Alpha-galactosidase and encoding gene and application thereof

A technology of galactosidase and gene, applied in the field of α-galactosidase and its coding gene and application

Active Publication Date: 2011-09-14
BEIJING CHALLENGE AGRI SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The genetically engineered strains of α-galactosidase are rarely reported, mainly in the stage of laboratory research

Method used

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  • Alpha-galactosidase and encoding gene and application thereof
  • Alpha-galactosidase and encoding gene and application thereof
  • Alpha-galactosidase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1, the acquisition and identification of bacterial strain WD, the discovery of α-galactosidase (LGAL protein) and its coding gene (lgal gene)

[0039] 1. Acquisition and identification of strain WD

[0040] 1. Obtaining strain WD

[0041] Strain WD was independently screened from soybean planting soil in September 2008.

[0042] 2. Identification of strain WD

[0043] (1) Morphological characteristics of strain WD

[0044] Plate culture is black velvet or powdery, liquid medium culture, mycelium is black mass flocculent, growing rapidly.

[0045] (2) Molecular identification of strain WD

[0046] The genomic DNA of strain WD was extracted, and the 18sRNA sequence was obtained by PCR amplification (see sequence 4 in the sequence listing). The 18sRNA sequence shown in Sequence 4 was compared with the 18sRNA sequence of existing strains, and it was found that the similarity with the 18sRNA of Alternaria was the highest, reaching 99%. The strain WD was confirm...

Embodiment 2

[0141] Embodiment 2, the preparation of recombinant bacteria

[0142] 1. Preparation of recombinant expression vector

[0143] 1. Extract the RNA of the bacterial strain WD, reverse transcribe it into cDNA; use the cDNA as a template, and perform PCR amplification with a primer pair composed of LYF and LYR to obtain a PCR amplification product.

[0144] LYF: 5'-GTCGACATGTTTGGCATGAAGGGTGTTTG-3' (Add Sal I recognition sequence);

[0145] LYR: 5'-GCGGCCGCTCAGACCTTGTTCAACCAAAATCA-3' (Add Not I recognition sequence).

[0146] 2. The PCR amplification product was recovered, connected to PEASY-T3 Cloning Vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number CT301), and the positive clones were picked for sequencing. The sequencing results showed that the sequence 2 containing the sequence listing was obtained The recombinant plasmid of the indicated DNA was named as the recombinant plasmid pT3-lgal.

[0147] 3. Digest the recombinant plasmid pT3-lgal wi...

Embodiment 3

[0154] Embodiment 3, preparation of LGAL protein (genetic engineering expression obtains α-galactosidase)

[0155] The recombinant bacteria R-pET-lgal and the recombinant bacteria R-pET-30a(+) were respectively operated as follows:

[0156] 1. Fermentation of recombinant bacteria

[0157] (1) The recombinant bacteria were inoculated in 10 ml of liquid LB medium (containing 100 μg / mL of kanamycin), cultured on a shaker at 37° C. (250 rpm, amplitude 26 mm) overnight.

[0158] (2) Transfer to 100ml liquid LB medium (containing 100μg / mL kanamycin), 250rpm, amplitude 26mm) to OD by 1% volume 600 0.6 to 0.8.

[0159] (3) Induced expression

[0160] Add IPTG (to make the final concentration 0.4mmol / L), and culture on a shaker at 28°C (250rpm, amplitude 26mm) for 4 hours to obtain 100ml fermentation broth.

[0161] 2. Protein extraction

[0162] (1) Get the 100ml fermented liquid obtained in step 1, and collect the thalline by centrifugation.

[0163](2) Add 20Mm Tris-HCl buffer...

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Abstract

The invention discloses alpha-galactosidase and an encoding gene and an application thereof. The alpha-galactosidase is as described in the following (a) or (b) or (c): (a) a protein constituted by amino acid residues from 20th site to 739th site of N terminal of sequence 1 in a sequence table; (b) the protein constituted by the amino acid sequence as shown in the sequence 1 in the sequence table; and (c) the protein with the alpha-galactosidase activity, which is derived by replacement and/or deletion and/or addition of one or more amino acid residues in the amino acid sequence of the sequence 1. The alpha-galactosidase can provide technical parameters for establishing a biological reactor with high-efficient expression in the future and be applied in different industrial production fields; furthermore, protein sources are enriched, the protein has low consistency with the known protein amino acid sequence, and new material is provided for taxonomy of the alpha-galactosidase.

Description

technical field [0001] The invention relates to an alpha-galactosidase, its coding gene and application. Background technique [0002] α-galactosidase (alpha-galactosidase, EC 3.2.1.22), also called melibiase, is an exoglycosidase that acts on α-galactosidic bonds and catalyzes α-galactosides oligosaccharides and polysaccharides containing α-galactosidic linkages. α-galactoside is widely distributed in plants, animals and microorganisms. [0003] As a new type of enzyme preparation, α-galactosidase plays an important role in feed industry, food industry, sugar industry, paper industry and medical field. Different fields have different pH action environments, and the requirements for the characteristics of α-galactosidase are also different. For example, acid α-galactosidase is currently not suitable for use in soybean milk processing, because the pH of soybean milk is about 6.2-6.4, while the pH of acid α-galactosidase is 4.0-4.5, which is easy to cause protein precipitat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/40C12N15/56C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N1/14C12P13/00C12R1/645
Inventor 段文娟杨禄良谢宁王海燕李卓夫
Owner BEIJING CHALLENGE AGRI SCI & TECH CO LTD
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