Clone and expression of alpha-glucosidase gene

A glucosidase and gene technology, which is applied to the DNA sequence encoding Aspergillus niger WX-07AGLU enzyme and the field of expression thereof, can solve problems such as the recovery and use of unfavorable recombinant α-glucosidase

Inactive Publication Date: 2009-05-20
JIANGNAN UNIV
View PDF0 Cites 17 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to domestic reports, recombinant α-glucosidase is generally located in Escherichia coli to form inclusion bodies or exist in the periplasm, which is not conducive to the recovery and use of recombinant α-glucosidase

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Clone and expression of alpha-glucosidase gene
  • Clone and expression of alpha-glucosidase gene
  • Clone and expression of alpha-glucosidase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] This example illustrates the extraction of Aspergillus niger (Aspergillus niger) WX-07 total DNA:

[0056] Aspergillus niger WX-07 strain was cultured in YPD liquid medium for 2 days, the mycelium was filtered, washed three times with sterile water, blotted dry with sterile filter paper, the mycelium was transferred into a mortar, and liquid nitrogen was added. Grind the bacterial powder, put the bacterial powder into a pre-weighed 50mL centrifuge tube, weigh 1g of the bacterial powder, and mix it with 8.1816g NaCl, 2g sodium dodecylsulfonate, 10mL 500mmoL / L EDTA, 5mL 1mol / L Tris- The extraction buffer composed of HCl buffer and water to make up to 100mL was preheated in a water bath at 65°C, and 3 mL / g of bacterial powder was added to the extraction buffer, sealed with a parafilm, and placed at 65°C for 1 hour to obtain the bacterial liquid; Put 1.5mL bacterial liquid in a sterilized 1.5mL centrifuge tube, parallel 2 tubes, centrifuge at 10000r / min for 5min, suck 0.75m...

Embodiment 2

[0058] This example illustrates the cloning procedure for the gene encoding the AGLU enzyme.

[0059] Using the total DNA of Aspergillus niger WX-07 as a template, since the aglu gene has four exons and three introns, the aglu gene was amplified by PCR and over-lap PCR, and the following eight nucleotide sequences As a primer, the restriction site Not I is underlined:

[0060] Primer 1: 5'-ATTAAT GCGGCCGC GTCCACCACTGCCCCTTCG-3'

[0061] Primer 2: 5'-CCGTAGAGGTTTTGGTCAATAGGTGTTC-3'

[0062] Primer 3: 5'-ACCTATTGACCAAAAACCTCTACGGCCA-3'

[0063] Primer 4: 5'-TCAATATCGGTCCAGATATATTCCAAC-3'

[0064] Primer 5: 5'-GAATATATCTGGACCGATATTGACTACATGCACG-3'

[0065] Primer 6: 5'-CGTAGCGTAGGCATCAGAGGCATTTTC-3'

[0066] Primer 7: 5'-GCCTCTGATGCCTACGCTACGTATGACA-3'

[0067] Primer 8: 5'-AGCACTA GCGGCCGC CCATTCCAATACCCAGTTTTCC-3'

[0068] The first round of PCR:

[0069] Using the total DNA of Aspergillus niger WX-07 as a template, using P1, P2; P3, P4; P5, P6; P7, P8 as upstream and...

Embodiment 3

[0086] This example illustrates the construction procedure of the aglu gene on the expression vector.

[0087] The plasmid used to construct the expression vector is pPIC 9K, with the α-Factor signal peptide. The pPIC 9K plasmid and the amplified aglu gene were digested with Not I, and the digested products were recovered by tapping and then ligated with T4 ligase at 16°C. Overnight, the ligation product was transformed into E.coli JM109 competent cells, cultured overnight at 37°C, and the transformants were selected for liquid culture on LB plates containing 100mg / L ampicillin, and then the plasmid was extracted to obtain the enriched aglu / pPIC 9K plasmid ;

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses clone and expression of an Alpha-glucosidase (abbreviated as AGLU enzyme) gene and belongs to the fields of enzyme gene engineering and enzyme engineering. The invention obtains the aglu gene with SEQ ID NO of 1 through an Aspergillus niger (A.niger) WX-07 total DNA, and aglu cDNA uses an expression vector of plasmid pPIC9K and an expression parasitifer of pichia stipitis (P.pastoris), thus realizing extracellular dissolubility expression of the aglu gene; and the aglu cDNA totally has 2880 nucleic acid and encoding of 960 amino acid, constructs eukaryotic expression plasmid and transforms Pichia pastoris KM71 for AGLU enzyme expression. The recombinant enzyme has transnucleosidation activity, can generate isomalto oligosaccharide through maltose transnucleosidation, is applicable to the requirements of industrial application including food, feeding stuffs and medicaments and the like, and can be applied into the industrial production of isomalto oligosaccharide.

Description

technical field [0001] The invention relates to the cloning and expression of an α-glucosidase (AGLU enzyme for short) gene, and the invention belongs to the fields of enzyme genetic engineering and enzyme engineering. Specifically, it relates to a DNA sequence encoding Aspergillus niger (Aspergillus niger) WX-07AGLU enzyme and its expression. Background technique [0002] α-glucosidase (α-glucosidase, referred to as AGLU enzyme, EC 3.2.1.20), also known as α-D-glucoside hydrolase, it can cut α-1,4 glycosidic bonds from the non-reducing end of oligosaccharides, Glucose is released, and free glucose groups are transferred to other sugar substrates in the form of α-1,6 glycosidic bonds to form non-fermentable functional oligosaccharides—isomaltooligosaccharides (referred to as IMOs, mainly including isomaltose, panose, isomaltotriose, etc.). Isomaltooligosaccharides are a class of oligosaccharides containing at least one α-(1,6) glycosidic bond, mainly isomaltose, panose, an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/81C12N1/19C12R1/84
Inventor 陈坚吴敬童星
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap