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Process for producing gene engineering immobilized enzyme N-glycoamidase

A technology of sugar amidase and immobilized enzyme, which is applied to other methods of inserting foreign genetic materials, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems that limit the widespread use of N-glycoamidase and large-scale preparation, and achieve The production method is simple and easy, the stability is high, and the effect of reducing production costs

Inactive Publication Date: 2006-03-01
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This will greatly limit the widespread utilization of N-glycan amidase and the large-scale preparation of N-glycan chains

Method used

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  • Process for producing gene engineering immobilized enzyme N-glycoamidase
  • Process for producing gene engineering immobilized enzyme N-glycoamidase
  • Process for producing gene engineering immobilized enzyme N-glycoamidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Construction of recombinant expression vector:

[0042] Using 5-ATATGAATTCGCTCCGCCAGATAATACCGT-3; 5-CACGTCTAGAGTTTGTAACTACCGGAG-3 as primers to clone the N-glycoamidase gene from Pseudomonas meningitidis ATCC 33958 (purchased from ATCC), PCR amplification conditions: 50 μl reaction system, 10×PCR buffer 5 μl , MgCl 2 (25mmol / L) 3μl, d NTP (25mmol / L) 1μl, primers 1 and 2 (20pmol / L) 1μl each, genomic DNA 1μl, Taq DNA polymerase 0.5U, pre-denaturation at 94°C for 5min, according to the following parameters: Denaturation at 94°C for 45 s, annealing at 56°C for 45 s, extension at 72°C for 2 min, 28 cycles, and the last cycle of extension at 72°C for 10 min. The XbaI and NotI sites inserted into the pBluescriptSK(+) vector after digestion with XbaI and NotI were named vector-PNG. Then use 5-TGCATCTAGACGCCAAAAGCTCTTTTATCT-3; 5-TCAGCGGCCGCTTTGATTATGTTCTTTCT-3 as primers to clone the Saccharomyces cerevisiae coagulation factor C-terminal anchor site gene, PCR amplification...

Embodiment 2

[0054] (1) Construction of recombinant expression vector:

[0055] Using 5-ATATGAATTCGCTCCGCCAGATAATACCGT-3; 5-CACGTCTAGAGTTTGTAACTACCGGAG-3 as primers to clone the N-glycoamidase gene from Pseudomonas meningitidis ATCC 33958 (purchased from ATCC), PCR amplification conditions: 50 μl reaction system, 10×PCR buffer 5 μl , MgCl 2 (25mmol / L) 3μl, d NTP (25mmol / L) 1μl, primers 1 and 2 (20pmol / L) 1μl each, genomic DNA 1μl, Taq DNA polymerase 0.5U, pre-denaturation at 94°C for 5min, according to the following parameters: Denaturation at 94°C for 45 s, annealing at 56°C for 45 s, extension at 72°C for 2 min, 28 cycles, and the last cycle of extension at 72°C for 10 min. The XbaI and NotI sites inserted into the pBluescriptSK(-) vector after digestion with XbaI and NotI were named vector-PNG. Then use 5-TGCATCTAGACGCCAAAAGCTCTTTTATCT-3; 5-TCAGCGGCCGCTTTGATTATGTTCTTTCT-3 as primers to clone the Saccharomyces cerevisiae coagulation factor C-terminal anchor site gene, PCR amplification...

Embodiment 3

[0067] (1) Construction of recombinant expression vector:

[0068] Using 5-ATATGAATTCGCTCCGCCAGATAATACCGT-3; 5-CACGTCTAGAGTTTGTAACTACCGGAG-3 as primers to clone the N-glycoamidase gene from Pseudomonas meningitidis ATCC 33958 (purchased from ATCC), PCR amplification conditions: 50 μl reaction system, 10×PCR buffer 5 μl , MgCl2 (25mmol / L) 3μl, d NTP (25mmol / L) 1μl, primers 1 and 2 (20pmol / L) 1μl each, genomic DNA 1μl, Taq DNA polymerase 0.5U, pre-denaturation at 94°C for 5min, according to the following parameters: Denaturation at 94°C for 45 s, annealing at 56°C for 45 s, extension at 72°C for 2 min, 28 cycles, and the last cycle of extension at 72°C for 10 min. The XbaI and NotI inserted into the pET22b vector after digestion with XbaI and NotI were named vector PNG. Then use 5-TGCATCTAGACGCCAAAAGCTCTTTTATCT-3; 5-TCAGCGGCCGCTTTGATTATGTTCTTTCT-3 as primers to clone the Saccharomyces cerevisiae coagulation factor C-terminal anchor site gene, PCR amplification conditions are th...

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Abstract

This invention discloses a kind of method of producing genetic engineering immobilized enzyme N-glycoamidase (PNGase). The N-glycoamidase is fused with yeast agglutination factor by the genetic engineering technique, Anchor it to the surface of methanol nutrition type yeast cell and get a kind of immobilized N-glycoamidase using cells as carrier. In this method, the activity of the enzyme during immobilizing process will not decrease. The produced immobilized enzymes have high stability and can use repeatedly. The method also leaves out purification and reduces the cost. The N-glycoamidase can be widely used in extracting oligosaccharide chain which has biological function from farm product remains (egg yolk powder, milk)

Description

technical field [0001] The present invention relates to a method for preparing immobilized enzymes, in particular to a method for immobilizing N-glycoamidase (including PNGase) capable of completely removing oligosaccharide chains on glycoproteins on the surface of methanolotrophic yeast cells by using genetic engineering technology A method to obtain a large number of whole-cell catalysts with N-glycoamidase anchored on the surface. Background technique [0002] Carbohydrates are the most abundant organic compounds in nature. Various glycoconjugates in cells such as glycoproteins, glycolipids and glycosamines play an extremely important role in many biological processes such as cell-cell recognition, signal transduction, immune response, cell differentiation and apoptosis. Role. [0003] N-glycosamidase (Peptide-N-(N-acetyl-β-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) can specifically break the amide bond between the sugar chain and the protein to generate a c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/81C12N1/19C12N15/87
Inventor 祁庆生苏移山王鹏
Owner SHANDONG UNIV
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