Engineering bacterium for producing human stomach ethanol dehydrogenase (ADH) and preparation method of ADH of engineering bacterium
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A technology of alcohol dehydrogenase and bacteria, applied in the biological field, can solve problems such as high cost, long process time, and complicated procedures, and achieve the effects of improving efficiency, reducing ethanol content, and simplifying operation steps
Active Publication Date: 2017-03-29
JIANGNAN UNIV RUGAO FOOD BIOTECH RES INST
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Although this method uses a low concentration of denaturant to dissolve inclusion bodies, the dissolved inclusion bodies still need to be refolded, and the process of obtaining an active target protein is time-consuming, complicated and costly.
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Embodiment 1
[0038] Embodiment 1: Construction of recombinant expression vector
[0039] In order to facilitate the subcloning of the target gene adh7, first connect it to the pMD19T-simple vector, transform E.coliJM109, select the positive clone for enzyme digestion verification, name the positive clone pMD19-T-adh7 and perform sequencing verification, and sequence The correct plasmid was digested with double enzymes to obtain the target gene (the amino acid sequence of the target gene is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2), and it was combined with pET-32a ( +) After ligation, transform E.coli BL21(DE3) and smear the bacterial solution on LB solid medium containing 100 μg / L ampicillin, culture at 37°C overnight, and select transformants for colony PCR verification. PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min and 20 s, 29 cycles; final extension ...
Embodiment 2
[0040] Example 2: Induced expression in Escherichia coli
[0041]Pick the recombinant expression plasmid (pET-32a-adh7) and inoculate it in 3 mL of LB liquid medium containing 100 μg / L ampicillin, culture overnight at 37°C with shaking at 200 r / min, and then expand the culture at an inoculum size of 1:100 , when the bacterial solution concentration reaches OD 600 When the value is 0.6, add IPTG to a final concentration of 0.1 mmol / L, and continue culturing at 30°C for 4h.
Embodiment 3
[0042] Example 3: Preparation of inclusion bodies
[0043] The bacterial solution was centrifuged to remove the supernatant, and the bacterial cells were resuspended with 50mmol / L Tris-HCl, 150mmol / LNaCl, 10mmol / L EDTA, pH8.0 disruption buffer and ultrasonically disrupted in an ice bath for 10min (ultrasound 3s, interval 6s, power 350W), centrifuge at 8000r / min for 15min at 4°C, and harvest the crude inclusion body precipitate. The precipitate was washed once with 50mmol / L Tris-HCl, 150mmol / LNaCl, 0.5% Triton X-100, 1mmol / L EDTA, pH 8.0 washing buffer, then washed once with water, centrifuged at 8000r / min for 15min at 4°C, The harvested precipitate is the purified δδ-ADH inclusion body.
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Abstract
The invention discloses an engineering bacterium for producing human stomach ethanol dehydrogenase (ADH) and a preparation method of the ADH of the engineering bacterium, and belongs to the technical field of biology. According to the engineering bacterium, a target gene is synthesized according to a gene sequence of the human stomach delta delta-ADH in a gene bank, and the target gene is connected with an expression vector pET-32a (+) and converted into E.coli BL21 (DE3). The preparation method comprises the steps that the E.coli BL21 (DE3) is induced by isopropylthio-beta-D-galactoside (IPTG) to express target protein existing in the form of inclusion bodies; thalluses are collected, crushed and centrifugalized, sediment is taken for obtaining the inclusion bodies, and a low-concentration urea solution or any of other buffer solutions is utilized for dissolving the inclusion bodies to obtain activated crude delta delta-ADH fluid; and the target protein is obtained by purifying HisTrap<TM>excel through affinity chromatographic columns, and the activity of the protein is 2.085 U/mg or over. By adoption of the delta delta-ADH produced by the engineering bacterium provided by the invention, high-concentration ethyl alcohol can be oxidized, and the delta delta-ADH exists in escherichia coli in the form of the inclusion bodies; the activated crude delta delta-ADH fluid can be obtained simply by dissolving the inclusion bodies through the dissolving solution, renaturation is not needed, the cost can be lowered, the operating steps can be simplified, and the efficiency can be improved.
Description
technical field [0001] The invention relates to an engineering bacterium producing human gastric alcohol dehydrogenase and a method for preparing the enzyme, belonging to the field of biotechnology. Background technique [0002] Alcohol dehydrogenase (alcohol dehydrogenases, ADH, EC1.1.1.1) is a kind of cytoplasmic dimer enzyme with zinc atom as prosthetic group and nicotinamide adenine dinucleotide (NAD) as coenzyme. key enzymes in metabolism. ADH can catalyze the conversion of ethanol into acetaldehyde, acetaldehyde dehydrogenase continues to catalyze the conversion of acetaldehyde into acetic acid, and acetic acid is finally converted into carbon dioxide and water to be excreted through the tricarboxylic acid cycle. Alcohol dehydrogenase has broad application prospects in disease diagnosis and medical analysis, bioelectrodes and ethanol biosensors, and food science. [0003] With the increase of alcohol consumption, people's demand for anti-alcoholic and liver-protectin...
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