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Disaccharide degrading enzyme gene and application thereof

A technology of gene and trehalase, which is applied in the field of biological enzymes, can solve the problems of no related reports on the application, and achieve the effects of increasing alcohol production, reducing residues, and reducing processing costs

Active Publication Date: 2019-01-11
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Most of the trehalase genes that have been reported so far are derived from insects, plants, animals, etc. The trehalase gene derived from Trichoderma reesei and its expression in Pichia pastoris, and the trehalase produced by the expression system of Aspergillus niger in alcohol There is no relevant report on the application in fermentation industry, etc.

Method used

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  • Disaccharide degrading enzyme gene and application thereof
  • Disaccharide degrading enzyme gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of embodiment 1 trehalase gene TH3155

[0039] 1. Experimental materials and reagents:

[0040] 1. Strains and vectors

[0041] Escherichia coli Topl0, Pichia pastoris X33, Aspergillus niger 319 strain, plasmid pPICzαA, plasmid PBC-gla3, and Zeocin antibiotics were all purchased from Invitrogen.

[0042] 2. Gene

[0043] The strain Trichoderma reesei was purchased from the Guangdong Microorganism Culture Collection Center, and a trehalase gene TH3155 was amplified from the genome of the strain.

[0044] 3. Enzymes and reagents

[0045] Ultra-fidelity 2×Master Mix PCR polymerase was purchased from NEB Company; universal DNA Purification Kit, TIANprep Mini Plasmid Kit, and restriction endonuclease were purchased from Shanghai Sangon Company.

[0046] 4. Medium

[0047] The culture medium for Escherichia coli was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0). LB+Amp medium is LB medium with ampicillin at a final concentration of 100ug / mL added....

Embodiment 2

[0062] Example 2 Pichia pastoris expression vector and host containing trehalase gene TH3155

[0063] Expression of trehalase gene TH3155 in Pichia pastoris: Digest the pPICzαA-TH3155 recombinant expression vector into linearized DNA fragments with Pme I restriction enzyme, purify the DNA fragments, and transform the linear pPICzαA-TH3155 DNA fragments by electroporation In the expression host Pichia pastoris X33 competent cells, spread on the YPD+Zeocin plate, culture upside down at 30°C for 3-4 days, pick a single colony into 2ml liquid YPD medium, culture at 30°C, 200rpm for 24 hours, 1% methanol was used to induce culture for 24 hours, the fermentation broth was collected, centrifuged, and the supernatant was taken as the crude enzyme solution.

Embodiment 3

[0064] Example 3 Aspergillus niger expression vector and host containing trehalase gene TH3155

[0065] Expression of trehalase gene TH3155 in Aspergillus niger: construct trehalase gene TH3155 in pPICzαA-TH3155 into Aspergillus niger expression vector PBC-gla3 with BglII restriction endonuclease and PmeI restriction endonuclease, transform Aspergillus niger 319 strain protoplasts. Coated on TZ + In the hygromycin plate, culture upside down at 32°C for 5-6 days, pick a single colony into 20mL liquid maltodextrin medium, culture at 32°C, 200rpm, and screen to obtain recombinant Aspergillus niger expressing trehalase.

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Abstract

The invention discloses a disaccharide trehalase gene and application thereof. The nucleotide sequence of the disaccharide trehalase gene TH3155 is represented by SEQ ID NO:1, the gene can be used forproducing trehalase TH3155 and contains 1056 amino acids, and the amino acid sequence of the gene is represented by SEQ ID NO:2. Trehalase is capable of degrading trehalose into glucose. By adding acertain amount of trehalase in the fermentation production process of alcohol, seaweed produced in a metabolism process can be hydrolyzed into glucose for use by alcohol yeast, so that the yield of alcohol is increased, meanwhile, the residue of trehalose in alcohol fermentation wastewater is reduced, and the treatment cost of the alcohol fermentation wastewater is lowered.

Description

technical field [0001] The invention belongs to the field of biological enzymes, in particular to a disaccharide degrading enzyme gene and its application. Background technique [0002] Trehalase (Trehalase) (EC3.2.1.28, α, α-trehalose glucohydrolase) was first discovered by Bourquelot in Aspergillus niger in 1893, and later research found that trehalase widely exists in microorganisms, insects, plants and In invertebrates, it plays an important role in glucose transport and energy storage. Trehalase is trehalose hydrolase, trehalase can decompose trehalose into two molecules of glucose. At present, researchers have isolated trehalase from many organisms. [0003] The catalytic residues of trehalase are distributed between the trehalase marker domain 1 and the C-terminus, and are not connected together as a whole but scattered in the sequence. In different trehalases, the distribution of these conserved domains is different. [0004] The mechanism of action of glycoside ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12P7/06
CPCC12N9/2402C12P7/06C12Y302/01028Y02E50/10
Inventor 李阳源黄江江民华陈丽芝王水生刘金山何小梅高芝
Owner GUANGDONG VTR BIO TECH
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