Carbonic anhydrase SaCA as well as encoding gene and application thereof
A carbonic anhydrase, gene technology, applied in the application, genetic engineering, carbon-oxygen lyase and other directions, to achieve the effect of major application prospects
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Embodiment 1
[0020] Example 1, Isolation, Identification and Preservation of Halospiral JH
[0021] 1. Separation
[0022] The sample was taken from an alkali lake in the Ordos area of Inner Mongolia. Take 50 μL of the alkali lake sample, add 450 μL of the alkali lake filtrate, and mix well by pipetting to obtain 10 -1 Concentration samples were diluted sequentially to obtain 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 concentrated samples. Take 20 μL, 40 μL of the original solution of the alkali lake water sample and the diluted sample, spread it on the LBH solid medium, culture it in a constant temperature incubator at 35°C for 3-4 days, and observe the growth.
[0023] 2. Identification
[0024] The purified strains were inoculated into LBH solid medium and cultured at a constant temperature of 30°C for 3 days, and the colony color, protrusion, edge regularity, size and transparency of the strains were recorded. The morphology, size and extracellular appendages of the strains were obser...
Embodiment 2
[0034] Embodiment 2, the preparation of carbonic anhydrase (SaCA protein)
[0035] After a lot of sequence analysis, alignment and functional verification, a new protein was found from Halospira JH, which was named SaCA protein, as shown in sequence 1 of the sequence list. The gene encoding the SaCA protein in Halospira JH was named SaCA gene, and its coding frame is shown in sequence 2 of the sequence listing.
[0036] 1. Construction of recombinant plasmids
[0037]1. Using the genomic DNA of Halospiral JH as a template, PCR amplification was carried out with a primer pair composed of CA-F and CA-R, and the PCR amplification product was recovered.
[0038] CA-F: 5'-CGGAATTCATGGACAACCTTAAC-3';
[0039] CA-R: 5'-CCCTCGAGTCAGCCGCC-3'.
[0040] 2. Take the PCR amplification product obtained in step 1 and connect it to the pET-28a vector to obtain the recombinant plasmid pET-28a-SaCA.
[0041] pET-28a Vector (pET-28a Vector): Novagen, catalog number 69864-3.
[0042] After s...
Embodiment 3
[0054] Embodiment 3, the enzymatic property of carbonic anhydrase (SaCA protein)
[0055] p-Nitrophenyl acetate (p-NPA) solution (freshly prepared before the experiment): Weigh 0.04g of p-nitrophenyl acetate and dissolve it in 0.01L of acetonitrile, and then dilute it 10 times.
[0056] PBS buffer (100mM, pH 6.0): Weigh 2.88g sodium dihydrogen phosphate, 0.48g potassium dihydrogen phosphate, 0.40g potassium chloride, 16.00g sodium chloride, dissolve in 800mL ultrapure water, adjust pH with HCl to 6.0, set the volume to 1L.
[0057] 1. The effect of pH on the activity of carbonic anhydrase
[0058] Take the SaCA protein solution prepared in Example 2, dilute it to 2 times the volume with buffer, and use the diluted solution as the test solution.
[0059] Detection method: Take a quartz cuvette, add 200 μL of test solution, 1 mL of p-nitrophenyl acetate (p-NPA) solution, and 1.8 mL of buffer solution, and measure the initial OD of the reaction 400nm The absorbance value at th...
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