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Clone, expression of beta-glucosidase gene, and preparation for gentian oligose

A technology of glucosidase and gentian oligosaccharide, which is applied in the field of β-glucosidase preparation and β-glucosidase conversion of glucose to prepare gentio oligosaccharide, yeast genetic engineering bacteria

Inactive Publication Date: 2009-07-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the production of gentian oligosaccharides by using the transglycosidase activity of recombinant β-glucosidase

Method used

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  • Clone, expression of beta-glucosidase gene, and preparation for gentian oligose
  • Clone, expression of beta-glucosidase gene, and preparation for gentian oligose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: This example illustrates the extraction of total RNA from Aspergillus niger WX-07.

[0055] Aspergillus niger WX-07 strain was cultured in PD liquid medium for 2 days, the mycelium was filtered, the bacteria were washed once with PBS buffer, 1 mL TRI REAGENT (purchased from Sigma Company) was added, and repeatedly blown with a pipette gun to lyse Cells; centrifuge at 12,000×g, 4°C for 10 min to remove insoluble matter; transfer the supernatant to a clean centrifuge tube, place the sample at room temperature for 5 min, add 0.2 mL of chloroform, shake vigorously for 15 s, and place at room temperature for 10 min, Centrifuge at 12,000×g at 4°C for 15 minutes, take the upper colorless aqueous phase into a clean centrifuge tube, add 500 μL of isopropanol, mix well, place at room temperature for 10 minutes, centrifuge at 12,000×g at 4°C for 10 minutes, discard the supernatant, Add 1mL by DEPC-H 2 O (purchased from Yubao Bioengineering Co., Ltd.) prepared 75% ethan...

Embodiment 2

[0056] Example 2: This example illustrates the cloning of the gene encoding β-glucosidase.

[0057] Using the total RNA of Aspergillus niger WX-07 as a template, the first strand of cDNA was synthesized by reverse transcription (the following PrimeScript RTase, 5×PrimeScript Buffer, RNase Inhibitor, dNTP Mixture, OligodT Primer, Random 6mers, RNase free H 2 O are from the kit "PrimeScript TM 1stStrand cDNA Synthesis Kit", purchased from Bao Biological Engineering Co., Ltd.),

[0058] ① Prepare the following template RNA / Primer mixture in a microcentrifuge tube:

[0059] 50 μM Oligo dT Primer or Random 6mers 1 μL,

[0060] 1 μL of 10mM dNTP Mixture,

[0061] Total RNA 1 μg,

[0062] and RNase free H 2 O was added to the total system 10 μL;

[0063] ②Quickly cool on ice after holding at 65°C for 5 minutes;

[0064] ③ Prepare the following cDNA synthesis reaction solution in the above microcentrifuge tube:

[0065] 10 μL of the RNA / Primer mixture prepared in step ①,

[0...

Embodiment 3

[0077] Example 3: This example illustrates the construction of the bg gene on the expression vector.

[0078] The plasmid used to construct the expression vector is pPIC 9K with a-Factor signal peptide. The pPIC 9K plasmid and the amplified bgl gene were digested with NotI and EcoRI. After ligation 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 in 100 mg / L ampicillin LB, and then the plasmid was extracted to obtain the enriched bgl / pPIC 9K plasmid.

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Abstract

A method for cloning and expressing a beta-glucosaccharase gene and application in preparing gentian oligose belongs to the field of enzyme gene engineering and enzyme engineering. In the invention, cDNA of beta-glucosaccharase gene (bgl)SEQ ID NO: 1 synthesized by the reverse transcription of the gross RNA of Aspergillus niger WX-07 uses plasmid pPIC9K as an expression vector and P.pastoris as an expression host to realize the solubility expression of the bgl gene outside a cell; the full length of the cDNA of the bgl is 2523 ribonucleotide and 841 amino acids are coded; the constructed bgl / pPIC9K transforms P.pastoris KM71 so that P.pastoris KM71 can express BGL enzyme. The BGL enzyme has the activity of transglycosylation and can generate dextrose into the gentian oligose by transglycosylation. The technique of preparing the gentian oligose by enzyme process is optimized; and resin cation is used to carry out separation and refining to achieve a better effect. The prepared gentian oligose product, as functional food ingredient, has the physiological functions of low energy, low decayed tooth and stimulating the gastrointestinal mucosa. The invention provides a new approach with commercial values for preparing the gentian oligose.

Description

technical field [0001] The invention relates to a DNA sequence encoding Aspergillus niger (Aspergillus niger) WX-07 BGL enzyme and its expression, constructing yeast genetically engineered bacteria expressing β-glucosidase efficiently, and β-glucosidase preparation and β-glucosidase A method for enzymatically converting glucose to prepare gentiooligosaccharides. The invention belongs to the fields of enzyme genetic engineering and enzyme engineering. Background technique [0002] Gentiooligosaccharides are a new type of functional oligosaccharides bound by glucose with β-1,6 glycosidic bonds, including gentiobiose, a small amount of trisaccharides and tetrasaccharides. As a functional food ingredient, in addition to low-calorie, low-cavity, and intestinal-regulating physiological functions, gentian oligosaccharides can better promote bifidobacteria and Propagation of lactic acid bacteria; and heat-resistant, acid-resistant, can be used in some other health food where oligo...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12P19/34C12N15/81C12P19/00C12R1/685C12R1/84
Inventor 吴敬陈坚刘玲玲
Owner JIANGNAN UNIV
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