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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: 2010-12-22
JIANGNAN UNIV
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  • 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

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  • 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 total DNA from Aspergillus niger WX-07:

[0056] The Aspergillus niger WX-07 strain was cultured in YPD liquid medium for 2 days, the mycelium was filtered, and then rinsed with sterile water three times, the water was absorbed by sterile filter paper, the mycelium was transferred into a mortar, and liquid nitrogen was added. Grind to obtain bacterial powder, put the bacterial powder into a 50mL centrifuge tube weighed in advance, weigh 1g bacterial powder, will be made of 8.1816g NaCl, 2g sodium dodecyl sulfonate, 10mL 500mmoL / L EDTA, 5mL 1mol / L Tris- The extraction buffer consisting of adding water to 100mL of HCl buffer solution is preheated in a 65℃ water bath, 3mL / g of bacterial powder is added to the extraction buffer solution, sealed with a sealing film, and placed at 65℃ for 1 hour to obtain the bacterial solution; 1.5mL bacterial solution was placed in a sterilized 1.5mL centrifuge tube, paralleled 2 tubes, centrifuged at...

Embodiment 2

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

[0059] Taking 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, with the following eight nucleotide sequences As a primer, the underline is the restriction site Not I:

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

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

[0062] Primer 3: 5’-ACCTATTGACCAAAACCTCTACGGCCA-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 the template, using P1, P2; P3, P4; P5, P6; P7, P8 as the upstream and downstream prime...

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 α-Factor signal peptide. The pPIC 9K plasmid and the amplified aglu gene are digested with Not I, and the digested product is tapped and recovered, 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, the transformants were selected for liquid culture on LB plates containing 100 mg / L ampicillin, and the plasmids were extracted to obtain enriched aglu / pPIC 9K plasmids ;

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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 cloning and expression of an alpha-glucosidase (AGLU enzyme for short) gene belongs to the field of enzyme genetic engineering and enzyme engineering. Specifically, it relates to a DNA sequence encoding Aspergillus niger WX-07AGLU enzyme and its expression. Background technique [0002] α-glucosidase (α-glucosidase, AGLU enzyme for short, EC 3.2.1.20), also known as α-D-glucoside hydrolase, can cleave α-1,4 glycosidic bonds from the non-reducing ends of oligosaccharides. Glucose is released and the free glucose groups are transferred to other sugar substrates in the form of α-1,6 glycosidic bonds to form non-fermentable functional oligosaccharides-isomalto-oligosaccharides (IMOs for short, mainly including Isomaltose, panose, isomaltotriose, etc.). Isomaltooligosaccharides are a kind of oligosaccharides containing at least one α-(1,6) glycosidic bond, mainly isomaltose, panose, isomaltose, and a small amount naturally exists in soy sauce and honey. It...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/81C12N1/19C12R1/84
Inventor 陈坚吴敬童星
Owner JIANGNAN UNIV
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