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Polypeptide having pNPPC hydrolase activity and coding gene, preparation method and application thereof

A technology for hydrolyzing activity and residues, which is applied in the field of polypeptides with pNPPC hydrolase activity, and can solve the problems of large interference, low activity, and easy false positives of the blank control

Active Publication Date: 2017-05-10
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These lead to the disadvantages of large interference of the blank control and prone to false positives when SEAP is used as a reporter gene
[0023] At present, there are genes with pNPPC activity reported in literature and patents, except that phospholipase C or lysophospholipase C can act on pNPPC, other reports can hydrolyze the compound to produce the chromogenic product p-nitrophenol The enzymes are very few and can The activity of hydrolyzed pNPPC is very low, and there will be no widespread interference such as SEAP

Method used

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  • Polypeptide having pNPPC hydrolase activity and coding gene, preparation method and application thereof
  • Polypeptide having pNPPC hydrolase activity and coding gene, preparation method and application thereof
  • Polypeptide having pNPPC hydrolase activity and coding gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Example 1: Fermentation of wild pNPPC hydrolase capable of hydrolyzing pNPPC to produce pNP

[0143] A ring 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 20mM Tris-HCl pH8.7 buffer. 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 collected solution wit...

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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Abstract

The invention relates to polypeptide having pNPPC hydrolase activity and a coding gene, a preparation method and application thereof. The separated polypeptide is selected from: (1) amino acid sequence shown as SEQ ID NO:4, 6, 8 or 10; (2), SEQ ID NO:21 or segment obtained by truncating at least 15 amino acid residues from a terminal N thereof and / or at least 29 residues from a terminal C; (3), polypeptide derived from (1) or (2) by substituting, deleting or adding one or multiple amino acids in the amino acid sequence mentioned in (1) or (2) and maintaining pNPPC hydrolysis activity that SEQ ID NO: 4, 6, 8 or 10 or SEQ ID NO:21 or segment thereof; (4), polypeptide containing the amino acid sequence mentioned in (1), (2) or (3). The polypeptide like MPPX has performances like high substrate specifity and is quite suitable for application of enzyme in enzyme-linked immunosorbent assay.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a polypeptide having pNPPC hydrolase activity, its coding gene, its preparation method and its application. Background technique [0002] p-nitrophenol phosphorylcholine (p-nitrophenyl phosphorylcholine, pNPPC) is a kind of artificially synthesized relatively stable compound, and its structure is as follows: [0003] [0004] The structure contains p-nitrophenol (pNP) structure, which is a yellow substance with strong absorption at 405-410nm. Numerous synthetic compounds have been developed with p-nitrophenol as the backbone to detect the activity of various hydrolases, such as p-nitrophenol phosphate is used to detect phosphatase activity, p-nitrophenol palmitate is used to detect lipase or Esterase activity, etc. High-purity pNPPC compounds are usually colorless, but their hydrolysis yields pNPP which is bright yellow. Because it has a phosphorylcholine structure, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/20C12N15/62C12N15/55G01N33/573C11B3/00C12N1/21C12N1/19C12N1/15C12R1/19C12R1/125C12R1/84C12R1/685C12R1/39
CPCC07K14/00C07K2319/00C11B3/003C12N9/20C12Y301/04003G01N33/573
Inventor 周美凤徐正军许骏牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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