is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in (c), where the codons 478 to 480 of the nucleotide sequence are mutated to one of CAT, ACC, AGC, GAA, or GAT.

4. A recombinant expression vector, characterized in that: Includes the gene encoding the alcohol dehydrogenase PhyADH as described in claim 3.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector has a vector backbone of pET30a.

6. An engineered bacterium, characterized in that: Includes the recombinant expression vector as described in claim 4 or 5.

7. A crystal of the alcohol dehydrogenase PhyADH, characterized in that: Formed by crystallization of the alcohol dehydrogenase PhyADH protein, the crystal has a square pyramidal morphology and a fine three-dimensional spatial structure containing 387 amino acids. The structure has a unit cell with a = 74.338 Å, b = 74.338 Å, c = 153.653 Å, α = β = γ = 90.0°, and the protein space group of the crystal is P43212.

8. The method for preparing the alcohol dehydrogenase PhyADH crystal according to claim 7, characterized in that... Includes the following steps: Take the purified alcohol dehydrogenase PhyADH protein solution, adjust the concentration to 12.5 mg / mL, and inoculate it into an equal volume of crystallization buffer. Place the resulting crystallization solution on a plastic coverslip, then drop the crystallization solution upside down onto a crystallization plate, seal the gap in the middle with Vaseline, and place it in a biological incubator at 16℃ for crystal culture. After the crystals grow, collect them.

9. The method for preparing PhyADH crystals of alcohol dehydrogenase according to claim 8, characterized in that: The crystal buffer solution is composed of PEG6000 and NaCl, wherein the mass percentage of PEG6000 is 10% and the concentration of NaCl is 2.0 mol / L.

10. The application of the alcohol dehydrogenase PhyADH according to claim 1 or 2 in catalyzing the conversion of alcohols into aldehydes and ketones.

Images