Amide hydrolase SaAH and coding gene and application thereof
A technology of amidohydrolase and gene, applied in the field of amidohydrolase SaAH and its coding gene and application, can solve the problems of few research reports on amidohydrolase, poor properties of amidohydrolase, poor thermal stability, etc.
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Embodiment 1
[0018] Example 1, Isolation, Identification and Preservation of Halospiral JH
[0019] 1. Separation
[0020] Take 50 μL of the alkali lake sample, add 450 μL of the alkali lake filtrate, and mix 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 samples, and spread them on solid medium such as YMAH, TSAH, LBH, 2216EH, etc., culture them in a constant temperature incubator at 35°C for 3-4 days, and observe the growth conditions. Colonies with different colors and shapes were picked, purified and cultivated by the three-section line method, and after repeating the line purification process 3-4 times, pure colonies were obtained.
[0021] 2. Identification
[0022] The purified strains were lined in three zones in LBH solid medium, cultured at 35°C for 2 days, and the morphology, size, prese...
Embodiment 2
[0033] Embodiment 2, the preparation of amidohydrolase (SaAH protein)
[0034] After a lot of sequence analysis, alignment and functional verification, a new protein was found from Halospira JH, which was named SaAH protein, as shown in sequence 1 of the sequence listing. The gene encoding SaAH protein in Halospira JH is named SaAH gene, and its coding frame is shown in sequence 2 of the sequence listing.
[0035] 1. Construction of recombinant plasmids
[0036] 1. Using the genomic DNA of Halospiral JH as a template, PCR amplification was performed with primer pairs composed of AH-F and AH-R, and the PCR amplification product was recovered.
[0037] AH-F: 5'-CGGATCCATGACCAGCCAAC-3';
[0038] AH-R: 5'-CCAAGCTTCTAAACCAACCGG-3'.
[0039] 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-SaAH.
[0040] pET-28a Vector (pET-28a Vector): Novagen, catalog number 69864-3.
[0041]After sequencin...
Embodiment 3
[0053] Embodiment 3, the enzymatic property of amidohydrolase (SaAH protein)
[0054] Tris-HCl buffer solution (50mM, pH 7.4): Weigh 6.06g Tris, dissolve it in ultrapure water, and adjust the pH to 7.5 with HCl.
[0055] Substrate solution (1.0 M butanamide): Weigh 8.7 g of butanamide, dissolve it in Tris-HCl buffer, and dilute to 100 mL.
[0056] 1. The effect of pH on the activity of amidohydrolase
[0057] 1. Optimal pH
[0058] Take the SaAH protein solution prepared in Example 2, Tris-HCl buffer solution (50mM, pH 7.4) to 2 times the volume, and use the diluted solution as the test solution.
[0059] Detection method: Add 100 μL of test solution and 900 μL of substrate solution, react at 40° C. for 30 min, then add 100 μL of TCA (15% trichloroacetic acid) solution to terminate the reaction. The reaction mixture was centrifuged at 10,000×g for 10 min, and 100 μL of the supernatant was taken, mixed with 100 μL of Nessler’s reagent and 800 μL of distilled water, and the O...
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