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Clone of novel beta-mannanase gene and preparation method of recombinase thereof

A mannanase, a new type of technology, applied in genetic engineering, DNA preparation, plant genetic improvement and other directions, can solve problems that have not been reported in research, and achieve the effects of large-scale industrial production, high catalytic activity and thermal stability

Inactive Publication Date: 2011-08-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although there are some literatures and patent reports on the cloning and expression of β-mannanase gene, there is no research on the cloning and expression of β-mannanase gene derived from Aspergillus usamii. There are reports

Method used

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  • Clone of novel beta-mannanase gene and preparation method of recombinase thereof
  • Clone of novel beta-mannanase gene and preparation method of recombinase thereof
  • Clone of novel beta-mannanase gene and preparation method of recombinase thereof

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Embodiment 1

[0038] Cloning of the 3' end mRNA sequence of embodiment 1β-mannanase gene

[0039] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer as primers; the first round of PCR was performed with M13 Primer M4 and Man5A-3F1 as primers, and the reaction conditions were: 94°C for 2min, 30 cycles (94 ℃ for 30s, 53℃ for 30s, 72℃ for 90s), 72℃ for 10min; use M13 Primer M4 and Man5A-3F2 as primers for the second round of PCR, and the reaction conditions are: 94℃ for 2min, 30 cycles (94℃ for 30s, 53 ℃ 30s, 72℃ 90s), 72℃ 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-man5A3′), transformed into JM109, and sent to Shanghai Sangong for sequencing after enzyme digestion and identification. Aus man5A 3' mRNA sequence.

Embodiment 2

[0040] Cloning of the 5' end mRNA sequence of embodiment 2β-mannanase gene

[0041] The first round of PCR was performed using the Outer Primer and Man5A-5R1 of the 5′-Full RACE Kit as primers. The reaction conditions were: 94°C for 3min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10 min; the second round of PCR was performed using Inner Primer and Man5A-5R2 as primers, and the reaction conditions were: 94°C for 3 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), and 72°C for 10 min. 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-man5A5′), transformed into JM109, and sent to Shanghai Sangong for sequencing after enzyme digestion and identification. The 5' end mRNA sequence of Aus man5A was obtained.

Embodiment 3

[0042] Cloning of the 5' end regulatory sequence of embodiment 3β-mannanase gene

[0043] Using the processed A.usamii E001 genomic DNA as a template, the first round of PCR used T-PrimerF and Man5A-5R1 as primers, and the reaction conditions were: 94°C for 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C 60s), 72°C for 10min; the second round of PCR uses T-PrimerF and Man5A-5R2 as primers, and the reaction conditions are: 94°C, 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 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-man5AP), transformed into JM109, and sent to Shanghai Sangong for sequencing after enzyme digestion and identification. Aus man5A 5' regulatory sequence.

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Abstract

The invention provides a cloning method for the complete mRNA and DNA sequences of a novel beta-mannanase gene derived from Aspergillus usamii E001, and the nucleotide sequences of the mRAN and the DNA are SEQ ID No.1 and SEQ ID No.2 respectively. Bioinformatic analysis indicates that beta-mannanase belongs to the fifth family of glycoside hydrolase and is named Aus Man5A. The amino acid sequence of the beta-mannanase is SEQ ID No.32 and the corresponding gene of the beta-mannanase is named Aus Man5A. The invention also discloses a construction method of beta-mannanase engineering bacteria and a high-efficiency expression and purification method for recombinant beta-mannanase. The optimal temperature and optimal pH value of the prepared recombinant beta-mannanase are 75 DEG C and 3.5 respectively, the recombinant beta-mannanase is stable in a pH value range from 3.0 to 7.0 and at a temperature of 70 DEG C. The methods have high industrial production potential and economic value.

Description

technical field [0001] The invention relates to the cloning of the complete mRNA and DNA sequence of a novel β-mannanase gene derived from Aspergillus usamii E001, the construction of a novel β-mannanase engineering bacterium, and the recombinant β-mannan The method for high-efficiency expression and purification of enzymes belongs to the technical field of microbial genetic engineering. Background technique [0002] Mannan is a polysaccharide with complex structure, which can be divided into homomannan and heteromannan. Homogeneous mannan is a linear polysaccharide linked by β-1,4-mannosidic bonds; if some mannose residues in the main chain are replaced by glucose or The residues are connected to form branches, which are called different mannans, mainly including glucomannan, galactomannan and galactoglucomannan. Mannan is a relatively abundant hemicellulose resource in nature, and its quantity is second only to xylan. Leguminous plant seeds, conifer wood, coffee beans, ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/10C12N15/09
Inventor 邬敏辰唐存多赵顺阁郭静韦敬土李剑芳
Owner JIANGNAN UNIV
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