Aspartate aminotransferase and preparation method thereof
An aspartate aminotransferase and amino acid technology, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, transferase, etc., can solve the problem of high temperature requirements, inability to exert activity, aspartate aminotransferase tolerance Problems such as poor ability, to achieve the effect of good tolerance, simple method and easy operation
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Embodiment 1
[0023] Acquisition of aspartate aminotransferase gene
[0024] 1) Obtain the strain containing the target gene
[0025] The present invention utilizes strains of the genus Cryobacterium, which are screened from the soil of Changbai Mountain. The specific screening process is like the Chinese invention patent with the patent number of 201710034491.5. The strain is named: Cryobacteriumbaishanse 02, and it is preserved in the China Type Culture Collection Center , deposit number CCTCC NO: M2016604. The date of deposit is October 31, 2016, and the deposit address is Wuhan University, Wuhan, China.
[0026] 2) Genome extraction
[0027] The genome of Cryobacterium baishanse 02 strain was extracted using a DNA extraction kit (TAKARA Dalian) as a template, and the extracted genome samples were stored at -20°C until use.
[0028] 3) PCR amplification of the target gene
[0029] Design primers: the forward primer is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO...
Embodiment 2
[0037] Construction of expression vector of aspartate aminotransferase
[0038] The PCR amplified product obtained in Example 1 was carried out to gel recovery, and the PCR amplified product was carried out double digestion reaction with restriction endonuclease EcoRI and NdeI; Carrier pET21a (+ ) for double enzyme digestion reaction, and then catalyzed by high-efficiency DNA ligase High Ligation (TOYOBO) to connect the pET21a(+) and PCR amplification product after the double enzyme digestion reaction; construct the expression vector pET21a-AT of aspartate aminotransferase , the expression vector pET21a-AT was constructed as image 3 shown.
Embodiment 3
[0040] Expression and purification of aspartate aminotransferase protein
[0041] The expression vector pET21a-AT obtained in Example 2 was transformed into the competent thallus of Escherichia coli BL21, and the preparation of the competent state of Escherichia coli BL21 was carried out using CaCl 2 method, CaCl 2 Preparation of the competent state of Escherichia coli BL21 by this method is a routine experimental method in the laboratory, and will not be repeated here. The obtained expression vector pET21a-AT was transformed into Escherichia coli BL21 to obtain the recombinant strain Ecoli BL21-pET21a-AT. Expand Ecoli BL21-pET21a-AT to OD 600 After = 0.6, add 1mMIPTG, induce culture and expression of aspartate aminotransferase protein at 28°C, collect the bacteria after the induction culture, and sequentially crush the bacteria and purify the Ni-NTA affinity chromatography column to obtain purified aspartate Acid transaminase proteins, such as figure 2 As shown, the prot...
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