Efficient xyloglucanase and application thereof

A xyloglucanase and high-efficiency technology, applied in the field of xyloglucanase, can solve the problem of not finding xyloglucanase, etc., and achieve the effects of excellent enzyme activity, high yield and high enzyme activity

Active Publication Date: 2021-06-29
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been reports of obtaining xyloglucanase from microorganisms, no efficient and acidic xyloglucanase has been found from microorganisms.

Method used

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  • Efficient xyloglucanase and application thereof
  • Efficient xyloglucanase and application thereof
  • Efficient xyloglucanase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Enzyme specific activity definition: under the set assay conditions, the enzyme activity per mg of protein (U / mg). Example 1 Obtaining of CvXEG1 gene cDNA

[0101] 1. Extraction of grape white rot fungus RNA

[0102] Inoculate Ascospora viticola on PDA medium, culture for 2-3 days, gently scrape the mycelia with a scalpel, and crush the mycelium with liquid nitrogen grinding method. The mortar, pestle and tweezers used must be subjected to high temperature Sterilization. Use the column type fungal total RNA extraction and purification kit to extract grape white rot fungus RNA, and follow the product instructions.

[0103] 2. Reverse transcription and amplification of CvXEG1 gene cDNA

[0104] According to the genome sequence analysis of Coniella vitis, the coding region of CvXEG1 gene is a single exon. Design the following primers:

[0105] 5'-GCTGAAGCTTACGTACACCCCCCAACCCCAC-3'

[0106] 5'-GAATTAATTCGCGGCATGATGATGATGATGATGCTCGGAACGCTTGCG-3'

[0107] Grape white r...

Embodiment 2

[0113] Example 2 Expression and purification of CvXEG1

[0114] 1. Construction of pPIC9K-CvXEG1 recombinant expression vector

[0115] Plasmid pPIC9K was double-digested with restriction endonucleases EcoRI and NotI, and the double-digested product was recovered with a DNA purification and recovery kit. The CvXEG1 cDNA obtained in Example 1 was connected to the pPIC9K vector plasmid using a vector cloning kit and transformed into JM109 competent cells in Escherichia coli. After ampicillin antibiotic screening, PCR was performed on positive transformants using universal primers 5'AOX and 3'AOX, and positive transformants with correct band sizes were obtained. The transformant with the correct size of the verified band was sent to the company for sequencing, and the sequence of this piece of DNA was consistent with the sequence of SEQ ID 2 in the sequence listing.

[0116] 2 Construction of yeast strain containing pPIC9K-CvXEG1 recombinant plasmid

[0117] The pPIC9K-CvXEG1 ...

Embodiment 3

[0144] Example 3 Research on the enzymatic properties of CvXEG1

[0145] 1. Substrate-specific detection of recombinant proteins

[0146]Prepare 0.5% xyloglucan, 0.5% sodium carboxymethylcellulose, 0.5% xylan, 0.5% filter paper, and 0.5% microcrystalline cellulose respectively, and use The aforementioned methods measure the enzymatic activity of recombinant proteins on different substrates.

[0147] The results showed that the recombinant protein had high enzymatic activity on xyloglucan (specific activity up to 229U / mg), and at the same time, the enzyme also had certain enzymatic activity on xylan (specific activity up to 105U / mg). The results are shown in Table 3 below:

[0148] Table 3 Substrate-specific detection of recombinant protein pPIC9K-CvXEG1

[0149]

[0150] 2. Optimum working temperature

[0151] Under the condition of pH 5.0, a 0.2% xyloglucan solution was prepared, and the enzyme activity of the recombinant protein was measured at 30°C, 40°C, 50°C, 60°C,...

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Abstract

The invention discloses efficient xyloglucanase and application thereof, and the efficient xyloglucanase is derived from Coniella vitis and is selected from one of the following: (a) a protein with the 19-390 amino acid sequence in SEQ ID NO: 3, (b) a protein with an amino acid complete sequence as shown in SEQ ID NO: 3; and (c) a protein which has the complete sequence as shown in SEQ ID NO: 3 or the 19-390th amino acid sequence which is subjected to conservative substitution, deletion or insertion by one or more amino acids and has the same function as the complete sequence as shown in SEQ ID NO: 3. The invention also provides a coding gene of the high-efficiency xyloglucanase, a recombinant expression vector containing the gene, an engineering bacterium and application thereof. The efficient xyloglucanase has excellent endo-xyloglucanase activity, the enzyme activity is kept stable under a strong acid condition, the characteristic completely meets the condition requirements of a synchronous saccharification and fermentation process, and the efficient xyloglucanase has a very high application prospect in many fields as the endo-xyloglucanase.

Description

technical field [0001] The invention relates to the field of xyloglucanase, in particular to a high-efficiency xyloglucanase and its application. Background technique [0002] Plant cell walls are mainly composed of lignocellulose (about 40%), hemicellulose (about 20-30%) and lignin (about 20-30%). Cellulose and hemicellulose are important renewable biomass resources, which have important uses in the production of fuel ethanol and other energy fields, and can greatly alleviate the tense situation of increasing international energy shortage. Our country is an important agricultural country in the world, and the plant solid waste produced in agriculture can provide very rich cellulose and hemicellulose resources. [0003] The cellulose in the plant cell wall is embedded by hemicellulose. This structural composition determines that the full degradation of the plant cell wall must first degrade the peripheral hemicellulose, so that the cellulose can be fully exposed before it c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12N1/14C12R1/84C12R1/645
CPCC12N9/2434C12N15/815C12Y302/01151
Inventor 周善跃孙佳宁李保华
Owner QINGDAO AGRI UNIV
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