A thermostable alcohol dehydrogenase ADH10C1, its gene, and its applications

Abstract

This invention relates to a thermostable alcohol dehydrogenase ADH10C1, its encoding gene, and its applications. Alcohol dehydrogenase ADH10C1 was obtained by mutating the sequence SEQ ID NO.2 of the alcohol dehydrogenase ADH10 from *Zygosacchariformis* CBS732, with mutation sites G201C, G207C, P52C, and E56C. This mutant retains 80% enzyme activity after being kept at 70°C for 5 minutes, while ADH10 retains only 60% activity after 5 minutes at 60°C. The optimal pH for this mutant is 8.5, and it exhibits high enzyme activity in the pH range of 7.5–10.5, demonstrating good pH stability. This mutant also exhibits good thermostability and protease resistance. This invention expands the resource of alcohol dehydrogenase genes and provides an excellent alcohol dehydrogenase for the conversion of ethanol to acetaldehyde during ethanol metabolism, showing potential application value in the production of hangover remedies.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to an alcohol dehydrogenase ADH10C1 with improved thermostability, its gene, and its applications. Background Technology

[0002] After alcohol enters the body, it is mainly broken down by the liver. This breakdown process consists of two steps: breaking down alcohol (ethanol) into acetaldehyde, which requires the participation of alcohol dehydrogenase; and converting acetaldehyde into acetic acid, which also requires the participation of enzymes, namely acetaldehyde dehydrogenase.

[0003] Biocatalysis is a technology that uses microbial cells or enzymes as catalysts for the transformation of substances. It boasts advantages such as mild conditions, few side reactions, high selectivity, low energy consumption, and environmental friendliness, and has achieved remarkable success. It has attracted widespread attention, particularly in the fields of green chemistry and medicine. Enzymes, as common biocatalysts, play a crucial role in biocatalytic processes.

[0004] Alcohol dehydrogenase (ADH) is abundant in the livers of humans and animals, as well as in plant and microbial cells. It is a cytoplasmic dimerase with zinc as a cofactor and nicotinamide adenine dinucleotide (NAD) as a coenzyme, catalyzing the reversible reaction between primary alcohols and aldehydes: CH3CH2OH + NAD + →CH3CHO+NADH+H + Alcohol dehydrogenase (ALH) is an important redox enzyme in living organisms, playing a crucial role in many physiological processes. In humans and mammals, ADH and aldehyde dehydrogenase (ALDH) constitute the alcohol dehydrogenase system, participating in ethanol metabolism: ADH catalyzes the oxidation of ethanol, removing a hydrogen atom from ethanol and transferring it to NAD+, converting it into the reduced form of NADH and producing acetaldehyde. Acetaldehyde and coenzyme NAD+ are then converted into acetic acid by ADH, producing NADH. Acetic acid is ultimately converted into acetyl-CoA and enters the tricarboxylic acid cycle, ultimately producing water, carbon dioxide, and a large amount of energy. The activity levels of ADH and ADH determine the rate of ethanol metabolism in the body. In addition to ethanol metabolism, ADH also participates in the metabolism of retinol, aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. ADH is used as an enzyme in ethanol biosensors and as a biocatalyst in chemical production to modify or synthesize various raw materials and chiral compounds.

[0005] Thousands of enzymes have been studied in laboratories, but only a very small number are actually used. This is mainly because while these enzymes are active under biological or natural conditions, their activity is extremely poor in actual production systems, rendering them unusable. In industrial production, almost all reaction systems are in acidic, alkaline, or solvent systems at relatively high temperatures, causing the vast majority of enzymes to denature under these conditions. Mutant enzymes are produced by controlled splicing, modification, or mutation of natural enzyme genes, thereby altering the catalytic properties, substrate specificity, or coenzyme specificity of these enzymes to better meet the needs of human society, production, and daily life.

[0006] Alcohol dehydrogenase (ADH), an essential enzyme for ethanol metabolism in the human body, has its activity used as an important indicator to observe whether hangover remedies induce ADH activity. Modifying microorganisms to produce ADH and studying its enzymatic properties has become a current research hotspot, and developing highly active ADH has become a key direction in hangover remedy research. Currently, various alcohol dehydrogenase genes have been isolated from microorganisms of different origins. However, most alcohol dehydrogenases exhibit poor pH and thermal stability, with their activity decreasing sharply above 40°C. Therefore, cloning and isolating stable acetaldehyde dehydrogenase genes with potential application value is of great significance both from the perspective of expanding gene resources and practical production applications. Summary of the Invention

[0007] To address the aforementioned issues, the alcohol dehydrogenase provided by this invention has an optimal pH of 8.5 and exhibits high enzyme activity between pH 7.5 and 10.5; it also demonstrates good pH stability and excellent resistance to proteases. Even after being kept at 70°C for 5 minutes, it retains 80% of its enzyme activity, demonstrating its thermostability and enabling its application in industrial production requiring high-temperature environments. This invention expands the resource of alcohol dehydrogenase genes and provides an excellent alcohol dehydrogenase for the conversion of ethanol to acetaldehyde during ethanol metabolism, showing potential application value in food and the production of hangover remedies.

[0008] This invention provides an alcohol dehydrogenase ADH10C1 with improved heat resistance, the amino acid sequence of which is SEQ ID NO.4.

[0009] Specifically, the alcohol dehydrogenase ADH10C1 is obtained by mutating the sequence SEQ ID NO.2 of alcohol dehydrogenase ADH10, with mutation sites G201C, G207C, P52C and E56C.

[0010] G201C, G207C, P52C and E56C respectively represent the mutation of glycine at position 201 to cysteine, glycine at position 207 to cysteine, proline at position 52 to cysteine, and glutamic acid at position 56 to cysteine ​​in SEQ ID NO.2.

[0011] Specifically, the N-terminus of the sequence SEQ ID NO.4 is linked to the signal peptide sequence SEQ ID NO.3.

[0012] On the other hand, the present invention also provides a gene sequence encoding the aforementioned alcohol dehydrogenase ADH10C1.

[0013] Specifically, the gene sequence is SEQ ID NO.5 or a sequence that has more than 90% identity with SEQ ID NO.5.

[0014] The aforementioned sequence with over 90% identity refers to the phenomenon where different gene sequences encode the same amino acid due to codon degeneracy, where one amino acid is encoded by more than one triplet codon. It is considered that a sequence with over 90% identity to gene SEQ ID NO.3 can encode the same amino acid.

[0015] On the other hand, the present invention provides a recombinant vector comprising the aforementioned alcohol dehydrogenase ADH10C1 gene sequence.

[0016] Specifically, the vector can be one of plasmids, bacteriophages, and viruses.

[0017] Preferably, the vector is a plasmid; more preferably, it is the pPIC9 plasmid.

[0018] Specifically, the alcohol dehydrogenase ADH10C1 gene is inserted between appropriate restriction enzyme sites on the expression vector.

[0019] Preferably, the alcohol dehydrogenase ADH10C1 gene is inserted between the EcoRI and NotI restriction sites on plasmid pPIC9.

[0020] In another aspect, the present invention provides cells comprising the aforementioned recombinant vector.

[0021] Specifically, the cells may be genetically engineered cells used to express proteins, including but not limited to: plant cells, animal cells, bacteria, and yeast.

[0022] Preferably, the cells are engineered bacteria.

[0023] More preferably, the engineered bacteria can be Escherichia coli, yeast, Bacillus, or Lactobacillus.

[0024] In another aspect, the present invention provides a method for preparing alcohol dehydrogenase, comprising the following steps:

[0025] (1) Transform host cells with the aforementioned recombinant vector to obtain recombinant strains;

[0026] (2) Culture recombinant strains and induce recombinant protease expression;

[0027] (3) The alcohol dehydrogenase ADH10C1 was recovered and purified.

[0028] Specifically, the host cell may be Pichia pastoris, brewer's yeast cells, or polymorphonuclear yeast cells.

[0029] Preferably, the host cell is Pichia pastoris.

[0030] More preferably, the host cell is Pichia pastoris GS115.

[0031] Furthermore, this invention provides the application of alcohol dehydrogenase ADH10C1 in the preparation of hangover remedies. The technical effects achieved by this invention are as follows:

[0032] (1) The alcohol dehydrogenase ADH10C1 provided by the present invention has an optimal pH of 8.5 and high enzyme activity in the pH range of 7.5-10.5; it also has good pH stability.

[0033] (2) The alcohol dehydrogenase ADH10C1 also has the characteristic of high thermal stability. After treatment at 70℃ for 5 min, the remaining enzyme activity is still more than 80%, while the control group recombinant alcohol dehydrogenase ADH10C loses most of its enzyme activity after treatment at 60℃ for 5 min. This makes it suitable for industrial production that requires a high-temperature environment.

[0034] (3) The ability of alcohol dehydrogenase ADH10C1 to resist pepsin and trypsin is improved, and it has a good ability to resist pepsin and trypsin. Attached Figure Description

[0035] Figure 1 The optimal pH for alcohol dehydrogenase and its mutants.

[0036] Figure 2 This refers to the pH stability of alcohol dehydrogenase and its mutants.

[0037] Figure 3 The optimal temperature for alcohol dehydrogenase and its mutants.

[0038] Figure 4 The thermostability of alcohol dehydrogenase and its mutants.

[0039] Figure 5 The thermal stability of ADH1, ADH2 and ADH10C1.

[0040] Figure 6 pH stability of ADH1, ADH2 and ADH10C1. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0042] Experimental materials and reagents

[0043] 1. Strains and vectors: The alcohol dehydrogenase mutant ADH10C1 of this invention was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and the Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

[0044] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, and ligases were purchased from Invitrogen. Ethanol and other reagents were domestically produced (available from common biochemical reagent companies).

[0045] 3. Culture medium:

[0046] (1) Yeast culture medium YPD (100mL): 1g peptone, 0.5g yeast extract, 1g glucose, 2g agar, pH 7.0.

[0047] (2) Escherichia coli culture medium LB (100mL): 1g peptone, 0.5g yeast extract, 1g NaCl, pH 7.0.

[0048] (3) BMGY medium (100mL): 1g yeast extract, 2g peptone, 1.34g YNB, 0.00004g Biotin, 1% glycerol (V / V).

[0049] (4) BMMY medium: except that 0.5% (V / V) methanol is used instead of glycerol, the other components are the same as BMGY, pH 4.0.

[0050] Note: Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0051] Example 1: Preparation of alcohol dehydrogenase ADH10C1

[0052] 1.1 Synthesis of ADH10C1 encoding gene of Zygosaccharomyces rouxi CBS732 alcohol dehydrogenase mutant

[0053] This invention uses the alcohol dehydrogenase ADH10 gene from Zygosaccharomycesrouxi CBS732 as a reference. The amino acid sequence of the obtained alcohol dehydrogenase ADH10 is SEQ ID NO.1. The enzyme gene encodes 405 amino acids, and the N-terminal 102 amino acids are its predicted signal peptide sequence SEQ ID NO.3.

[0054] The theoretical molecular weight of the mature alcohol dehydrogenase ADH10 is 39.6 kDa, and its amino acid sequence is SEQ ID NO.2. The following mutations were performed on its amino acid sequence (SEQ ID NO.2) (G201C, G207C, P52C, E56C), and EcoRI and NotI restriction enzyme sites were added to the 5' and 3' ends of the mutated gene sequence, respectively. The sequence was then sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for artificial gene synthesis. The amino acid sequence of the artificially synthesized alcohol dehydrogenase mutant ADH10C1 is shown in SEQ ID NO.4, and its nucleotide sequence is shown in SEQ ID NO.5.

[0055] 1.2 Cloning of the alcohol dehydrogenase mutant encoding gene ADH10C1

[0056] Extracting gene vectors carrying mannanase mutants

[0057] The synthesized gene vector was preserved in the form of puncture bacteria. The puncture bacteria were picked up with a sterile toothpick in a clean bench and placed in an LB shaker containing antibiotic Amp (working concentration: 100 μg / mL). The culture was carried out overnight at 37°C and 220 rpm. The next day, the vector containing the mutant gene was extracted according to the instructions of the Kangwei Century Plasmid Extraction Kit PurePlasmid Mini Kit (CW0500).

[0058] Primers SEQ ID NO.6 and SEQ ID NO.7 were designed and synthesized based on the gene sequence of alcohol dehydrogenase ADH10.

[0059] PCR amplification was performed using the extracted vector as a template. The PCR reaction parameters were: denaturation at 94℃ for 5 min; followed by denaturation at 94℃ for 30 sec, annealing at 45℃ for 30 sec, extension at 72℃ for 1 min, and 30 cycles, followed by incubation at 72℃ for 10 min. A fragment of approximately 1122 bp was obtained. This fragment was recovered, ligated into the pMD19 vector, and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0060] Based on the nucleotide sequences obtained from sequencing, the nucleotide sequences were compared with the ADH10 gene sequence using DNAMan software, confirming that the mutations at the four positions of G201C, G207C, P52C, and E56C were correct.

[0061] 1.3 Preparation of recombinant alcohol dehydrogenase

[0062] The expression vector pPIC9 was double-digested (EcoRI+NotI), and the gene encoding the alcohol dehydrogenase mutant, ADH10C1, was also double-digested (EcoRI+NotI). The resulting fragment encoding the mature alcohol dehydrogenase was ligated into the expression vector pPIC9. The alcohol dehydrogenase gene was inserted between the EcoRI and NotI restriction sites on the plasmid pPIC, placing the nucleotide sequence downstream of and regulated by the AOX1 promoter, resulting in the recombinant yeast expression plasmid pPIC9-ADH10C1. This plasmid was then transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / ADH10C1.

[0063] The GS115 strain containing the recombinant plasmid and the control strain (i.e., the unmutated strain GS115 / ADH10) were inoculated into 300 mL of BMGY medium and cultured at 30°C with shaking at 250 rpm for 48 h. The cells were then collected by centrifugation. The cells were then resuspended in 150 mL of BMGY medium and cultured at 30°C with shaking at 250 rpm. After induction for 72 h, the supernatant was collected by centrifugation. The activity of alcohol dehydrogenase was measured. SDS-PAGE results showed that the recombinant alcohol dehydrogenase was expressed in Pichia pastoris.

[0064] Example 2 Activity analysis of recombinant alcohol dehydrogenase ADH10C1

[0065] The specific method is as follows: Under pH 8.5 and 35℃ conditions, 0.03 mol / L pH 8.5 Tris-HCl buffer, 1 mM NAD solution, and 100 mM ethanol solution were mixed in a ratio of 70 μL:10 μL:10 μL. The mixture was placed in a quartz cuvette, and 10 μL of enzyme solution was added and quickly mixed. The absorbance was immediately measured at a wavelength of 340 nm. One unit of enzyme activity (U) was defined as an increase of 0.001 absorbance per minute. The expression level of recombinant alcohol dehydrogenase was 10.44 U / mL, while the expression level of mannanase in the control group was 10.20 U / mL. SDS-PAGE results showed that recombinant alcohol dehydrogenase was expressed in Pichia pastoris.

[0066] Example 3: Determination of the properties of recombinant alcohol dehydrogenase ADH10C1

[0067] 1. The method for determining the optimal pH and pH stability of recombinant alcohol dehydrogenase ADH10C1 is as follows:

[0068] When measuring the optimal pH of recombinant alcohol dehydrogenase, the concentrations of the two substrates, ethanol and NAD, and the amount of alcohol dehydrogenase must be kept constant. Only the pH of the buffer solution in the reaction system is changed. The buffer solution used is as follows:

[0069] We used KH₂PO₄-NaOH buffer (pH 6.5-7.5), Tris-HCl buffer (pH 8.0-8.5), Glycine-NaOH buffer (pH 9.0-10.5), and Na₂HPO₄-NaOH buffer (pH 11.0-11.5) to determine the optimal pH for alcohol dehydrogenase by comparing its activity in different pH buffers. Results ( Figure 1 The results showed that the optimal pH for alcohol dehydrogenase was 8.5, and it maintained a relative enzyme activity of over 60% in the pH range of 7.5-10.5. The enzyme solution was treated with buffer solutions of different pH values ​​for 60 min, and the enzyme activity was measured to study the enzyme's pH tolerance. Figure 2 The results showed that the recombinant alcohol dehydrogenase was very stable between pH 7.0 and 10.5. After treatment within this pH range for 60 min, the remaining enzyme activity was more than 70%, while the remaining enzyme activity of the control group recombinant alcohol dehydrogenase ADH10CH was about 50% after treatment within the same pH range for 60 min. This indicates that the enzyme has good pH stability in alkaline and neutral ranges.

[0070] 2. The optimal temperature and thermal stability determination method for alcohol dehydrogenase are as follows:

[0071] The optimal temperature for alcohol dehydrogenase was determined by conducting enzymatic reactions in a Tris-HCl buffer (pH 8.5) system at different temperatures. Thermostability was determined by treating alcohol dehydrogenase at different temperatures for the same amount of time, followed by enzyme activity assays at 35°C. Results of the optimal temperature determination for the enzyme reaction (…) Figure 3 Its optimal temperature is 35℃. Thermostability tests of the enzyme show that... Figure 4 The recombinant alcohol dehydrogenase ADH10C1, after treatment at 70℃ for 5 min, still retained over 80% of its original enzyme activity. Figure 4 The recombinant alcohol dehydrogenase ADH10C in the control group lost most of its enzyme activity after being treated at 60℃ for 5 min, indicating that the thermostability of recombinant alcohol dehydrogenase ADH10C was significantly improved compared with that of the control group.

[0072] 3. The anti-trypsin ability of alcohol dehydrogenase was determined as follows:

[0073] 0.1 mg / mL trypsin was prepared using pH 8.5 Tris-HCl buffer. 0.5 mL of the purified enzyme solution, diluted with pH 8.5 Tris-HCl buffer, was added to 0.5 mL of trypsin and mixed. The ratio of trypsin to alcohol dehydrogenase (w / w) was approximately 0.1. Samples were taken after incubation at 35°C for 60 and 120 min, and enzyme activity was measured at pH 8.5 and 35°C. The results showed that after treatment with trypsin for 120 min, the enzyme activity of alcohol dehydrogenase ADH10C1 increased by 23% compared to before treatment.

[0074] Comparative Example

[0075] A comparative experiment was designed according to the method in Example 3 to compare the thermostability and pH stability of ADH10C1 with two alcohol dehydrogenases from different sources. When comparing the thermostability and pH stability of ADH10C1 with alcohol dehydrogenase ADH1 derived from horse liver and alcohol dehydrogenase ADH2 derived from corn, the results were as follows: Figure 5 The results showed that the recombinant alcohol dehydrogenase ADH10C1 maintained an activity of over 80% between 40-70℃. In contrast, the activity of ADH1 and ADH2 began to decline at 40℃, and by 60℃, most of their activity had been lost. Treatment with different pH buffers for 60 min followed by enzyme activity assays yielded different results. Figure 6 The results showed that ADH10C1 was more stable than the other two enzymes between pH 7.0 and 10.5, with more than 70% of its remaining enzyme activity remaining. ADH1 showed good stability between pH 8.6 and 9.5, but its activity decreased sharply at pH 10. ADH2 showed a relatively rapid decrease in activity between pH 9 and 10, with only 50% of its activity remaining. These findings indicate that the mutant ADH10C1 has better thermal and pH stability.

Claims

1. An alcohol dehydrogenase ADH10C1 with improved heat resistance, characterized in that, Its amino acid sequence is SEQ ID NO.

4.

2. The alcohol dehydrogenase ADH10C1 according to claim 1, characterized in that, The N-terminal signal peptide sequence SEQ ID NO.4 of the sequence is linked to SEQ ID NO.

3.

3. The gene sequence encoding the alcohol dehydrogenase ADH10C1 as described in claim 1.

4. The gene sequence according to claim 3, characterized in that, The gene sequence is SEQ ID NO.

5.

5. A recombinant vector comprising the gene sequence according to any one of claims 3-4.

6. The recombinant vector according to claim 5, characterized in that, The vector is either a plasmid or a bacteriophage.

7. The recombinant vector according to claim 6, characterized in that, The carrier is pPIC9 Plasmid.

8. Cells comprising the recombinant vector according to any one of claims 5-7.

9. The cell according to claim 8, characterized in that, It is an engineered bacterium.

10. The cell according to claim 9, characterized in that, It consists of Escherichia coli, yeast, Bacillus, or Lactobacillus.

11. A method for preparing alcohol dehydrogenase, characterized in that, Includes the following steps: (1) Transform host cells with the recombinant vector according to any one of claims 5-7 to obtain recombinant strains; (2) Culture recombinant strains and induce recombinant protease expression; (3) The alcohol dehydrogenase ADH10C1 was recovered and purified.

12. The preparation method according to claim 11, characterized in that, The host cell is Pichia pastoris, brewer's yeast, or polymorphonuclear yeast.

13. The preparation method according to claim 12, characterized in that, The host cell is Pichia pastoris.

14. The preparation method according to claim 13, characterized in that, The host cell is Pichia pastoris GS115.

15. The application of the alcohol dehydrogenase ADH10C1 according to claim 1 in the preparation of hangover relief beverages.

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