Alcohol dehydrogenase mutant and application thereof

Abstract

The invention relates to the field of biochemical engineering, in particular to an alcohol dehydrogenase mutant and application thereof. The mutant is obtained by carrying out mutation on one or more sites selected from 111Y, 258V, 283L, 274L, 107K and 108N aiming at alcohol dehydrogenase, and the mutation is that amino acid at the sites is mutated into one of W, Q, V, G and A. The enzyme activity of the alcohol dehydrogenase mutant is higher than that of a wild type; the enzyme specific activity (36.42) of the mutant A-111-107-274 taking FAL as a substrate is 2.4 times that of a wild type, and the enzyme specific activity (22.16) of the mutant A-111-107-274 taking HMF as a substrate is 9.2 times that of the wild type. Good industrial application performance is shown.

Description

Technical Field

[0001] This invention relates to the field of biochemical engineering, specifically to an alcohol dehydrogenase mutant and its applications. Background Technology

[0002] Alcohol dehydrogenase (ADH) is a key enzyme widely found in animals, plants, and microorganisms, especially highly expressed in the human liver. Using NAD / NADP as coenzymes, this enzyme catalyzes the oxidation of alcohols to aldehydes or ketones, a core step in alcohol metabolism. In humans, ADH works synergistically with aldehyde dehydrogenase to convert ethanol to acetaldehyde, which is then degraded into acetic acid, maintaining homeostasis. Abnormal ADH activity is closely related to alcohol tolerance and liver disease. ADH has diverse biological functions, promoting the mobilization of stored energy substances in plant seed germination and participating in fermentation metabolism regulation in microorganisms. Clinically, serum ADH activity can serve as an early diagnostic indicator of liver damage, with changes in its level reflecting alterations in hepatocyte membrane permeability. In industry, ADH enables the synthesis of chiral alcohols through asymmetric catalysis, making it highly valuable in drug development. Genetically engineered ADH mutants can significantly improve catalytic efficiency and substrate specificity, promoting the development of green chemistry. With advancements in enzyme engineering and synthetic biology, the application potential of ADH in metabolic engineering, environmental monitoring, and medical diagnostics will continue to expand, becoming a bridge between basic research and industrial transformation.

[0003] Lignocellulosic biomass (such as agricultural and forestry waste) produces aldehydes during pretreatment. These compounds are highly toxic to subsequent microbial fermentation, inhibiting microbial growth and product formation. Aldehyde removal primarily involves physical, chemical, and enzymatic methods. While adsorption and extraction methods offer mild conditions, they result in low separation purity and high solvent consumption. Distillation is efficient but easily damages heat-sensitive hydroxymethylfurfural (HMF). In contrast, enzymatic methods are inexpensive, readily available, and more environmentally friendly. Alcohol dehydrogenases can convert alcohols into aldehydes. In studies of microbial tolerance, alcohol dehydrogenases ADH6 and ADH7 have been found to reduce furfural and 5-hydroxymethylfurfural to less toxic alcohols. This process is an effective way for microorganisms (such as yeast) to resist toxic inhibitors in biomass hydrolysates. Summary of the Invention

[0004] To overcome the limitation of low specific activity of existing alcohol dehydrogenases, the enzyme was modified to obtain an alcohol dehydrogenase mutant with increased specific activity.

[0005] This invention uses wild-type alcohol dehydrogenase as the starting enzyme. The amino acid sequence of the wild-type alcohol dehydrogenase is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:2. Mutations are made on one or more amino acid residues at positions 111, 258, 283, 274, 107, and 108 of its amino acid sequence to obtain an alcohol dehydrogenase mutant. Specifically, the mutant includes one or more of the following mutations: 1) Mutate tyrosine (Y) at position 111 to tryptophan (W); 2) Mutate valine (V) at position 258 to glutamine (Q); 3) Mutate leucine (L) at position 283 to valine (V); 4) Mutate leucine (L) at position 274 to glycine (G); 5) Mutate lysine (K) at position 107 to glutamine (Q); 6) Mutate the 108th position of asparagine (N) to alanine (A).

[0006] Preferably, the alcohol dehydrogenase is a single-point mutation among the above mutations. Specifically, the amino acid sequence of the alcohol dehydrogenase mutant Y111W is SEQ ID NO:3, the amino acid sequence of the alcohol dehydrogenase mutant V258Q is SEQ ID NO:4, the amino acid sequence of the alcohol dehydrogenase mutant L283V is SEQ ID NO:5, the amino acid sequence of the alcohol dehydrogenase mutant L274G is SEQ ID NO:6, the amino acid sequence of the alcohol dehydrogenase mutant K107Q is SEQ ID NO:7, and the amino acid sequence of the alcohol dehydrogenase mutant N108A is SEQ ID NO:8.

[0007] The alcohol dehydrogenase is a mutation of two amino acid residues in the above mutations. Specifically, the amino acid sequence of the alcohol dehydrogenase mutant A-111-107 is SEQ ID NO:9, the amino acid sequence of the alcohol dehydrogenase mutant A-111-108 is SEQ ID NO:10, the amino acid sequence of the alcohol dehydrogenase mutant A-111-258 is SEQ ID NO:11, the amino acid sequence of the alcohol dehydrogenase mutant A-111-274 is SEQ ID NO:12, the amino acid sequence of the alcohol dehydrogenase mutant A-107-274 is SEQ ID NO:13, the amino acid sequence of the alcohol dehydrogenase mutant A-108-274 is SEQ ID NO:14, the amino acid sequence of the alcohol dehydrogenase mutant A-258-274 is SEQ ID NO:15, and the amino acid sequence of the alcohol dehydrogenase mutant A-283-274 is SEQ ID NO:16.

[0008] The alcohol dehydrogenase is a mutation of three amino acid residues in the above mutations. Specifically, the amino acid sequence of the alcohol dehydrogenase mutant A-111-107-274 is SEQ ID NO:17, and the amino acid sequence of the alcohol dehydrogenase mutant A-111-107-283 is SEQ ID NO:18.

[0009] The present invention also provides the encoding gene of the alcohol dehydrogenase mutant.

[0010] The present invention provides a recombinant vector carrying the gene encoding the above-mentioned alcohol dehydrogenase mutant.

[0011] The present invention provides a recombinant vector carrying the gene encoding the above-mentioned alcohol dehydrogenase mutant and recombinant bacteria transformed / transfected with the recombinant vector.

[0012] The specific mutation method and preparation method of alcohol dehydrogenase mutant of this invention are as follows:

[0013] 1. The alcohol dehydrogenase was simulated using molecular docking analysis and molecular dynamics simulation software to obtain the spatial structure of the alcohol dehydrogenase and the key sites affecting the product (111Y, 258V, 283L, 274L, 107K, 108N).

[0014] 2. Design primers for site-directed mutagenesis, amplify the alcohol dehydrogenase gene by PCR, and construct a recombinant vector with the expression vector glucose dehydrogenase GDH, which is then transformed into Escherichia coli BL21.

[0015] 3. The above recombinant bacteria were cultured and induced to express by IPTG. The bacterial cells were collected, the cells were disrupted by sonication, the supernatant and precipitate were collected by centrifugation, and the protein was purified by Ni-NTA affinity chromatography to obtain the purified alcohol dehydrogenase mutant.

[0016] This invention further provides the application of the above-mentioned alcohol dehydrogenase mutant in the reduction of aldehyde compounds, using FAL and HMF as substrates, respectively.

[0017] The enzyme activity of the alcohol dehydrogenase mutant of this invention is higher than that of the wild type; the specific enzyme activity of mutant A-111-107-274 with furfural (FAL) as substrate (36.42) is 2.4 times that of the wild type, and the specific enzyme activity with 5-hydroxymethylfurfural (HMF) as substrate (22.16) is 9.2 times that of the wild type. It exhibits good performance for industrial applications. Attached Figure Description

[0018] Figure 1 Flowchart for site-directed mutagenesis of alcohol dehydrogenase AdhP;

[0019] Figure 2 This is a polyacrylamide gel electrophoresis image of a single mutant alcohol dehydrogenase.

[0020] Figure 3 This is a polyacrylamide gel electrophoresis image of a double mutant alcohol dehydrogenase. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0022] In the embodiments of the present invention, unless otherwise specified, conventional molecular biology experimental methods are used. The PCR, plasmid construction, transfection / transformation and other processes involved in the embodiments can be understood and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore will not be described in detail.

[0023] Example 1

[0024] This invention relates to an alcohol dehydrogenase mutant and its construction method. Specifically, it involves identifying key amino acid sites that affect the degree of polymerization of the product through substrate channel analysis and performing site-directed saturation mutagenesis on these sites to obtain an alcohol dehydrogenase mutant with improved catalytic performance.

[0025] Using a high-activity alcohol dehydrogenase preserved in the early stages of the experiment as the starting enzyme, its amino acid sequence was submitted to the SWISS-MODEL platform to construct a three-dimensional structural model of the enzyme. Based on this model, substrate channel analysis technology was used to identify key amino acid sites in the substrate channel that may affect catalytic activity and product polymerization degree, namely: 111Y, 258V, 283L, 274L, 107K, and 108N.

[0026] Table 1. Primers for the mutant library

[0027] For the aforementioned key sites, saturation mutation primers were designed using SnapGene software, with an annealing temperature of 70℃. Long primers F and R were used for PCR amplification, and KOD Plus Nco enzyme was employed for the PCR reaction to obtain DNA fragments carrying the mutation sites. The PCR reaction system and procedure are shown in Tables 2 and 3, respectively.

[0028] Table 2 Two-step PCR reaction system

[0029] Table 3 Two-step PCR reaction procedure

[0030] After PCR, the product was purified and recovered, and the amplification success was verified by agarose gel electrophoresis. The purified PCR product was template-free, ligated using the T5 homologous recombination system, and then transformed into *E. coli* TOP10 competent cells. After culture, single clones were selected for sequencing verification. The mutant plasmid confirmed by sequencing was extracted and transformed into the expression host BL21. In TB medium, the expression of the mutant enzyme protein was induced by an inducer. The expressed protein samples were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) to verify the expression of the target protein.

[0031] The protein samples obtained from expression were quantitatively analyzed, and the protein concentration was determined by the Coomassie brilliant blue method (based on the standard curve). Based on this, the catalytic activity of the mutant enzyme was determined using two key biomass-derived inhibitors, FAL (furfural) and HMF (5-hydroxymethylfurfural), as substrates. The enzyme activity assay methods are shown in Table 4. The enzyme activities of the mutant enzyme, using FAL and HMF as substrates respectively, are shown in Table 5.

[0032] Table 4. Methods for Enzyme Activity Assay

[0033] The above system was thoroughly mixed, and the change in absorbance at 340 nm was detected. Enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1 μmol of NADH per minute.

[0034] Calculation formula: Enzyme activity (U) = EW * V * 1000 / (6220 * L) EW: Change in OD340 over 1 minute; V: Total volume of the reaction system (mL); L: Optical path distance (cm); 6220: Molar extinction coefficient (L*mol) -1 *cm -1 ).

[0035] Specific activity (U*mg⁻¹) = Enzyme activity (U) / Protein mass (mg)

[0036] Protein content was determined using the Coomassie Brilliant Blue method. Specifically, BSA (bovine serum albumin) standard protein was diluted to different concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg / mL). 0.1 mL of the standard protein solution was added to 2.5 mL of Coomassie Brilliant Blue working solution, mixed thoroughly, and incubated at 30 ℃ for 15 min. The absorbance at 595 nm was measured using a spectrophotometer to obtain the protein concentration standard curve.

[0037] Table 5. Enzyme activity of mutant enzymes

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, Mutating one or more amino acid residues at positions 111, 258, 283, 274, 107, and 108 of the wild-type alcohol dehydrogenase amino acid sequence yields an alcohol dehydrogenase mutant, wherein the wild-type alcohol dehydrogenase amino acid sequence is shown in SEQ ID NO:1, and the mutation includes one or more of the following mutations: 1) Mutate tyrosine at position 111 to tryptophan; 2) Mutate valine at position 258 to glutamine; 3) Mutate leucine at position 283 to valine; 4) Mutate leucine at position 274 to glycine; 5) Mutate lysine at position 107 to glutamine; 6) Mutate the 108th position of asparagine to alanine.

2. The alcohol dehydrogenase mutant according to claim 1, characterized in that the amino acid sequence of the alcohol dehydrogenase mutant is as shown in SEQ ID NO:3~SEQ ID NO:

18.

3. A gene encoding the alcohol dehydrogenase mutant as described in claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector carries the gene of the alcohol dehydrogenase mutant as described in any one of claims 1-2.

5. A recombinant bacterial strain, characterized in that, The recombinant strain is a host bacterium containing the gene as described in claim 3 or the recombinant vector as described in claim 4.

6. The use of the alcohol dehydrogenase mutant of claim 1 or 2, the gene encoding the alcohol dehydrogenase mutant of claim 3, the recombinant vector of claim 4, or the recombinant strain of claim 5 in the catalytic conversion of aldehyde compounds.

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