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Clone and expression of beta-1, 4-inscribe xylanase catalysis domain (Aor Xyn10BC) gene

A technology of xylanase catalytic domain, xyn10bc-f1, applied in the field of bioengineering, can solve the problem that the enzymatic properties and yield of xylanase cannot meet industrial production, and achieve large industrial production and application potential as well as economic value, The effect of good pH stability

Inactive Publication Date: 2012-06-13
JIANGNAN UNIV
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Problems solved by technology

[0004] At present, xylanase has shown important application value in the fields of papermaking, food, energy, feed, and environment. Although a large number of new xylanases are discovered every year, in fact most of the microorganisms in xylanase The enzymatic properties and yield of glycanase cannot meet the requirements of industrial production

Method used

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  • Clone and expression of beta-1, 4-inscribe xylanase catalysis domain (Aor Xyn10BC) gene
  • Clone and expression of beta-1, 4-inscribe xylanase catalysis domain (Aor Xyn10BC) gene
  • Clone and expression of beta-1, 4-inscribe xylanase catalysis domain (Aor Xyn10BC) gene

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Effect test

Embodiment 1Ao

[0024] The cloning of embodiment 1Aor xyn10BC cDNA sequence

[0025] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer; the first round of PCR amplification was performed with M13 Primer M4 and Xyn10BC-F1 as primers (94°C 2min; 30 cycles, 94°C 30s, 51 ℃ for 30s, 72℃ for 90s; 72℃ for 10min), use Xyn10BC-F1 and Xyn10BC-R1 as primers for the second round of PCR amplification (94℃ for 2min; 30 cycles, 94℃ for 30s, 51℃ for 30s, 72℃ for 1min; 72°C for 10 minutes). The PCR amplified product was analyzed by 1% agarose gel electrophoresis, the target band was recovered by slicing the gel and ligated with pUCm-T (pUCm-T-xyn10BC), transformed into JM109, and sent to Shanghai Sangon for sequencing.

Embodiment 2

[0026] Example 2 Construction of expression plasmid containing mature peptide gene encoding xylanase catalytic domain

[0027]Use Oligo dT-Adaptor Primer as primers to reverse transcribe the first strand of cDNA synthesized; use M13 Primer M4 and Xyn10BC-F1 as primers for the first round of PCR (94°C 2min; 30 cycles, 94°C 30s, 51°C 30s , 72°C 90s; 72°C 10min); the second round of PCR using Xyn10BC-F1 and Xyn10BC-R1 as primers (94°C 2min; 30 cycles, 94°C 30s, 51°C 30s, 72°C 1min; 72°C 10min). The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-xyn10BC), transformed into JM109, and sent to Shanghai Sangon for sequencing. The sequenced correct pUCm-T-xyn10BC and pPIC9K M Plasmids were double digested with Xho I and Not I, and the recovered digested products were ligated under the action of T4DNA ligase to obtain recombinant plasmid pPIC9K M -xyn10BC, and sequenced the re...

Embodiment 3

[0028] Example 3 Construction, expression, product purification and activity determination of GS115 / xyn10BC

[0029] pPIC9K with Sal I M -xyn10BC was linearized, electrotransformed and screened according to the Pichia expression manual, and a high-copy Pichia recombinant GS115 / xyn10BC was obtained. The engineered bacterium was induced with 0.5% methanol for 96 hours, and the recombinant xylanase activity in the fermentation broth was measured by DNS method to reach 20IU / mL. The centrifuged supernatant is the recombinant xylanase crude enzyme solution, which is concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10kDa, and then purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. After purification, It was detected as a single band by SDS-PAGE, and showed that the molecular weight of the recombinant xylanase was 50kDa. The optimum action temperature of the recombinant xylanase is 60° C., a...

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Abstract

The invention provides clone and analysis of a complementary deoxyribose nucleic acid (cDNA) sequence, plasmid construction and expression method of a novel tenth family xylanase catalysis domain gene stemming from aspergillus oryzae CICC 40186. A nucleotide sequence is SEQ ID NO:1. Bioinformatics analysis shows that the xylanase catalysis domain belongs to the tenth family of glycoside hydrolase and is named as Aor Xyn10BC, an amino acid sequence of the xylanase catalysis domain is SEQ ID NO:2, and the corresponding gene is named as Aor Xyn10BC. A theoretical basis is established for heterologous expression and industrial production of the gene. The invention further comprises construction of xylanase engineering bacteria and a method of efficient expression and purification of recombination xylanase. As a novel enzyme preparation, the xylanase has high industrial production and application potentials and economic value and establishes a theoretic basis for research of other xylanases.

Description

technical field [0001] The present invention relates to the cloning and analysis of the complete cDNA sequence of a novel 10-family xylanase catalytic domain (Aor Xyn10BC) gene derived from Aspergillus oryzae CICC 40186 strain, and the construction of xylanase catalytic domain genetically engineered bacteria The invention relates to a method for high-efficiency expression and purification of recombinant xylanase, belonging to the technical field of bioengineering. Background technique [0002] In nature, the plant cell wall is a huge carbohydrate storehouse, and hemicellulose accounts for about 33% of the dry weight of plants, and is the most abundant polysaccharide besides cellulose. Xylan is one of the most important components of hemicellulose, which widely exists in the cell wall and intercellular layer of plant cells. The structure of xylan is complex, and its main chain is composed of xylose residues linked by β-1,4-glycosidic bonds. When the main chain is replaced, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/10C12N15/81C12N1/19C12R1/84
Inventor 邬敏辰殷欣李剑芳刘晓彤罗秋玲
Owner JIANGNAN UNIV
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