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Alpha-cyclodextrin glucosyltransferase gene and application thereof

A glucosyl, cyclodextrin technology, applied in the directions of transferase, application, genetic engineering, etc., can solve the problems of unfavorable industrial production of CGT, complex activation requirements, etc.

Inactive Publication Date: 2014-05-14
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the expression products of CGT in E.coli exist in the form of insoluble inclusion bodies, and the activation of such inclusion bodies requires a complicated process, which is not conducive to the industrial production of recombinant CGT

Method used

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  • Alpha-cyclodextrin glucosyltransferase gene and application thereof
  • Alpha-cyclodextrin glucosyltransferase gene and application thereof
  • Alpha-cyclodextrin glucosyltransferase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning of α-cyclodextrin glucosyltransferase gene

[0030] Extract the total genomic DNA of Paenibacillus macerans thalline with proteinase K-phenol extraction method, use this genomic DNA as template, in primer 1 (5'-CCATATGT CCATGG ATTCACCCGATACGAGC-3’) and Primer 2 (5’-CTCGAGA GAATTC GGATTTTGCCAGTCCAC-3’) for PCR amplification, the amount of each component in the PCR reaction system (total volume 50 μL): 10×Taq DNA Polymerase Buffer 5 μL (Mg 2+ ), 5 μL of 10 mM dNTP mixture (dATP, dCTP, dGTP, and dTTP each 2.5 mM), 1 μL each of cloning primer 1 and primer 2 at a concentration of 10 μM, 1 μL of genomic DNA, 0.25 μL of Taq DNA polymerase, and add nucleic acid-free water to 50 μL.

[0031] The PCR instrument of Biorad was used, and the PCR reaction conditions were: pre-denaturation at 94°C for 4min, followed by a temperature cycle of 94°C for 30s, 55°C for 30s, and 72°C for 2min, a total of 35 cycles, and finally an extension at 72°C for 10min, with a term...

Embodiment 2

[0033] Example 2: Acquisition of α-cyclodextrin glucosyltransferase gene

[0034] The total genomic DNA of Paenibacillus macerans JFB05-01 cells was extracted by proteinase K-phenol extraction method, and the genomic DNA was used as a template in primer 3:5'-CAATGTGGGTCCCACaATGGGCCAGCCG-3' and primer 4:5 '-CGGCTGGCCCATtGTGGGACCCACATTG-3') for PCR amplification. The amount of each component in the PCR reaction system (total volume 50 μL): 10×Taq DNA Polymerase Buffer 5 μL (Mg 2+ ), 5 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 1 μL of each primer 3 and primer 4 at a concentration of 10 μM, 1 μL of template DNA, 0.25 μL of Taq DNA polymerase, and add nucleic acid-free water to 50 μL.

[0035] The PCR instrument of Biorad was used, and the PCR reaction conditions were: pre-denaturation at 94°C for 4min, then entering into a temperature cycle of 94°C for 30s, 55°C for 30s, and 72°C for 6min, a total of 35 cycles, and finally extending at 72°C for 6min, w...

Embodiment 3

[0072] Example 3: Preparation and Transformation of Recombinant Escherichia coli BL21 / pET-20b(+) / cgt

[0073] According to Example 2, the vector pMD18-T simple / cgt2 that has eliminated the NcoI site in the cgt gene was double digested with NcoI and EcoRI, and the target fragment containing sticky ends was recovered, and then used the same endonuclease and alkaline phosphatase The digested pET-20b(+) plasmid fragment was ligated with T4 DNA ligase, and the ligated product was transformed into E.coliJM109, and the transformed recipient bacteria were spread on the LB solid plate containing 100 μg / mL ampicillin, and cultured overnight at 37°C. A single clone was picked and cultured overnight in LB medium containing 100 μg / mL ampicillin, the plasmid was extracted and identified by restriction electrophoresis and sequencing, and finally the expression vector pET-20b(+) / cgt containing the correct sequence was obtained, and its E.coli BL21(DE3) was transformed to obtain genetically en...

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Abstract

The invention discloses an alpha-cyclodextrin glucosyltransferase gene and the application thereof and relates to an alpha-cyclodextrin glucosyltransferase gene, an encoding enzyme, a carrier and an engineering bacterium which are extracted from paenibacillus macerans and the application thereof. The alpha-cyclodextrin glucosyltransferase gene can be connected with an expression vector to construct an expression recombinant plasmid (pET-20b(+) / cgt) containing the gene, the expression recombinant plasmid is then transferred into escherichia coli E.coil BL21, then recombinant escherichia coli is obtained, and the recombinant escherichia coli can secrete expression recombinant starch cyclodextrin glucosyltransferase, so that cyclodextrin is prepared through biotransformation reaction with glucose polymer such as starch, glycogen and oligosaccharide as substrate.

Description

technical field [0001] The invention relates to an alpha-cyclodextrin glucosyltransferase (CGTase) gene extracted from Paenibacillus macerans, an encoded enzyme, a carrier, an engineering bacterium and applications. Background technique [0002] Cyclodextrin glucosyltransferase (EC2.4.1.19, CGTase) is an important member of the glycosyl hydrolase α-amylase family (family 13), and it is a key extracellular expressed enzyme, which can convert starch into Or starch derivatives can generate cyclodextrin (CGT for short) through cyclization reaction. According to the number of D-glucofuranose units, the most commonly used cyclodextrins are α-, β-, and γ-cyclodextrin, and the corresponding cyclodextrin glucosyltransferases also have three types: α-, β -, γ-CGTases. [0003] Cyclodextrin glucosyltransferase is a multifunctional enzyme that catalyzes four different reactions: three transglycosylation reactions (disproportionation, cyclization, and coupling) and hydrolysis. Cyclode...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/63C12N1/21C12N15/70C12P19/18C12P19/04
Inventor 杨轶囡
Owner JILIN AGRICULTURAL UNIV
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