N-carbamyl-L-cysteine (L-NCC) amidohydrolase, encoding gene and application of recombinant expressed protein of L-NCC amidohydrolase
An amidohydrolase and encoding gene technology, which can be applied in the directions of hydrolase, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of few reports of L-cysteine-related enzymes, and achieve good industrialization The effect of value, low fermentation cost and high catalytic efficiency
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Embodiment 1
[0025] Enzymatic activity determination method of L-NCC amidohydrolase
[0026] (1) Principle: The acid ninhydrin method is used. L-NCC amidohydrolase decomposes L-NCC to form L-cysteine. L-cysteine reacts with an acidic ninhydrin reagent under boiling conditions to form a red substance, which has a maximum absorption at 560 nm, and its color is similar to proportional to the amount of L-cysteine.
[0027] (2) Method: Accurately add 6% K to the solution of 1% L-NCC 2 HPO 4 To pH 7.5, a solution with a final substrate concentration of 0.75% was prepared, then 1 mL of the substrate solution was taken, 0.5 mL of enzyme solution was added, and the reaction was carried out in a water bath at 37°C for 10 min.
[0028] The content of L-cysteine in the reaction solution was determined by the acidic ninhydrin method, 0.2 mL of the reaction solution was taken, 0.2 mL of glacial acetic acid was added, and 0.2 mL of acidic ninhydrin reagent was added (we weighed 250 mg of ninhydrin...
Embodiment 2
[0032]Cloning and Primary Structure Characterization of L-NCC Amidohydrolase
[0033] The present inventor extracted genomic DNA from Pseudomonas sp.QR-101, then digested genomic DNA with Hind III, and recovered 2-9kb Hind III from 1.2% agarose gel with gel recovery kit Digest the fragments, connect the recovered fragments to the Pucl8 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.
[0034] The above-mentioned recombinant white single colony containing the insert fragment was picked into each well containing 50 μL LB medium (peptone: 10g / L, yeast powder: 5g / L, sodium chloride: 10g / L), and after overnight culture at 37°C, Add 100 μL of 0.75% L-NCC as a substrate, shake at 35° C. for 30 min, and add acidic ninhydrin reagent for color reaction.
[0035] The recombinants with high activity were re-screened through the microwell plate, and the plasmids ...
Embodiment 3
[0037] Construction of Escherichia coli Genetic Engineering Bacteria and Expression of Recombinant L-NCC Amidehydrolase
[0038] According to the sequencing results of Example 2, primers P1 and P2 were designed according to the two-terminal sequences of the protein-coding gene, wherein upstream P1: 5'-CCG GAA TTC ATG AGT GGA GTC AAC AGC ATG AA-3' contains an EcoR I restriction site ; Downstream primer P2: 5'-CCC AAG CTT TCA GCC GGT GCG GCT GTC CGC CA-3' contains a HindIII restriction site.
[0039] Using the genome of strain Pseudomonas sp.QR-101 as a template, DNA amplification was performed according to the following PCR procedure:
[0040] 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.
[0041] The PCR amplified product was double digested with EcoR I and Hind III, and connected to the vector pET21a(+) after the same digestion to construct the recombinant pET-21a(+) / atcC.
[0042] The o...
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