A gene expressing acid β-mannanase, its carrier and application

A technology of mannanase and expression vector, which is applied in the field of genetic engineering, can solve the problems of low yield of β-mannanase, difficulty in meeting the needs of industrial production, and the survival rate of enzyme activity is only 32%, so as to achieve high-efficiency expression , important industrial application value, good temperature resistance

Active Publication Date: 2020-10-02
TIANJIN BIOFEED TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the mannanase produced by the isolated Aspergillus niger can better meet the production needs, but the yield of β-mannanase produced by it is low, and it is difficult to meet the needs of industrial production. - The optimum temperature for mannanase action is 50°C, and the optimum action pH is 5.50. The β-mannanase of the strain before optimization was treated at pH 3.0 for 30 minutes, and the enzyme activity retention rate was only 32%.

Method used

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  • A gene expressing acid β-mannanase, its carrier and application
  • A gene expressing acid β-mannanase, its carrier and application
  • A gene expressing acid β-mannanase, its carrier and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Optimization of β-mannanase gene and construction of recombinant expression vector

[0041] 1) Optimization and synthesis of β-mannanase gene

[0042]The original β-mannanase gene Man was derived from a strain of Aspergillus niger screened and isolated from natural soil. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. On the premise of not changing the encoded amino acid sequence, the Man gene was codon-optimized according to the codon preference of Pichia pastoris, and the whole gene sequence ManT of acid β-mannanase was synthesized by the whole gene synthesis method. The nucleotide sequence of the artificially synthesized acid β-mannanase gene ManT is shown in SEQ ID NO.3. Both ends of the synthetic ManT gene also have EcoRI and Not I restriction sites for easy connection with expression vectors. Using the ManT gene sequence as a template to amplify the β-mannanase ManT gene, the primers used a...

Embodiment 2

[0048] Example 2 Acquisition and Identification of Acidic β-Mannanase Engineering Bacteria

[0049] Pichia pastoris P. pastoris GS115 was transformed, and the main operation procedures of transformation and screening were referred to the Pichi expression operation manual of Invitrogen Company. The recombinant plasmid pPICZαA-ManT was linearized with Sac I, transformed into Pichia pastoris GS115 by electroporation, and the transformed cells were spread to YPD culture containing 100 μg / ml Zeocin antibiotic and cultured at 30°C for two days. With the transformants obtained by screening, the genome was extracted according to the yeast genomic DNA extraction kit, and PCR amplification was carried out with a-factor / 3'AOX primers. After the reaction was completed, the PCR amplification products were identified by 1% agarose gel electrophoresis (such as figure 1 shown). Cut the gel to recover and purify the PCR product, and send it to BGI for sequencing to confirm that the sequence i...

Embodiment 3

[0052] Example 3 High-efficiency expression of acidic β-mannanase recombinant strain

[0053] 1) Shake Flask Seeds

[0054] Medium formula: yeast powder 10g / L, peptone 20g / L, glucose 20g / L, natural pH.

[0055] Culture conditions: culture temperature 30°C, shaker rotation speed 220rpm, liquid volume of shaker flask 15%, natural pH, culture period 20h. After the seeds are mature, the primary seed pots are flame inoculated.

[0056] 2) Level 1 seed tank

[0057] Medium formula: glycerol 40g / L, ammonium dihydrogen phosphate 30g / L, potassium dihydrogen phosphate 5g / L, potassium sulfate 10g / L, magnesium sulfate 5g / L, calcium sulfate 0.5g / L, potassium hydroxide 1.5g / L L, PTM1 4.5g / L.

[0058] Culture conditions: inoculum size 0.5%, ventilation rate 40m3 / h, stirring speed 300rpm, ammonia water was used to maintain the pH value at 5.0 during the cultivation process, and the cultivation temperature was 30°C. The cultivation period is 20h. After the seeds are mature, they are all ...

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Abstract

The invention discloses a gene expressing acid beta-mannase. A nucleotide sequence of the encoding gene is SEQ ID NO:3. The invention further discloses an expression cassette containing the gene, a recombinant carrier or a recombinant microorganism and application thereof, and a method for preparing the acid beta-mannase by fermentation of a recombinant strain. According to the gene, a whole-genome sequence of the beta-mannase obtained by a whole-genome synthesis method can be stably and efficiently expressed and inherited after being transferred into a host cell, the expressed acid beta-mannase has good temperature and acid resistance, the enzyme activity in a pot can reach 32000 U / ml, and is twice of the enzyme activity before optimization.

Description

technical field [0001] This application relates to the field of genetic engineering, specifically, the present invention relates to a gene for encoding acid β-mannanase, its vector and application. Background technique [0002] β-mannan is a major hemicellulose found in cell walls and plant seeds. β-mannanase is an endoglycoside hydrolase that hydrolyzes the β-1,4-D-mannosidic linkages of mannan to generate mannan-oligosaccharides. β-Mannanase is rich in sources, which can be divided into plant sources (such as beans, black locust, straw, etc.), animal sources (such as snails, marine molluscs, etc.) and microbial sources. Due to the wide range of microbial resources, easy cultivation, simple operation and extraction process, and the relatively high activity of β-mannanase produced, β-mannanase with good tolerance to temperature and acidity can be screened in extreme environments , so the β-mannanase derived from microorganisms has a wider scope of application, and has also...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/24C12N15/81C12N1/19C12R1/84
CPCC12N9/2491C12N15/815C12N2800/22C12Y302/01025
Inventor 李爽曹春红王海燕张广民蔡辉益李阳
Owner TIANJIN BIOFEED TECH CO LTD
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