Method for producing soluble humanized acetaldehyde dehydrogenase 2 in escherichia coli

Abstract

The scheme relates to a method for producing soluble human acetaldehyde dehydrogenase 2 in escherichia coli in the field of enzyme engineering, and the method comprises the following steps: firstly, inserting a human ALDH2 gene into a pET30a (+) plasmid through Nde I and Xho I restriction enzyme cutting sites to form a recombinant expression vector; then, transforming the recombinant vector into escherichia coli by utilizing a heat shock method, and screening out recombinant engineering bacteria which are correctly sequenced; and finally, inoculating and culturing the engineering bacteria under proper conditions, adding lactose as an inducer (the final concentration is 0.2-0.3 mM), inducing for 4-8 hours under the conditions of 25-35 DEG C and 100r / min, and then collecting thalli. The thalli are suspended by a buffer solution, treated by a cell crusher and centrifuged to obtain crude enzyme, and the crude enzyme is further purified to obtain the soluble recombinant human-derived ALDH2. According to the method disclosed by the invention, the production of the soluble humanized acetaldehyde dehydrogenase 2 from the escherichia coli is realized.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to a method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli. Background Art

[0002] After alcohol enters the human body, its metabolism mainly depends on alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Specifically, alcohol is first oxidized into acetaldehyde under the catalysis of alcohol dehydrogenase, and acetaldehyde is further oxidized into acetic acid under the catalysis of aldehyde dehydrogenase. Acetic acid is finally metabolized into carbon dioxide and water and excreted from the body. The content and enzyme activity of alcohol dehydrogenase in different individuals are basically the same, so the step of alcohol being oxidized into acetaldehyde after entering the human body is basically unrestricted. There are many types of acetaldehyde dehydrogenase in the human body, among which the main one involved in alcohol metabolism is liver mitochondrial acetaldehyde dehydrogenase 2 (ALDH2). In the population of China and other Asian countries, about 35-40% of individuals carry acetaldehyde dehydrogenase 2 mutants. The 487th glutamic acid of this mutant is replaced by lysine, resulting in a basic loss of activity. People who carry the acetaldehyde dehydrogenase 2 mutant often experience symptoms such as flushing, headache, and vomiting after drinking because they cannot eliminate acetaldehyde in time. In severe cases, they may even develop cardiovascular diseases, alcoholic liver disease and other diseases, causing great harm to human health.

[0003]

[0004] For people who are carriers of acetaldehyde dehydrogenase 2 mutants, exogenous supplementation of highly active acetaldehyde dehydrogenase can make up for the lack of endogenous acetaldehyde dehydrogenase activity and fundamentally solve the harm of acetaldehyde to the human body. Therefore, acetaldehyde dehydrogenase 2 has received great attention in the development of alcohol relieving drugs. At present, most commercial acetaldehyde dehydrogenases are extracted from animal livers, which are difficult to industrially mass produce and are relatively expensive. In order to obtain a large amount of active acetaldehyde dehydrogenase 2, researchers began to study the use of engineered bacteria to produce human acetaldehyde dehydrogenase 2. Escherichia coli has the advantages of fast growth, low cost, and high expression level. However, in the process of using Escherichia coli to produce human acetaldehyde dehydrogenase 2, relevant researchers found that human acetaldehyde dehydrogenase 2 mostly forms inclusion bodies. The formation of this inclusion body makes it difficult for us to directly obtain active acetaldehyde dehydrogenase 2, which to some extent hinders the research on acetaldehyde dehydrogenase 2. Therefore, it is of great theoretical and practical significance to study the mass production of soluble human acetaldehyde dehydrogenase 2 in the Escherichia coli system. Summary of the invention

[0005] The present invention is intended to provide a method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli, so as to realize large-scale production of soluble human acetaldehyde dehydrogenase 2 from the Escherichia coli system.

[0006] A method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli in this scheme comprises the following steps:

[0007] Step 1, construction of a recombinant expression vector: insert the human acetaldehyde dehydrogenase 2 gene into the plasmid pET30a(+) through the Nde I and Xho I restriction enzyme sites, and the resulting recombinant expression vector is named pET30a(+)-ALDH2;

[0008] Step 2, culturing recombinant engineered bacteria: transforming the recombinant expression vector into Escherichia coli by heat shock method and culturing, collecting the cultured bacteria and extracting the plasmid pET30a(+)-ALDH2 and sequencing, and the bacteria with correct sequencing are recombinant engineered bacteria;

[0009] Step 3, purification of acetaldehyde dehydrogenase 2: inoculate and culture the recombinant engineered bacteria, then add lactose with a final concentration of 0.2-0.3 mM, and induce expression at 25-35° C., 100 r / min for 4-8 hours; after the induction, collect the bacteria by centrifugation, suspend the bacteria with buffer, and then break the cells with a cell disruptor, collect the supernatant by centrifugation to obtain a crude enzyme solution of human acetaldehyde dehydrogenase 2, and purify the crude enzyme solution of human acetaldehyde dehydrogenase 2 to obtain the human acetaldehyde dehydrogenase 2.

[0010] Furthermore, the Escherichia coli is selected from Escherichia coli Rosetta (DE3) or Escherichia coli BL21 (DE3).

[0011] Furthermore, when the recombinant expression vector is transformed into Escherichia coli by the heat shock method and cultured, an LB solid plate containing 45 to 55 μg / mL kanapencillin is used for screening; a single clone is picked from the screening plate and inoculated into an LB liquid culture medium, and kanapencillin with a final concentration of 45 to 55 μg / mL is added, and cultured at 35 to 40°C and 180 to 220 r / min for 10 to 15 hours.

[0012] Furthermore, when the recombinant engineered bacteria are inoculated and cultured, the recombinant engineered bacteria are inoculated into LB liquid culture medium, kanapencillin with a final concentration of 45-55 μg / mL is added, and cultured at 35-40°C, 180-220 r / min for 10-15 hours, and then transferred to fresh LB liquid culture medium, kanapencillin with a final concentration of 45-55 μg / mL is added, and cultured at 35-40°C, 180-220 r / min for 4-6 hours.

[0013] Furthermore, when the recombinant engineered bacteria were transferred to fresh LB liquid culture medium, lactose was added for induction after culturing for 5.5 hours.

[0014] Furthermore, the final concentration of lactose is 0.25 mM.

[0015] Furthermore, the lactose induction temperature is 30°C.

[0016] Furthermore, the lactose induction time is 6 hours.

[0017] Further, the crude enzyme solution of human acetaldehyde dehydrogenase 2 was purified by nickel column affinity chromatography, and the specific steps were as follows:

[0018] (1) Nickel column equilibration: Add 5 column volumes of Lysis buffer to the nickel column and allow gravity to flow out to equilibrate the column;

[0019] (2) Loading: Add the crude enzyme solution of human acetaldehyde dehydrogenase 2 to the above-mentioned equilibrated nickel column and let it flow out by gravity;

[0020] (3) Washing: Add 10 column volumes of washing buffer (50 mM Tris, 300 mM NaCl, 30 mM imidazole) to the nickel column and allow gravity flow to wash out the impurities bound to the nickel column;

[0021] (4) Elution: Add 2 column volumes of Elution buffer (50 mM Tris, 300 mM NaCl, 150 mM imidazole) to the nickel column and allow gravity to flow out. At this time, the target protein is displaced and eluted, and this portion of the eluate is collected;

[0022] (5) Dialysis: The eluate was transferred to a 14 kD dialysis bag, which was placed in a 20 mM Tris-HCl buffer and dialyzed overnight to reduce the imidazole concentration in the enzyme solution.

[0023] Furthermore, during elution, the composition of the elution buffer was 50 mM Tris, 300 mM NaCl, and 150 mM imidazole.

[0024] Beneficial effects:

[0025] 1. This patent uses the E. coli system to directly obtain soluble human acetaldehyde dehydrogenase 2. After purification, the concentration of human acetaldehyde dehydrogenase 2 reaches 3.7 mg / mL. This method effectively avoids the cumbersome and complicated refolding process, and does not require the removal of the fusion tag, which greatly simplifies the purification process of human acetaldehyde dehydrogenase 2, making it more operable and efficient;

[0026] 2. This patent uses cheap lactose as an inducer to induce the expression of human aldehyde dehydrogenase 2 gene. Lactose is not only non-toxic to cells, but also has a better induction effect than IPTG. In addition, its significant advantage of low cost has laid a certain foundation for the realization of industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram for constructing a recombinant expression vector;

[0028] Figure 2 This is the electrophoresis diagram of human acetaldehyde dehydrogenase 2 after purification by nickel column;

[0029] Figure 3 The electrophoresis diagram of human acetaldehyde dehydrogenase 2 expressed in Rosetta (a) and BL21 (DE3) (b);

[0030] Figure 4 This is the electrophoresis diagram of the effect of induction temperature on the expression of human acetaldehyde dehydrogenase 2;

[0031] Figure 5 The electrophoresis diagram shows the effect of the concentration of inducer IPTG on the expression of human acetaldehyde dehydrogenase 2;

[0032] Figure 6 The electrophoresis diagram shows the effect of the concentration of inducer lactose on the expression of human acetaldehyde dehydrogenase 2;

[0033] Figure 7 This is the electrophoresis diagram of the effect of lactose induction time on the expression of human acetaldehyde dehydrogenase 2;

[0034] Figure 8 This is the electrophoresis diagram of the effect of induced rotation speed on the expression of human acetaldehyde dehydrogenase 2;

[0035] Fig. 9 This is the electrophoresis diagram of the effect of induction time on the expression of human acetaldehyde dehydrogenase 2;

[0036] Fig.10 This is the electrophoresis diagram of the effect of lactose addition on the expression of human acetaldehyde dehydrogenase 2;

[0037] Fig.11 The electrophoretic diagram shows the effect of the fragmentation time on the target protein content in the supernatant;

[0038] Fig.12 This is the electrophoresis diagram of the elution results of human acetaldehyde dehydrogenase 2 at different imidazole concentrations;

[0039] Fig.13 This is the effect curve of temperature on the activity of human acetaldehyde dehydrogenase 2;

[0040] Fig.14 This is the effect curve of pH on the activity of human acetaldehyde dehydrogenase 2;

[0041] Fig.15 This is a bar graph showing the effect of metal ions on the activity of human acetaldehyde dehydrogenase 2;

[0042] Fig.16 This is the acetaldehyde oxidation curve catalyzed by human acetaldehyde dehydrogenase 2. DETAILED DESCRIPTION

[0043] The following is further described in detail through specific implementation methods:

[0044] Example 1: A method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli, comprising the following steps:

[0045] Step 1: Sequence analysis of human aldehyde dehydrogenase 2

[0046] The human acetaldehyde dehydrogenase 2 encoding gene SEQ ID NO1 and the human acetaldehyde dehydrogenase 2 amino acid sequence SEQ ID NO2 in the present invention are as follows:

[0047] The sequence of SEQ ID NO1 is as follows:

[0048] TCAGCCGCCGCCACCCAGGCCGTGCCTGCCCCCAACCAGCAGCCCGAGGTCTTTCTGCAACCAGATTTTCATAAAC

[0049] AATGAATGGCACGATGCCGTCAGCAGGAAAACATTCCCCACCGTCAATCCGTCCACTGGAGAGGTCATCTGTCAGGTAG

[0050] CTGAAGGGGACAAGGAAGATGTGGACAAGGCAGTGAAGGCCGCCCGGGCCGCCTTCCAGCTGGGCTCACCTTGCGCCG

[0051] CATGGACGCATCACACAGGGGCCGGCTGCTGAACCGCCTGGCCGATCTGATCGAGCGGGACCGGACCTACCTGGCGGCC

[0052] TTGGAGACCCTGGACAATGGCAAGCCCTATGTCATCTCCTACCTGGTGGATTTGGACATGGTCCTCAAATGTCTCCGGT

[0053] ATTATGCCGGCTGGGCTGATAAGTACCACGGGAAAACCATCCCCATTGACGGAGACTTCTTCAGCTACACACGCCATGA

[0054] ACCTGTGGGGGTGTGCGGGCAGATCATTCCGTGGAATTTCCCGCTCCTGATGCAAGCATGGAAGCTGGGCCCAGCCTTG

[0055] GCAACTGGAAACGTGGTTGTGATGAAGGTAGCTGAGCAGACACCCCTCACCGCCCTCTATGTGGCCAACCTGATCAAGG

[0056] AGGCTGGCTTTCCCCCTGGTGTGGTCAACATTGTGCCTGGATTTGGCCCCACGGCTGGGGCCGCCATTGCCTCCCATGA

[0057] GGATGTGGACAAAGTGGCATTCACAGGCTCCACTGAGATTGGCCGCGTAATCCAGGTTGCTGCTGGGAGCAGCAACCTC

[0058] AAGAGAGTGACCTTGGAGCTGGGGGGGAAGAGCCCCAACATCATCATGTCAGATGCCGATATGGATTGGGCCGTGGAAC

[0059] AGGCCCACTTCGCCCTGTTCTTCAACCAGGGCCAGTGCTGCTGTGCCGGCTCCCGGACCTTCGTGCAGGAGGACATCTA

[0060] TGATGAGTTTGTGGAGCGGAGCGTTGCCCGGGCCAAGTCTCGGGTGGTCGGGAACCCCTTTGATAGCAAGACCGAGCAG

[0061] GGGCCGCAGGTGGATGAAACTCAGTTTAAGAAGATCCTCGGCTACATCAACACGGGGAAGCAAGAGGGGGCGAAGCTGC

[0062] TGTGTGGTGGGGGCATTGCTGCTGACCGTGGTTACTTCATCCAGCCCACTGTGTTTGGAGATGTGCAGGATGGCATGAC

[0063] CATCGCCAAGGAGGAGATCTTCGGGCCAGTGATGCAGATCCTGAAGTTCAAGACCATAGAGGAGGTTGTTGGGAGAGCC

[0064] AACAATTCCACGTACGGGCTGGCCGCAGCTGTCTTCACAAAGGATTTGGACAAGGCCAATTACCTGTCCCAGGCCCTCC

[0065] AGGCGGGCACTGTGTGGGTCAACTGCTATGATGTGTTTGGAGCCCAGTCACCCTTTGGTGGCTACAAGATGTCGGGGAG

[0066] TGGCCGGGAGTTGGGCGAGTACGGGCTGCAGGCATACACTGAAGTGAAAACTGTCACAGTCAAAGTGCCTCAGAAGAAC

[0067] TCA

[0068] The sequence of SEQ ID NO2 is as follows:

[0069] SAAATQAVPAPNQQPEVFCNQIFINNEWHDAVSRKTFPTVNPSTGEVICQVAEGDKEDVDKAVKAARAAFQLGSPWRRMDASHRGRLLNRLADLIERDRTYLAALETLDNGKPYVISYLVDLDMV LKCLRYYAGWADKYHGKTIPIDGDFFSYTRHEPVGVCGQIIPWNFPLLMQAWKLGPALATGNVVVMKVAEQTPLTALYVANLIKEAGFPPGVVNIVPGFGPTAGAAIASHEDVDKVAFTGSTEIG RVIQVAAGSSNLKRVTLELGGKSPNIIMSDADMDWAVEQAHFALFFNQGQCCCAGSRTFVQEDIYDEFVERSVARAKSRVVGNPFDSKTEQGPQVDETQFKKILGYINTGKQEGAKLLCGGGIAA DRGYFIQPTVFGDVQDGMTIAKEEIFGPVMQILKFKTIEEVVGRANNSTYGLAAAVFTKDLDKANYLSQALQAGTVWVNCYDVFGAQSPFGGYKMSGSGRELGEYGLQAYTEVKTVTVKVPQKNS

[0070] Step 2: Construction of recombinant expression vector

[0071] According to the sequence of human aldehyde dehydrogenase 2 encoding gene SEQ ID NO1, we commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize it and connected it to plasmid pET30a(+) through Nde I and Xho I restriction enzyme cutting sites. The obtained recombinant expression vector was named pET30a(+)-ALDH2 (see Appendix Figure 1 ).

[0072] Step 3: Cultivation of recombinant engineered bacteria

[0073] The above recombinant expression vectors were transformed into Escherichia coli Rosetta (DE3) and BL21 (DE3) by heat shock method, and screened using LB solid plates containing 50 μg / mL kanapencillin. Single clones were picked from the screening plate and inoculated into LB liquid culture medium, and kanapencillin with a final concentration of 50 μg / mL was added, and cultured at 37°C 200r / min for 12h. After the culture was completed, the bacteria were collected, and the plasmid pET30a(+)-ALDH2 was extracted using a plasmid extraction kit. The clones with correct sequencing were selected and stored in glycerol tubes. The recombinant engineered bacteria were named Rosetta-pET30a(+)-ALDH2 and BL21-pET30a(+)-ALDH2.

[0074] Step 4: Preparation of soluble human acetaldehyde dehydrogenase 2 crude enzyme solution

[0075] The above recombinant engineered bacteria Rosetta-pET30a(+)-ALDH2 stored in the glycerol tube was inoculated into LB liquid culture medium, and kanamycin with a final concentration of 50μg / mL was added. After overnight culture at 37℃, 200r / min, it was transferred to fresh LB liquid culture medium, and cultured at 37℃, 200r / min for 5.5h, and then lactose with a final concentration of 0.25mM was added, and expression was induced at 30℃, 100r / min for 6h. After the induction, the bacteria were collected by centrifugation to obtain at least 1L of induced cells, and the bacteria were suspended in Lysis buffer (50mM Tris, 300mM NaCl, 10mM imidazole), and the cells were then broken with a cell disruptor, and the supernatant was collected by centrifugation at 12000r / min to obtain the crude enzyme solution of human acetaldehyde dehydrogenase 2.

[0076] Step 5. Purification of soluble human acetaldehyde dehydrogenase 2

[0077] Human acetaldehyde dehydrogenase 2 was purified by nickel column affinity chromatography. The specific steps are as follows:

[0078] (1) Nickel column equilibration: Take a nickel column and add 5 times the column volume of Lysis buffer to it, allowing gravity to flow out to equilibrate the column.

[0079] (2) Sample loading operation: Add the crude enzyme solution of human acetaldehyde dehydrogenase 2 to the balanced nickel column. Under the action of gravity, the crude enzyme solution slowly flows out. Since the carboxyl end of human acetaldehyde dehydrogenase 2 contains a histidine tag, it can produce an affinity binding effect with the nickel ions in the nickel column and then adsorb on the nickel column.

[0080] (3) Washing step: Add 10 times column volume of washing buffer (50mM Tris, 300mM NaCl, 30mM imidazole) into the nickel column and let it flow out by gravity. During this process, the impurities bound to the nickel column will be washed out. Use Coomassie Brilliant Blue to detect the washing effect.

[0081] (4) Elution process: Add 2 column volumes of Elution buffer (50 mM Tris, 300 mM NaCl, 150 mM imidazole) to the nickel column and allow it to flow out under gravity. At this time, the target protein, human acetaldehyde dehydrogenase 2, is washed out due to the displacement of imidazole. Collect this part of the effluent, which is rich in high-purity human acetaldehyde dehydrogenase 2.

[0082] (5) Dialysis operation: The collected eluate is transferred to a 14kD dialysis bag, which is then placed in a 20mM Tris-HCl buffer and dialyzed overnight. This dialysis process can effectively reduce the imidazole concentration in the enzyme solution, further improving the purity and stability of the enzyme solution.

[0083] The results of purification of human aldehyde dehydrogenase 2 are shown in Figure 2 , M represents protein marker, and lane 1 is the purified human acetaldehyde dehydrogenase 2. The electrophoresis diagram shows that after purification, human acetaldehyde dehydrogenase 2 presents a relatively single and clear band, and its band size is within the range of 50-70 kD, which is consistent with the expected size.

[0084] According to the protein concentration measurement, the concentration of purified human acetaldehyde dehydrogenase 2 can reach 3.7 mg / mL.

[0085] Optimization of aldehyde dehydrogenase 2 gene expression conditions

[0086] 1. Selection of competent cells

[0087] The glycerol-preserved recombinant engineered bacteria Rosetta-pET30a(+)-ALDH2 and BL21-pET30a(+)-ALDH2 obtained in Example 1 were inoculated into LB liquid culture medium, and kanamycin with a final concentration of 50 μg / mL was added. The culture was continued at 37°C 200r / min for 12h, and then transferred to fresh LB liquid culture medium. Kana penicillin with a final concentration of 50 μg / mL was added. The culture was continued at 37°C 200r / min for about 2.5h, and IPTG with a final concentration of 0.5mM was added. The expression was induced at 16°C 150r / min for 16-20h. After the induction, the cells were collected by centrifugation, and the cells were suspended in 1×PBS buffer. The cells were then broken with a cell disruptor. The conditions for the cell disruptor were: break for 2s, stop for 5s, and the total time was 15min. After the break, the supernatant and precipitate were separated by centrifugation, and the expression was detected by SDS-PAGE.

[0088] The expression results of human aldehyde dehydrogenase 2 gene in Rosetta (DE3) and BL21 (DE3) are shown in Figure 3 , M is a protein marker, lane 1 is whole cells before induction, lane 2 is whole cells after induction, lane 3 is supernatant of cell disruption after induction, and lane 4 is precipitate of cell disruption after induction. Figure 3It can be seen that the molecular weight of human acetaldehyde dehydrogenase 2 is in the range of 50-70kD, and its theoretical relative molecular weight is 55.6kD. It can be seen that the experimental results are consistent with expectations. Further observation shows that acetaldehyde dehydrogenase 2 is mainly concentrated in the crushed precipitate, while the content in the crushed supernatant is relatively small. Comparing the expression of the human acetaldehyde dehydrogenase 2 gene in the two competent states, when expressed in Rosetta (DE3), its content in the crushed supernatant was slightly increased compared to when expressed in BL21 (DE3). Based on this, the Rosetta (DE3) competent state was selected for the expression of the human acetaldehyde dehydrogenase 2 gene, and Rosetta-pET30a (+) -ALDH2 was determined as the target strain for subsequent condition optimization work.

[0089] 2. Induction temperature

[0090] The glycerol-preserved recombinant engineered bacteria Rosetta-pET30a(+)-ALDH2 obtained in Example 1 was inoculated into LB liquid culture medium, and according to the above-mentioned recombinant engineered bacteria induction expression steps, a final concentration of 0.5 mM IPTG was added, and the expression was induced at 16°C, 20°C, 30°C, and 37°C for 6 hours. The induction expression temperature of the subsequent experiment was determined according to the content of human acetaldehyde dehydrogenase 2 in the supernatant;

[0091] Figure 4 This is an electrophoresis diagram of the effect of induction temperature on the expression of human acetaldehyde dehydrogenase 2. Lane 1 is the whole cell before induction, lanes 2-5 are the whole cells after induction at 16°C, 20°C, 30°C, and 37°C, respectively. M is a protein marker, lanes 6-9 are the supernatants of cell fragmentation after induction at 16°C, 20°C, 30°C, and 37°C, respectively, and lanes 10-13 are the precipitates of cell fragmentation after induction at 16°C, 20°C, 30°C, and 37°C, respectively. It can be seen from the electrophoresis diagram that with the increase of the induction temperature, the content of human acetaldehyde dehydrogenase 2 in the supernatant shows a trend of gradual increase. When the induction temperature is 30°C, the content of human acetaldehyde dehydrogenase 2 in the supernatant is the highest. Based on this, the subsequent induction temperature is determined to be 30°C.

[0092] 3. Concentration of inducer IPTG

[0093] According to the above-mentioned recombinant engineering bacteria induction expression steps, IPTG was added at a final concentration of 0.25mM, 0.5mM, 0.75mM, and 1.0mM for induction, and the induction temperature was 30°C. The concentration of the inducer IPTG in subsequent experiments was determined according to the content of human acetaldehyde dehydrogenase 2 in the supernatant;

[0094] Figure 5This is an electrophoresis diagram of the effect of the concentration of inducer IPTG on the expression of human acetaldehyde dehydrogenase 2. Lane 1 is the whole cell before induction, lanes 2-5 are the whole cells after induction with final concentrations of 0.25mM, 0.5mM, 0.75mM, and 1.0mM IPTG, respectively. M is a protein marker, lanes 6-9 are the supernatants of cell fragmentation after induction with final concentrations of 0.25mM, 0.5mM, 0.75mM, and 1.0mM IPTG, respectively, and lanes 10-13 are the precipitates of cell fragmentation after induction with final concentrations of 0.25mM, 0.5mM, 0.75mM, and 1.0mM IPTG. It can be seen from the electrophoresis diagram that when the concentration of inducer IPTG is 0.25mM, the induction effect is already good, and the change of IPTG concentration has little effect on the content of human acetaldehyde dehydrogenase 2 in the supernatant. Based on the important principle of economic saving, the final concentration of inducer IPTG in subsequent experiments is determined to be 0.25mM.

[0095] 4. Concentration of inducer lactose

[0096] According to the above-mentioned recombinant engineering bacteria induction expression steps, the induction was carried out at a final concentration of 0.25mM IPTG and a final concentration of 0.25mM, 0.5mM, 1mM, 2mM, and 4mM lactose, respectively, and the induction temperature was 30°C. The type and concentration of the inducer for subsequent experiments were determined according to the content of human acetaldehyde dehydrogenase 2 in the supernatant;

[0097] Figure 6 This is an electrophoresis diagram of the effect of the inducer lactose concentration on the expression of human acetaldehyde dehydrogenase 2. Lane 1 is the whole cell before induction, lane 2 is the whole cell after induction with a final concentration of 0.25mM IPTG, lanes 3-7 are the whole cells after induction with final concentrations of 0.25mM, 0.5mM, 1mM, 2mM, and 4mM lactose, respectively. M is a protein marker, lane 8 is the supernatant of cell disruption after induction with a final concentration of 0.25mM IPTG, and lanes 9-13 are the supernatants of cell disruption after induction with final concentrations of 0.5mM, 1mM, 2mM, and 4mM lactose, respectively. It can be seen from the electrophoresis diagram that when lactose is used as an inducer, the content of human acetaldehyde dehydrogenase 2 in the cell disruption supernatant is higher than when IPTG is used as an inducer. With the increase of lactose concentration, the content of human acetaldehyde dehydrogenase 2 in whole cells and disrupted supernatants remains basically unchanged. Based on the significant advantages of lactose over IPTG, such as low cost and non-toxicity to cells, lactose was selected as the inducer for subsequent experiments with a final concentration of 0.25 mM.

[0098] 5. Timing of lactose induction

[0099] According to the above-mentioned recombinant engineering bacteria induction expression steps, the glycerol bacteria were cultured in LB medium at 37°C and 200 r / min overnight, and then transferred to fresh LB liquid medium. After culturing at 37°C and 200 r / min for 0 h, 1 h, 2.5 h, 4.0 h, and 5.5 h, lactose with a final concentration of 0.25 mM was added and induced at 30°C. The induction timing of subsequent experiments was determined according to the content of human acetaldehyde dehydrogenase 2 in the supernatant.

[0100] Figure 7 This is an electrophoresis diagram of the effect of lactose induction timing on the expression of human acetaldehyde dehydrogenase 2; lane 1 is the whole cell before induction, lanes 2-6 are the whole cells after induction at 0h, 1h, 2.5h, 4.0h, and 5.5h, respectively, M is a protein marker, and lanes 7-11 are the supernatants of cell fragmentation after induction at 0h, 1h, 2.5h, 4.0h, and 5.5h, respectively. It can be seen from the electrophoresis diagram that even if lactose is added immediately after the transfer for induction, human acetaldehyde dehydrogenase 2 can still be expressed in a soluble form. Between 0-5.5h, the content of human acetaldehyde dehydrogenase 2 in the supernatant increases with the increase of the induction timing. Based on time cost considerations, the induction timing is no longer extended, and the induction timing is selected to be 5.5h after the transfer.

[0101] 6. Induced speed

[0102] According to the above-mentioned recombinant engineering bacteria induction expression steps, 5.5 hours after transfer, lactose with a final concentration of 0.25mM was added, and the induction was carried out at a speed of 50r / min, 100r / min, 150r / min, and 200r / min, respectively. The induction temperature was 30°C, and the induction speed of subsequent experiments was determined according to the content of human acetaldehyde dehydrogenase 2 in the crushing supernatant;

[0103] Figure 8 This is an electrophoresis diagram of the effect of rotation speed on the expression of human acetaldehyde dehydrogenase 2; lane 1 is the whole cell before induction, lanes 2-5 are the whole cells after induction at rotation speeds of 50r / min, 100r / min, 150r / min, and 200r / min, respectively, M is a protein marker, lanes 6-9 are the supernatant of cell disruption after induction at rotation speeds of 50r / min, 100r / min, 150r / min, and 200r / min, respectively, and lanes 10-13 are the precipitates of cell disruption after induction at rotation speeds of 50r / min, 100r / min, 150r / min, and 200r / min, respectively. It can be seen from the electrophoresis diagram that when the rotation speed is 100r / min, the content of human acetaldehyde dehydrogenase 2 in the disruption supernatant is the highest, and it is determined that the induction speed in subsequent experiments is 100r / min.

[0104] 7. Induction duration

[0105] According to the above-mentioned recombinant engineering bacteria induction expression steps, 5.5 hours after transfer, lactose with a final concentration of 0.25mM was added, and the induction was carried out at 30°C and 100r / min. The induction time was 4h, 6h, 8h, and 10h respectively. The induction time of subsequent experiments was determined according to the content of human acetaldehyde dehydrogenase 2 in the supernatant;

[0106] Fig. 9 This is an electrophoresis diagram of the effect of lactose induction time on the expression of human acetaldehyde dehydrogenase 2. Lane 1 is the whole cell before induction, lanes 2-5 are the whole cells after induction for 4h, 6h, 8h, and 10h, respectively. M is a protein marker, lanes 6-9 are the supernatants of cell fragmentation after induction for 4h, 6h, 8h, and 10h, respectively, and lanes 10-13 are the precipitates of cell fragmentation after induction for 4h, 6h, 8h, and 10h. It can be seen from the electrophoresis diagram that when the induction time reaches 6h, if the induction time is further extended, the content of acetaldehyde dehydrogenase 2 in the whole cell and the supernatant is almost the same, with no obvious increase. In view of the fact that time cost is an extremely critical factor in the scientific research and production process, in order to maximize the benefits, the induction time is set to 6h.

[0107] 8. How lactose is added

[0108] According to the above-mentioned recombinant engineering bacteria induction expression steps, 5.5 hours after transfer, induce at 30°C, 100r / min for 6 hours. The inducer lactose is added in one time or in four times. The final concentration of lactose is 0.25mM. The addition method of lactose in subsequent experiments is determined according to the content of human acetaldehyde dehydrogenase 2 in the supernatant.

[0109] Fig.10 The electrophoresis diagram of the effect of lactose addition on the expression of human acetaldehyde dehydrogenase 2; Lane 1 is the whole cell before induction, Lanes 2-3 are the whole cells after induction with lactose added once and added in batches, M is the protein marker, Lanes 4-5 are the supernatants of cell fragmentation after induction with lactose added once and added in batches, Lanes 6-7 are the precipitates of cell fragmentation after induction with lactose added once and added in batches. It can be seen from the electrophoresis diagram that the addition of lactose once and added in batches have no obvious effect on the content of human acetaldehyde dehydrogenase 2 in whole cells and fragmentation supernatants. For the convenience of operation, it was determined that the induction method of adding lactose once was used in the subsequent experiments.

[0110] 9. Broken Time

[0111] According to the above-mentioned steps of inducing expression of recombinant engineered bacteria, 1L of bacterial solution was prepared. 5.5 hours after transfer, lactose with a final concentration of 0.25mM was added once, and induced at 30°C and 100r / min for 6 hours to obtain the induced recombinant engineered bacteria. The induced recombinant engineered bacteria were broken for 5min, 10min, 15min, 20min, 25min, and 30min respectively. The breaking time of subsequent experiments was determined according to the content of human acetaldehyde dehydrogenase 2 in the supernatant.

[0112] Fig.11 The electrophoresis diagram shows the effect of the fragmentation time on the amount of supernatant enzyme; M is a protein marker, lanes 1-6 are the fragmentation supernatants with fragmentation time of 5min, 10min, 15min, 20min, 25min, and 30min, respectively, and lanes 7-12 are the fragmentation precipitates with fragmentation time of 5min, 10min, 15min, 20min, 25min, and 30min, respectively. It can be seen from the electrophoresis diagram that when the fragmentation time is 5min, the content of human acetaldehyde dehydrogenase 2 in the fragmentation supernatant is already high. Considering that the heat generated during the fragmentation process may have an adverse effect on the activity of the enzyme, the fragmentation time in subsequent experiments was finally determined to be 5-10min.

[0113] 10. Imidazole concentration in eluent

[0114] According to the above-mentioned steps of inducing expression of recombinant engineered bacteria, 1L of bacterial solution was prepared. 5.5 hours after transfer, lactose with a final concentration of 0.25mM was added once, and induced at 30°C and 100r / min for 6 hours to obtain the induced recombinant engineered bacteria. After breaking for 10 minutes, the supernatant was obtained by centrifugation. The supernatant was passed through a nickel column, and the column was washed with eluents with imidazole concentrations of 30mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, and 500mM, respectively. The eluate was collected and the concentration of imidazole in the eluate in subsequent experiments was determined by electrophoresis.

[0115] Fig.12 This is an electrophoretic diagram showing the effect of imidazole concentration in the eluent on the elution of human acetaldehyde dehydrogenase 2. Lane 1 is whole cells before induction, lane 2 is whole cells after induction, M is protein marker, and lanes 3-10 are eluents with imidazole concentrations of 30mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, and 500mM, respectively. It can be seen from the electrophoretic diagram that human acetaldehyde dehydrogenase 2 can be basically washed out when the imidazole concentration in the eluent is 150mM. Considering that high salt concentration may affect enzyme activity, the imidazole concentration of the eluent in subsequent experiments was determined to be 150mM.

[0116] Enzymatic properties of soluble human aldehyde dehydrogenase 2

[0117] 1. Effect of temperature on acetaldehyde dehydrogenase activity

[0118] Mix the following ingredients: 30 μL of 1 mol / L Tris-HCl (pH 8.5), 30 μL of 20 mmol / L β-NAD, 20 μL of 100 mmol / L acetaldehyde, 10 μL of 3 mol / L KCl, 3 μL of 1 mol / L β-mercaptoethanol, and 206.5 μL of H2O, add to a cuvette, preheat at the corresponding temperature, and finally add 0.5 μL of purified human acetaldehyde dehydrogenase 2. React at 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, and 60°C for 5 minutes. After the reaction, immediately measure the absorbance of the reaction solution at 340 nm. NAD + It is a coenzyme in the catalytic reaction of ALDH2. In the catalytic reaction, NAD + It accepts hydrogen and becomes NADH. NADH has a maximum absorption value at 340nm. Therefore, the amount of NADH generated can be calculated by calculating the change in absorbance at 340nm within a specific time.

[0119] Fig.13 The figure is a curve chart showing the effect of temperature on the activity of human acetaldehyde dehydrogenase 2. It can be seen from the curve chart that with the increase of temperature, the activity of acetaldehyde dehydrogenase 2 shows a trend of first increasing and then decreasing, and the enzyme activity reaches the maximum at 37°C.

[0120] 2. Effect of pH on aldehyde dehydrogenase activity

[0121] The reaction system in 1 was followed, and the pH values ​​of the reaction system were adjusted to 4, 5, 6, 7, 8, 9, 10, and 11, respectively, wherein the buffer used for pH 4-8 was sodium hydrogen phosphate-citric acid buffer, and the buffer used for pH 8-11 was glycine-sodium hydroxide buffer. The reaction solutions with different pH values ​​were reacted at 37° C. for 5 min, and the absorbance of the reaction solution at 340 nm was measured immediately after the reaction.

[0122] Fig.14 This is a curve chart showing the effect of pH on the activity of human acetaldehyde dehydrogenase 2. It can be seen from the curve chart that with the increase of pH, the activity of acetaldehyde dehydrogenase shows a trend of first increasing and then decreasing, and the enzyme activity reaches the maximum at pH 8.0.

[0123] 3. Effects of metal ions on acetaldehyde dehydrogenase activity

[0124] The reaction system in 1 was followed, the pH of the reaction system was adjusted to 8, and EDTA and Ca were added to the reaction system at a final concentration of 5 mM. 2+ , K + 、Na + Mg 2+ , Mn 2+ 、Zn2+ , Fe 3+ , react at 37°C for 5 min, and immediately measure the absorbance of the reaction solution at 340 nm after the reaction.

[0125] Fig.15 This is a bar graph showing the effect of metal ions on the activity of human acetaldehyde dehydrogenase 2. It can be seen from the bar graph that no metal ions can promote the activity of acetaldehyde dehydrogenase 2.

[0126] 4. Acetaldehyde dehydrogenase 2 oxidation curve of acetaldehyde

[0127] The reaction system in 1 was followed, the pH value of the reaction system was adjusted to 8, the reaction was carried out at 37°C, and the absorbance at 340 nm was measured at regular intervals.

[0128] Fig.16 This is the curve of acetaldehyde oxidation by human acetaldehyde dehydrogenase 2. As can be seen from the curve, as the reaction time increases, OD 340nm It showed an increasing trend and basically reached the maximum value at 35 minutes. This shows that within 0-35 minutes, as time goes on, NADH in the reaction system increases. That is, after the expression conditions are optimized, the soluble human acetaldehyde dehydrogenase 2 produced in Escherichia coli by lactose induction has the corresponding enzyme activity and can catalyze the conversion of acetaldehyde into acetic acid.

[0129] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli, characterized in that: The following steps are involved: Step 1, construction of a recombinant expression vector: insert the human acetaldehyde dehydrogenase 2 gene into the plasmid pET30a(+) through the Nde I and Xho I restriction enzyme sites, and the resulting recombinant expression vector is named pET30a(+)-ALDH2; Step 2, culturing recombinant engineered bacteria: transforming the recombinant expression vector into Escherichia coli by heat shock method and culturing, collecting the cultured bacteria and extracting the plasmid pET30a(+)-ALDH2 and sequencing, and the bacteria with correct sequencing are recombinant engineered bacteria; Step 3, purification of acetaldehyde dehydrogenase 2: inoculate and culture the recombinant engineered bacteria, then add lactose with a final concentration of 0.2-0.3 mM, and induce expression at 25-35° C., 100 r / min for 4-8 hours; after the induction, collect the bacteria by centrifugation, suspend the bacteria with buffer, and then break the cells with a cell disruptor, collect the supernatant by centrifugation to obtain a crude enzyme solution of human acetaldehyde dehydrogenase 2, and purify the crude enzyme solution of human acetaldehyde dehydrogenase 2 to obtain the human acetaldehyde dehydrogenase 2.

2. The method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 1, characterized in that: The Escherichia coli is selected from Escherichia coli Rosetta (DE3) or Escherichia coli BL21 (DE3).

3. The method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 2, characterized in that: When the recombinant expression vector is transformed into Escherichia coli by the heat shock method for culture, LB solid plates containing 45-55 μg / mL kanapencillin are used for screening; a single clone is picked from the screening plate and inoculated into LB liquid culture medium, kanapencillin with a final concentration of 45-55 μg / mL is added, and cultured at 35-40° C. and 180-220 r / min for 10-15 hours.

4. The method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 3, characterized in that: When the recombinant engineered bacteria are inoculated and cultured, the recombinant engineered bacteria are inoculated into LB liquid culture medium, kanapenicillin with a final concentration of 45 to 55 μg / mL is added, and cultured at 35 to 40° C., 180 to 220 r / min for 10 to 15 hours, and then transferred to fresh LB liquid culture medium, kanapenicillin with a final concentration of 45 to 55 μg / mL is added, and cultured at 35 to 40° C., 180 to 220 r / min for 4 to 6 hours.

5. A method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 4, characterized in that: When the recombinant engineered bacteria were transferred to fresh LB liquid culture medium, lactose was added for induction after culturing for 5.5 hours.

6. The method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 5, characterized in that: The final concentration of lactose was 0.25 mM.

7. The method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 6, characterized in that: The lactose induction temperature is 30°C.

8. The method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 7, characterized in that: The lactose induction time is 6h.

9. A method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to any one of claims 1 to 8, characterized in that: The crude enzyme solution of human acetaldehyde dehydrogenase 2 was purified by nickel column affinity chromatography. The specific steps are as follows: (1) Nickel column equilibration: Add 5 column volumes of Lysis buffer to the nickel column and allow gravity to flow out to equilibrate the column; (2) Loading: Add the crude enzyme solution of human acetaldehyde dehydrogenase 2 to the above-mentioned equilibrated nickel column and let it flow out by gravity; (3) Washing: Add 10 column volumes of washing buffer to the nickel column and let gravity flow out to wash out the impurities bound to the nickel column; (4) Elution: Add 2 column volumes of elution buffer to the nickel column, let gravity flow out, and collect the eluate containing the target protein; (5) Dialysis: The eluate was transferred to a 14 kD dialysis bag, which was placed in 20 mM Tris-HCl buffer and dialyzed overnight.

10. The method for producing soluble human acetaldehyde dehydrogenase 2 in Escherichia coli according to claim 9, characterized in that: During elution, the composition of the elution buffer was 50 mM Tris, 300 mM NaCl, and 150 mM imidazole.

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