ATC racemase and coding gene thereof, and application of recombinant expression protein thereof
A racemase and protein technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of few reports of L-cysteine-related enzymes, achieve good industrial value, high catalytic efficiency, The effect of convenient separation and purification
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Embodiment 1
[0026] Cloning and Primary Structure Characterization of ATC Racemase
[0027] The present inventor extracted genomic DNA from Pseudomonas sp.QR-101, then digested genomic DNA with Hind III, and recovered 2-9kb Hind III enzymes from 1.2% agarose gel with a gel recovery kit Cut the fragments, connect the recovered enzyme-digested fragments to the pUC18 vector that has been treated with the same restriction enzymes, transfer them into E.coli JM109, and perform blue-white primary screening on a plate coated with X-gal and IPTG.
[0028] Recombinant white single colonies containing insert fragments were picked into each well containing 50 μL of LB medium, and after overnight culture at 37°C, 100 μL of 0.6% D was added, L-ATC was used as a substrate, and L-ATC hydrolase (the For the gene sequence, see SEQ ID No.3 in the sequence table) and 0.5 U each of L-NCC amidohydrolase (see the sequence table, SEQ ID No.4 for the gene sequence), shake at 35°C for 30 min, and add 150 μL of acid...
Embodiment 2
[0031] Construction of Genetic Engineering Bacteria of Escherichia coli and Expression of Recombinant ATC Racemase
[0032] According to the sequencing results, primers P1 and P2 were designed according to the two-end sequences of the protein-coding gene, wherein the upstream P1: 5'-CCGGAA TTC ATG AAG CAT CAT CAG ACG GGC AT-3' contains the EcoR I restriction site; the downstream primer P2 : 5'-CCC AAGCTT CTA GCC CAA CAG TTT TCC CAGGC-3' contains a HindIII restriction site.
[0033] Using the genome of strain Pseudomonas sp.QR-101 as a template, DNA amplification was performed according to the following PCR procedure:
[0034] Denaturation at 94°C for 1 min, renaturation at 66°C for 1 min, extension at 72°C for 1 min, and 30 cycles of amplification reaction.
[0035] The PCR amplified product was double digested with EcoR I and Hind III, and connected to the vector pET-21a(+) after the same digestion to construct the recombinant pET21-a(+) / atcA.
[0036] The obtained recombin...
Embodiment 3
[0038] Example 3 Verification of Recombinant ATC Racemase Activity
[0039] Take the enzyme source cell suspension in Example 2, adjust the final concentration to 20g / L, and add 3mL of 0.5% substrate DL-ATC and 1.5mL of mixed enzyme solution in sequence to a 5mL reaction tube, wherein, in each group The composition of the mixed enzyme solution is shown in Table 1.
[0040] Table 1 Composition of Mixed Enzyme Solution
[0041]
[0042] In a water bath at 35°C for 2 hours, each reaction solution was freeze-dried, then dissolved in 300 μL of isopropanol, and the contents of D-ATC and L-ATC were determined by high-performance liquid chromatography.
[0043] High performance liquid chromatography adopts CHIRALPAK IC column (0.46cm I.D.×25cm L) (Daicel chiral technologies CO., LTD., Shanghai, CHINA), the liquid phase conditions are: ultraviolet detector SPD-10A, detection wavelength is 220nm, flow The phase was n-hexane-isopropanol (85:15), containing 0.2% trifluoroacetic acid ...
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