A kind of alcohol detoxifying enzyme protein capable of decomposing ethanol and acetaldehyde and its application

Abstract

The present invention discloses an alcohol decomposition enzyme protein capable of decomposing ethanol and acetaldehyde and its application. The alcohol decomposition enzyme protein comprises at least one protein region for decomposing ethanol and at least one protein region for decomposing acetaldehyde, wherein one protein region for decomposing ethanol is derived from human ethanol dehydrogenase, and the amino acid sequence is shown in SEQ ID NO: 1; and / or one protein region for decomposing acetaldehyde is derived from human acetaldehyde dehydrogenase, and the amino acid sequence is shown in SEQ ID NO: 2 or 3; or further comprises two protein tags, one protein tag Strep II, and the amino acid sequence is shown in SEQ ID NO: 4; and one protein tag HIS6, and the amino acid sequence is shown in SEQ ID NO: 5. The present invention obtains an alcohol decomposition enzyme protein by modern molecular biology methods, which can decompose ethanol and acetaldehyde, accelerate ethanol metabolism, and provide a new option for reducing the harm of drinking to the human body and protecting human health.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to an alcohol detoxifying enzyme protein capable of decomposing ethanol and acetaldehyde and an application thereof. Background Art

[0002] Wine has a long history and is indispensable in people's daily lives. Alcohol (i.e. ethanol), the main component of wine, has many negative effects on the human body due to its unique properties, causing great physical and social damage. The effects of alcohol on the body can be divided into acute and chronic effects. The acute effects are mainly manifested as acute gastric and esophageal bleeding, etc. The chronic effects refer to long-term and large-scale drinking, which causes damage to various organs, manifested in the central and peripheral nervous systems, muscles, heart, liver, pancreas, digestive tract, etc. In daily life, the mental and physical state of drunkenness that is different from that of ordinary people is the best example of the negative effects of alcohol on the human body.

[0003] Alcohol can be directly absorbed by the human body without being digested, so it can quickly enter the blood and reach the whole body. After alcohol enters the human body, it will be decomposed into acetaldehyde under the action of alcohol dehydrogenase. Acetaldehyde is decomposed into acetic acid under the action of acetaldehyde dehydrogenase. Acetic acid is then decomposed into carbon dioxide and water, and finally excreted from the body. When the human body drinks alcohol at a rate that exceeds the rate of ethanol metabolism and decomposition, it will cause ethanol or acetaldehyde accumulation, which is harmful to the human body. In order to reduce the harm of drinking to the human body, the human body can be protected by accelerating the decomposition of ethanol. However, only accelerating the decomposition of ethanol may cause ethanol accumulation, which is also harmful to the human body, and the accumulation of acetaldehyde will also inhibit the decomposition of ethanol. Therefore, accelerating the decomposition of ethanol while accelerating the decomposition of acetaldehyde can truly reduce the harm of alcohol to the human body when drinking. Summary of the invention

[0004] In view of this, the main purpose of the present invention is to provide an alcohol decomposition enzyme protein that can decompose ethanol and acetaldehyde. The alcohol decomposition enzyme protein obtained by drawing on modern molecular biology methods can decompose both ethanol and acetaldehyde, providing a new option for reducing the harm of drinking to the human body and protecting human health.

[0005] Another object of the present invention is to provide an application of an alcohol decomposing enzyme protein capable of decomposing ethanol and acetaldehyde in decomposing ethanol and acetaldehyde.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides an alcohol decomposition enzyme protein capable of decomposing ethanol and acetaldehyde, comprising at least one protein region for decomposing ethanol and at least one protein region for decomposing acetaldehyde.

[0008] Preferably, the protein region that decomposes ethanol is derived from human alcohol dehydrogenase, and its amino acid sequence is STAGKVIKCKAAVLWEVKKPFSIEDVEVAPPKAYEVRIKMVAVGICHTDDHVVSGNLVTPLPVILGHEAAGIVESVGEGVTTVKPGDKVIPLFTPQCGKCRVCKNPESNYCLKNDLGNPRGTLQDGTRRFTCRGKPIHHFLGTSTFSQYTVVDENAVAKIDAASPLEKVCLIGCGFSTGYGSAVNVAKVTPGSTCAVFGLGGVGLSAVMMGCKAAGAARIIAVDINKDKFAKAKELGATECINPQDYKKPIQEVLKEMTDGGVDFSFEVIGRLDTMMASLLCCHEACGTSVIVGVPPASQNLSINPMLLLTGRTWKGAVYGGFKSKEGIPKLVADFMAKKFSLDALITHVLPFEKINEGFDLLHSGKSIRTVLTF (SEQ ID NO: 1).

[0009] Preferably, the protein region for decomposing acetaldehyde is derived from human acetaldehyde dehydrogenase, and its amino acid sequence is SAAATQAVPAPNQQPEVFCNQIFINNEWHDAVSRKTFPTVNPSTGEVICQVAEGDKEDVDKAVKAARAAFQLGSPWRRMDASHRGRLLNRLADLIERDRTYLAALETLDNGKPYVISYLVDLDMVLKCLRYYAGWADKYHGKTIPIDGDFFSYTRHEPVGVCGQIIPWNFPLLMQAWKLGPALATGNVVVMKVAEQTPLTALYVANLIKEAGFPPGVVNIVPGFGPTAGAAIASH EDVDKVAFTGSTEIGRVIQVAAGSSNLKRVTLELGGKSPNIIMSDADMDWAVEQAHFALFFNQGQCCCAGSRTFVQEDIYDEFVERSVARAKSRVVGNPFDSKTEQGPQVDETQFKKILGYINTGKQEGAKLLC GGGIAADRGYFIQPTVFGDVQDGMTIAKEEIFGPVMQILKFKTIEEVVGRANNSTYGLAAAVFTKDLDKANYLSQALQAGTVWVNCYDVFGAQSPFGGYKMSGSGRELGEYGLQAYTEVKTVTVKVPQKNS(SEQ ID NO:2).

[0010] Preferably, the protein region for decomposing acetaldehyde is derived from human acetaldehyde dehydrogenase, and its amino acid sequence is SSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQAFQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLNDLAGCIKTLRYCAGWADKIQGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIKEAGFPPGVVNIVPGYGPTAGAAISSH MDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLADADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQYDKILDLIESGKKEGAKLEC GGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKRANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVKTVTVKISQKNS(SEQ ID NO:3).

[0011] Preferably, the alcohol decomposition enzyme protein capable of decomposing ethanol and acetaldehyde comprises two protein tags, a protein tag Strep II, whose amino acid sequence is WSHPQFEK (SEQ ID NO: 4), and a protein tag HIS6, whose amino acid sequence is HHHHHH (SEQ ID NO: 5). The design of the double protein tags is conducive to the purification and identification of the binding protein.

[0012] Preferably, a signal peptide sequence is added to the N-terminus of the alcohol decomposition enzyme protein capable of decomposing ethanol and acetaldehyde, so that the dual-binding protein can be secreted and expressed by engineered cells, thereby facilitating protein production and purification.

[0013] Preferably, the amino acid sequence of the alcohol dehydrogenase protein capable of decomposing ethanol and acetaldehyde is MSTAGKVIKCKAAVLWEVKKPFSIEDVEVAPPKAYEVRIKMVAVGICHTDDHVVSGNLVTPLPVILGHEAAGIVESVGEGVTTVKPGDKVIPLFTPQCGKCRVCKNPESNYCLKNDLGNPRGTLQDGTRRFTCRGKPIHHFLGTSTFSQYTVVDENAVAKIDAASPLEKVCLIGCGFSTGYGSAVNVAKVTPGSTCAVFGLGGVGLSAVMGCKAAGAARIIAVDINKDKFAKAKELGATECINPQDYKKPIQEVLKEMTDGGVDFSFEVIGRLDTMMASLLCCHEACGTSVIVGVPPASQNLSINPMLLLTGRTWKGAVYGGFKSKEGIPKLVADFMAKKFSLDALITHVLPFEKINEGFDLLHSGKSIRTVLTFGGGGSGGGGSGGGGSSAAATQAVPAPNQQPEVFCNQIFINNEWHDAVSRKTFPTVNPSTGEVICQVAEGDKEDVDKAVKAARAAFQLGSPWRRMDASHRGRLLNRLADLIERDRTYLAALETLDNGKPYVISYLVDLDMVLKCLRYYAGWADKYHGKTIPIDGDFFSYTRHEPVGVCGQIIPWNFPLLMQAWKLGPALATGNVVVMKVAEQTPLTALYVANLIKEAGFPPGVVNIVPGFGPTAGAAIASHEDVDKVAFTGSTEIGRVIQVAAGSSNLKRVTLELGGKSPNIIMSDADMDWAVEQAHFALFFNQGQCCCAGSRTFVQEDIYDEFVERSVARAKSRVVGNPFDSKTEQGPQVDETQFKKILGYINTGKQEGAKLLCGGGIAADRGYFIQPTVFGDVQDGMTIAKEEIFGPVMQILKFKTIEEVVGRANNSTYGLAAAVFTKDLDKANYLSQALQAGTVWVNCYDVFGAQSPFGGYKMSGSGRELGEYGLQAYTEVKTVTVKVPQKNSWSHPQFEKHHHHHH(SEQ ID NO:6).

[0014] Preferably, the amino acid sequence of the alcohol dehydrogenase protein that can decompose ethanol and acetaldehyde is MSTAGKVIKCKAAVLWEVKKPFSIEDVEVAPPKAYEVRIKMVAVGICHTDDHVVSGNLVTPLPVILGHEAAGIVESVGEGVTTVKPGDKVIPLFTPQCGKCRVCKNPESNYCLKNDLGNPRGTLQDGTRRFTCRGKPIHHFLGTSTFSQYTVVDENAVAKIDAASPLEKVCLIGCGFSTGYGSAVNVAKVTPGSTCAVFGLGGVGLSAVMGCKAAGAARIIAVDINKDKFAKAKELGATECINPQDYKKPIQEVLKEMTDGGVDFSFEVIGRLDTMMASLLCCHEACGTSVIVGVPPASQNLSINPMLLLTGRTWKGAVYGGFKSKEGIPKLVADFMAKKFSLDALITHVLPFEKINEGFDLLHSGKSIRTVLTFGGGGSGGGGSGGGGSSSSGTPDLPVLLTDLKIQYTKIFINNEWHDSVSGKKFPVFNPATEEELCQVEEGDKEDVDKAVKAARQAFQIGSPWRTMDASERGRLLYKLADLIERDRLLLATMESMNGGKLYSNAYLNDLAGCIKTLRYCAGWADKIQGRTIPIDGNFFTYTRHEPIGVCGQIIPWNFPLVMLIWKIGPALSCGNTVVVKPAEQTPLTALHVASLIKEAGFPPGVVNIVPGYGPTAGAAISSHMDIDKVAFTGSTEVGKLIKEAAGKSNLKRVTLELGGKSPCIVLADADLDNAVEFAHHGVFYHQGQCCIAASRIFVEESIYDEFVRRSVERAKKYILGNPLTPGVTQGPQIDKEQYDKILDLIESGKKEGAKLECGGGPWGNKGYFVQPTVFSNVTDEMRIAKEEIFGPVQQIMKFKSLDDVIKRANNTFYGLSAGVFTKDIDKAITISSALQAGTVWVNCYGVVSAQCPFGGFKMSGNGRELGEYGFHEYTEVKTVTVKISQKNSWSHPQFEKHHHHHH(SEQ ID NO:7).

[0015] The present invention also provides the use of the alcohol decomposing enzyme protein capable of decomposing ethanol and acetaldehyde in decomposing ethanol and acetaldehyde.

[0016] The invention also provides a hangover-resolving product, which comprises the hangover-resolving enzyme protein capable of decomposing ethanol and acetaldehyde.

[0017] Preferably, the alcohol sobering product is an alcohol sobering enzyme.

[0018] Compared with the prior art, the beneficial effect of the present invention is that: the present invention proposes an alcohol decomposition enzyme protein that can decompose both ethanol and acetaldehyde, which has both the function of decomposing ethanol and the function of decomposing acetaldehyde, can accelerate ethanol metabolism, and reduce the harm of drinking to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the plasmid map for overexpressing AADH01 constructed in Example 1.

[0020] Figure 2 This is the plasmid map for overexpressing AADH02 constructed in Example 1.

[0021] Figure 3 This is a silver staining result diagram of the target protein after extraction and purification via the His tag in Example 1.

[0022] Figure 4 This is a silver staining result diagram of the target protein after double-tag extraction and purification in Example 1. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with embodiments and drawings, but the protection scope of the present invention is not limited thereto.

[0024] Example 1: Target protein expression

[0025] (I) Synthesis of overexpression plasmid

[0026] The corresponding codons were designed according to sequence 6 (SEQ ID NO: 6) and named AADH01. Codon optimization, sequence synthesis and plasmid construction were performed by GenScript Biotech Co., Ltd. The plasmid construction results are shown in the figure. Figure 1 shown.

[0027] The corresponding codons were designed according to sequence 7 (SEQ ID NO: 7) and named AADH02. Codon optimization, sequence synthesis and plasmid construction were performed by GenScript Biotech Co., Ltd. The plasmid construction results are shown in the figure. Figure 2 shown.

[0028] (II) Expression and purification of target protein

[0029] 1. Transform the plasmids overexpressing the target proteins AADH01 and AADH02 into Rosetta (DE3) competent cells (Adamas life, C8182-1 mL) and culture them in an incubator at 37°C with shaking for 1 hour.

[0030] 2. The competent cells transformed with the plasmids overexpressing the target proteins AADH01 and AADH02 were spread on different ampicillin-resistant LB culture plates and inverted for overnight culture, and were marked as AADH01 and AADH02, respectively.

[0031] 3. Select monoclonal colonies and place them into different ampicillin-resistant LB liquid culture media, marked as AADH01 and AADH02 respectively. Add two other monoclonal colonies into the same ampicillin-resistant LB liquid culture medium as a control group, marked as AADH00, and culture them in an air bath constant temperature oscillator at 37°C for 8 hours.

[0032] 4. Add IPTG (Adamas life, 52119M) to the bacterial solution of each transformed colony to a concentration of 1 mM. Do not add IPTG to the bacterial solution of the control group. Incubate the culture overnight at 37°C in an air bath constant temperature oscillator.

[0033] 5. Collect the bacterial precipitate by centrifugation and purify the protein using a His-tag protein purification kit (Beyotime, P2226).

[0034] 6. Use Bradford protein concentration determination kit (Beyotime, P0006) to determine the concentration of the purified target protein, then use SDS-PAGE gel electrophoresis and silver staining kit (Beyotime, P0017S) for silver staining.

[0035] 7. The protein purified by His tag was purified by Strep tag using streptavidin magnetic beads (Adamas life, F8029-1 mL) according to the instructions to obtain the double-tag purified expressed protein.

[0036] 8. Use Bradford protein concentration determination kit (Beyotime, P0006) to determine the concentration of the purified target protein, then use SDS-PAGE gel electrophoresis and silver staining kit (Beyotime, P0017S) for silver staining.

[0037] Silver staining results are as follows Figure 3 and 4 As shown, it can be seen that after double-tag purification, the purity of the target protein is greatly improved and the miscellaneous bands in electrophoresis are significantly reduced.

[0038] Example 2: Functional identification of target protein

[0039] (I) Identification of the ethanol decomposition function of the target protein

[0040] Alcohol dehydrogenase catalyzes NAD+ to oxidize ethanol to produce acetaldehyde and NADH. NADH has an absorption peak at 340nm, while NAD+ does not. The absorbance change at 340nm is measured to determine the ability of the target protein to catalyze the decomposition of ethanol.

[0041] 1. Preparation of solution: Take 0.05 g of NAD+ (Adamas, 63713A) and dissolve it in ultrapure water to 5 mL to obtain NAD+ solution; take 0.1 mL of anhydrous ethanol (Greagent, G73537F) and add it to 9.9 mL of ultrapure water to obtain a 1% ethanol solution; take 0.25 mL of 40% acetaldehyde (Greagent, G80994A) and add it to 9.75 mL of ultrapure water to obtain a 1% acetaldehyde solution.

[0042] 2. Add 560uL of anticoagulant human plasma, 70uL of 1% ethanol solution, and 35uL of NDA+ solution into the same test tube and mix well to obtain a mixed solution.

[0043] 3. Select 6 wells in a 96-well plate and add 95uL of the mixed solution obtained in the previous step to each well. Take 10uL of the control protein, AADH01 protein, and AADH02 protein purified by double tags and add them to the selected 6 wells respectively. Do one replicate for each sample.

[0044] 4. Use an enzyme-labeled instrument to measure the absorbance value at 340 nm, and take the average value for each sample. The results are shown in Table 1.

[0045] Table 1

[0046] Group Control group AADH01 AADH02 Absorbance value 0.7086 0.7163 0.7182

[0047] From the results, the absorbance values ​​of the target proteins AADH01 and AADH02 at 340nm were greater than that of the control group proteins, indicating that the target proteins AADH01 and AADH02 have the ability to catalyze the decomposition of ethanol.

[0048] (II) Identification of the target protein’s ability to decompose acetaldehyde

[0049] Acetaldehyde dehydrogenase catalyzes NAD+ to oxidize ethanol to produce acetic acid and NADH. NADH has an absorption peak at 340nm, while NAD+ does not. The absorbance change at 340nm is measured to determine the ability of the target protein to catalyze the decomposition of acetaldehyde.

[0050] 1. Preparation of solution: Take 0.05 g of NAD+ (Adamas, 63713A) and dissolve it in ultrapure water to 5 mL to obtain NAD+ solution; take 0.1 mL of anhydrous ethanol (Greagent, G73537F) and add it to 9.9 mL of ultrapure water to obtain a 1% ethanol solution; take 0.25 mL of 40% acetaldehyde (Greagent, G80994A) and add it to 9.75 mL of ultrapure water to obtain a 1% acetaldehyde solution.

[0051] 2. Add 560uL of anticoagulant human plasma, 70uL of 1% acetaldehyde solution, and 35uL of NDA+ solution into the same test tube and mix well to obtain a mixed solution.

[0052] 3. Select 6 wells in a 96-well plate and add 95uL of the mixed solution obtained in the previous step to each well. Take 10uL of the control protein, AADH01 protein, and AADH02 protein purified by double tags and add them to the selected 6 wells respectively. Do one replicate for each sample.

[0053] 4. Use an enzyme-labeled instrument to measure the absorbance value at 340 nm, and take the average value for each sample. The results are shown in Table 2.

[0054] Table 2

[0055] Group Control group AADH01 AADH02 Absorbance value 0.7264 0.7581 0.7584

[0056] From the results, the absorbance values ​​of the target proteins AADH01 and AADH02 at 340nm were greater than that of the control group proteins, indicating that the target proteins AADH01 and AADH02 have the ability to catalyze the decomposition of acetaldehyde.

[0057] In summary, the alcohol detoxifying enzyme protein designed in the present invention can catalyze the decomposition of both ethanol and acetaldehyde in the presence of NAD+, and has the function of accelerating alcohol metabolism and reducing the harm of alcohol to the human body.

[0058] The above is a preferred embodiment of the present invention, but the present invention should not be limited to the contents disclosed in the embodiment. Therefore, any equivalent or modification completed without departing from the spirit disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. Application of an alcohol decomposition enzyme protein capable of decomposing ethanol and acetaldehyde in decomposing ethanol and acetaldehyde; The amino acid sequence of the alcohol decomposition enzyme protein capable of decomposing ethanol and acetaldehyde is shown in SEQ ID NO: 6 or SEQ ID NO: 7; The method for preparing the alcohol detoxifying enzyme protein capable of decomposing ethanol and acetaldehyde comprises the following steps:

1. Synthesis of overexpression plasmid Codons were designed according to the sequence shown in SEQ ID NO: 6, named AADH01, and codon optimization, sequence synthesis and plasmid construction were performed; Codons were designed according to the sequence shown in SEQ ID NO: 7, named AADH02, and codon optimization, sequence synthesis and plasmid construction were performed; 2. Target protein expression and purification 1) Transform the plasmids overexpressing the target proteins AADH01 and AADH02 into competent cells and culture at 37°C with shaking for 1 hour; 2) The competent cells transformed with the plasmids overexpressing the target proteins AADH01 and AADH02 were spread on culture plates and inverted for overnight culture, and were labeled as AADH01 and AADH02 respectively; 3) Select monoclonal colonies and add them to liquid culture medium, label them as AADH01 and AADH02 respectively, place them in an air bath constant temperature oscillator, and culture them at 37°C for 8 hours; 4) Add IPTG to the bacterial solution of each transformed colony to a concentration of 1 mM, place AADH01 and AADH02 in an air bath constant temperature oscillator, and culture at 37°C overnight; 5) Centrifuge, collect the precipitate, and purify the protein using the His tag; 6) The protein purified by His tag is purified by Strep tag; 7) Determine the concentration of the purified target protein and identify it by SDS-PAGE gel electrophoresis and silver staining; The alcohol detoxifying enzyme protein + Under the conditions of its existence, it can catalyze the decomposition of both ethanol and acetaldehyde.

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