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Cloning and expression of beta-1, 4-endo-mannanase (An Man5A) gene

A mannanase and gene technology, applied in the field of bioengineering, can solve problems that have not been reported in research, and achieve the effects of large industrial production and application potential, economic value, high catalytic activity and thermal stability.

Inactive Publication Date: 2012-03-28
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 the β-mannanase gene, there is no research on the cloning and expression of the β-mannanase gene derived from Aspergillus niger. There are reports

Method used

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  • Cloning and expression of beta-1, 4-endo-mannanase (An Man5A) gene
  • Cloning and expression of beta-1, 4-endo-mannanase (An Man5A) gene
  • Cloning and expression of beta-1, 4-endo-mannanase (An Man5A) gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0038] Cloning of embodiment 1An man5A 3' end mRNA sequence

[0039] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer; the first round of PCR was performed with M13 Primer M4 and ManF1 as primers (94°C for 2min; 30 cycles, 94°C for 30s, 53°C for 30s, 72 ℃90s; 72℃10min); the second round of PCR was performed with the first round PCR product as template and M13Primer M4 and ManF2 as primers (94℃2min; 30 cycles, 94℃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 by slicing the gel and ligated with pUCm-T (pUCm-T-Anman5A3′), transformed into JM109, and sent to Shanghai Sangon for sequencing.

Embodiment 2A

[0040] Cloning of Example 2An man5A 5' end mRNA sequence

[0041] The first round of PCR was performed with 5′RACE Outer Primer (TaKaRa Company) and ManR1 as primers (94°C 3min; 30 cycles, 94°C 30s, 55°C 30s, 72°C 1min; 72°C 10min); with 5′RACE Inner Primer and ManR2 were used as primers for the second round of PCR (94°C for 3min; 30 cycles, 94°C for 30s, 53°C for 30s, 72°C for 1min; 72°C for 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-Anman5A5′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 3A

[0042] Cloning of Example 3 An man5A 5' end promoter sequence

[0043] Using the processed A.niger LW-1 genomic DNA as a template, the first round of PCR uses LD and ManR1 as primers, and the reaction conditions are: 94°C for 4min; 30 cycles, 94°C for 30s, 53°C for 30s, and 72°C for 30s; 10 min at 72°C; the second round of PCR was performed using LD and ManR2 as primers (4 min at 94°C; 30 cycles, 30 s at 94°C, 30 s at 55°C, 30 s at 72°C; 10 min at 72°C). The two rounds of PCR products were analyzed by 2% agarose gel electrophoresis, the target band was recovered by slicing the gel and ligated with pUCm-T (pUCm-T-Anman5AP), transformed into JM109, and sent to Shanghai Sangon for sequencing.

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Abstract

The invention puts forward a cloning method for complete mRNA and DNA sequences of a novel acidic beta-mannanase gene deriving from the Aspergillus niger LW-1 strain. Nucleotide sequences of the gene are respectively SEQ ID NO:1 and SEQ ID NO:2. Bioinformatics analyses show that the xylanase belongs to the 5th family of glycoside hydrolases and is named as An Man5A, with an amino acid sequence of SEQ ID NO:3 and a corresponding gene named as An man5A. The invention also discloses a method for An Man5A construction as well as high expression and purification of recombinant GS115 / man5A. The optimal temperature and pH of the prepared recombinant Man5A are respectively 70DEG C and 4.0, and the recombinant Man5A can be stable under pH of 2.5-7.5 and a temperature lower than 60DEG C, thus boasting great industrial production potential and economic value.

Description

technical field [0001] The present invention relates to the cloning and analysis of the complete mRNA and DNA of a novel acidic β-1,4-mannanase gene derived from Aspergillus niger (Aspergillus niger) LW-1 strain, β-1,4-mannan The construction of enzyme engineering bacteria and the high-efficiency expression and purification methods of recombinant β-1,4-mannanase belong to the technical field of bioengineering. Background technique [0002] Mannan is a general term for a class of polysaccharides composed of specific monosaccharide residues (mainly mannose, glucose and galactose) connected by α- and / or β-glycosidic bonds. It is abundant in nature, second only to xylan as the second largest component of plant hemicellulose. Due to the complexity of its structure, the complete degradation of mannan requires the joint action of β-1,4-mannanase, β-mannosidic bond, β-glucosidase and α-galactosidase. [0003] β-1,4-mannanase (β-1,4-D-mannanase EC 3.2.1.78) is a class of endonuclea...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/10C12N1/19C12N15/81C12R1/685C12R1/84
Inventor 李剑芳赵顺阁邬敏辰唐存多魏喜换
Owner JIANGNAN UNIV
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