Polypeptide with pnppc hydrolase activity, its coding gene, preparation method and application
A coding and residue technology, applied in the biological field, can solve the problems of large interference of blank control, prone to false positives, and low vitality
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Embodiment 1
[0142] Example 1: Fermentation of wild pNPPC hydrolase capable of hydrolyzing pNPPC to produce pNP
[0143] A loop of activated Bacillus licheniformis CGMCC No.7878 grown on PDA or LB agar medium was inoculated into a seed shaker flask, and cultured at 28° C. and 180 rpm for 24 hours. The seed medium is LB liquid medium, which consists of 1% tryptone, 0.5% yeast extract, 1% sodium chloride, and pH 7.0.
[0144] After the liquid seeds are cultured, they are inoculated into 30 ml fermentation medium with an inoculation amount of 1%, and cultured at 28° C. and 180 rpm for 24 hours. The composition of the fermentation medium is: 0.5% sucrose, 0.5% tryptone, 1% yeast extract powder, 0.5% beef extract, 0.5% corn steep liquor, K 2 HPO 4 0.05%, MgSO 4 0.05%, CaCl 2 0.05%, ZnSO 4 ·7H 2 O0.05%, manganese sulfate 0.1%, pH 7.0.
[0145] After the cultivation, the culture solution was centrifuged at 1200 rpm for 10 minutes, and about 800 ml of centrifuged supernatant was collected, ...
Embodiment 2
[0146] Embodiment 2: Separation and purification of wild pNPPC hydrolase
[0147] About 800ml of the centrifuged supernatant in Example 1 was microfiltered with a 0.22 μm microfiltration membrane, then concentrated by ultrafiltration with a 10KDa ultrafiltration membrane, and replaced with a buffer solution of 20 mM Tris-HCl pH8.7. Collect ultrafiltration retentate about 100ml.
[0148] Use a 5ml DEAE column (Hitrap TM DEAE FF, GE Healthcare) carried out anion exchange chromatography separation on the ultrafiltrate of above-mentioned about 100ml volume. The elution buffer was 20 mM Tris-HCl pH 8.7 buffer containing 1 M NaCl. The conditions of ion exchange chromatography are as follows:
[0149] Buffer A: 20mM Tris-HCl, pH8.7;
[0150] Buffer B: 20mM Tris-HCl, 1M NaCl, pH8.7;
[0151] Flow rate: 2ml / min;
[0152] Elution method: Gradient elution 0-100% B, 120min;
[0153] Collection: 2ml / tube.
[0154] The collected eluate was tested for pNPPC activity, and the collect...
Embodiment 3
[0163] Example 3: Mass spectrometry identification of wild pNPPC hydrolase protein
[0164] Figure carefully cut out with a clean blade figure 1 The protein band in lane 3 pointed to by the oblique arrow was then identified by LC-MS / MS mass spectrometry. After data analysis, it was found that the protein with the highest score in this band was a protein in the Bacillus licheniformis genome that was speculated to be 2',3'-cyclic nucleotide 2'-phosphodiesterase, and its GENE BANK number was WP_016886260. The polynucleotide sequence of the protein is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:12. The predicted protein has a length of 1445 amino acids and a molecular weight of 158.2 KDa, which is much higher than the molecular size of the wild pNPPC hydrolase prepared in Example 2.
[0165] At the same time, the mass spectrometric identification results of LC-MS / MS also gave several very high-scoring peptides, and their amino acid sequence informati...
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