Alpha-galactosidase gene, its coding protein, production and use

A galactosidase and coding technology, applied in the field of genes and their encoded proteins, can solve problems such as unsatisfactory decomposition ability

Active Publication Date: 2007-11-21
WUHAN SUNHY BIOLOGICAL
View PDF0 Cites 19 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing α-galactosidase is still unsatisfactory for the decomposing ability of α-galactoside oligosaccharides in soybean, so a new low-lying e

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
  • Alpha-galactosidase gene, its coding protein, production and use
  • Alpha-galactosidase gene, its coding protein, production and use
  • Alpha-galactosidase gene, its coding protein, production and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The separation and purification of embodiment 1 Penicillium α-galactosidase

[0063] Penicillium (Penicillium) F63 was inoculated in the enzyme-producing medium to induce the expression of α-galactosidase. After 7 days of induction, centrifuge the thalli to obtain the culture supernatant, and the culture supernatant was washed with 20-95% (NH 4 ) 2 SO 4 For precipitation, the pellet was resuspended in 20 mM Tris-HCl, pH 8.0, and dialyzed overnight.

[0064] Dialyzed samples were concentrated using ultrafiltration tubes. A 2 mL sample was loaded onto a HiTrap Q Sepharose XL anion column (Amersham Pharmacia Biotech) pre-equilibrated with 20 mM Tris-HCl, pH 8.0. The protein bound to the column was eluted with a NaCl salt gradient, and the protein containing the α-galactosidase component was eluted under a 0.3M salt gradient.

[0065] Sephacryl S-200 HR molecular sieves (Amersham Pharmacia Biotech) were applied to 500uL concentrated samples containing α-galactosidase c...

Embodiment 2

[0069] Example 2 Sequencing of α-galactosidase protein.

[0070] The α-galactosidase purified by molecular sieves in Example 1 was concentrated, subjected to SDS-PAGE electrophoresis, and a single band of electrophoretic pure α-galactosidase was subjected to in-gel digestion. The peptides were sequenced with a mass spectrometer to obtain 6 internal peptides. The amino acid sequences of these 6 internal peptides were LFVLDDGWFK, QSEGYTVSEFQYK, VNPLVLTGDMWR, DNAGLGDWLPNP, LEGLDENALYK and PEVQDFLLK, respectively. After sequence comparison, it was found that 4 of the endopeptides had high homology with known α-galactosidases, and no homologous α-galactosidase sequences were found for the other two endopeptides . Among them, the homology of LFVLDDGWFK and the corresponding peptides of Aspergillus niger, Aspergillus nidulans, Trichoderma, Absidia α-galactosidase is 100%, 88%, 100% and 88%; The homology of the corresponding peptides of Aspergillus and Trichoderma α-galactosidase is...

Embodiment 3

[0071] Example 3 Cloning of Penicillium α-galactosidase coding gene Agl1

[0072] Genomic DNA extraction of Penicillium sp (Penicillium sp) F63: take the Penicillium sp F63 bacterial liquid cultured at 30°C for 7 days and centrifuge at 60000rpm for 10min. Take 100mg of mycelia and add 500μL of sterile water to wash, centrifuge to get the precipitate.

[0073] The precipitate was resuspended in 500 μL extract mixture, incubated at 37°C for 60 min, and centrifuged at 10,000 rpm for 10 min to remove the precipitate. The supernatant was extracted sequentially with equal volumes of phenol, phenol:chloroform, and chloroform. Take the upper layer solution and add 0.6-1 times the volume of isopropanol to precipitate at room temperature for 10 minutes. Centrifuge at 12000rpm for 15min. The precipitate was washed with 70% ethanol, centrifuged slightly, dried and dissolved in 30 μL sterile water for later use.

[0074] According to the internal peptide sequence of sequenced α-galacto...

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

An alpha-galactosidase gene, its encoded protein, production and use are disclosed. The process separating out alpha-galactosidase from (Penicillium sp.) F63 (CGMCC No. 1669) while purifying, re-separating, cloning to obtain gene for encoding the enzyme ( SEQ ID No: 1 or SEQ ID No: 2 nucleotide sequence ), constructing recombinant expression plasmid containing SEQ ID No: 1 nucleotide sequence, culturing host cell to obtain recombinant strain, inducing recombinant alpha-galactosidase expression, recovering, purifying and comparing to obtain final product. It can hydrolyze melitose and lupeose in bean. It can be used to feed and food industries.

Description

technical field [0001] The present invention relates to a gene encoding glycosidase and its encoded protein, in particular to a gene encoding α-galactosidase isolated and cloned from Penicillium sp.; The gene recombinant expression vector, the host cell containing the gene of the present invention and their preparation method, in addition, the present invention also relates to the preparation of recombinant α-galactosidase and the use of the α-galactosidase. Background technique [0002] α-galactosidase (α-galactosidase, EC3.2.1.22) is melibiase, which belongs to exoglycosidases, and can specifically catalyze the hydrolysis of α-galactosidic bonds at the non-reducing end of sugar chains. It not only It can hydrolyze oligosaccharides containing α-galactosidic bonds, and can also catalyze polysaccharides containing such bonds. Raffinose, stachyose, and verbascose are oligosaccharides widely present in legumes. Under the action of α-galactosidase, these oligosaccharides can be...

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
IPC IPC(8): C12N9/40C12N15/56C12N15/63C12N15/81C12N1/19C12R1/84
Inventor 姚斌王亚茹罗会颖密士军史秀云袁铁铮柏映国杨培龙孟昆
Owner WUHAN SUNHY BIOLOGICAL
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