Alpha-L-rhamnosidase gene and application thereof

A rhamnosidase and gene technology, applied in the field of bioengineering, can solve the problems of difficult expression and low protein expression, and achieve the effects of increasing production, improving catalytic efficiency, and reliable biological safety

Inactive Publication Date: 2017-10-24
JIANGSU UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The author tried to display the rhA gene derived from Aspergillus niger A. niger on the surface of Saccharomyces cerevisiae cells, but the protein expression level was low. After analysis, it may be that some factors have an inhibitory effect on gene expression, such as codon usage preference etc., making it difficult to express in foreign hosts (Biotech J, 2011, 6(6):650-659)

Method used

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  • Alpha-L-rhamnosidase gene and application thereof
  • Alpha-L-rhamnosidase gene and application thereof
  • Alpha-L-rhamnosidase gene and application thereof

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Embodiment 1

[0025] Acquisition of the target gene

[0026] In the present invention, the genome derived from Aspergillus niger (A. niger) is used as a template, and the primers in the following table are used for PCR amplification.

[0027] The PCR reaction system is: 2×PCR Buffer (containing Mg 2+ ) 25 μL, dNTP (25 mM) 5 μL, upstream primer coRHA-F and downstream primer coRHA-R (10 μM) 1 μL each, template 1 μL, KOD Fx DNA polymerase (purchased from TOYOBO, catalog number KFX-101) 0.5 μL, plus no Bacterial water to a final volume of 50 μL.

[0028] The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 90 s, reaction for 30 cycles, and extension at 72°C for 10 min.

[0029] Sequencing verification was performed after the PCR was completed, and codon optimization was performed after the verification was correct to obtain a new α-L-rhamnosidase gene corhA nucleotide sequence, the sequence of wh...

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Abstract

The invention relates to an alpha-L-rhamnosidase gene, an application thereof and a method for utilizing a brewer yeast surface display system to express alpha-L-rhamnosidase and utilizing the enzyme to catalyze flavonoid hydrolysis. According to the invention, the alpha-L-rhamnosidase gene (rhA) derived from Aspergillus niger is subjected to codon optimization, a brand-new alpha-L-rhamnosidase gene (corhA) is acquired, an expression carrier containing corhA is established and a brewer yeast strain containing the expression carrier is acquired. According to the invention, the brewer yeast surface display system is utilized to display and express the alpha-L-rhamnosidase gene (coRHA) on the brewer yeast cell surface, the extracellular substrate is in full contact with the enzyme, the catalytic efficiency is increased, the single isomer with physiological activity can be acquired, the separating and purifying steps are avoided and the production cost is lowered.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to gene optimization and construction, in particular to an α-L-rhamnosidase gene and its application. Background technique [0002] Yeast-based cell surface display is an emerging protein processing system suitable for expressing foreign proteins from eukaryotic sources. In 2012, Xiao et al. used cell surface display technology to anchor α-L-rhamnosidase RhaL1 from Alternaria sp.L1 to the cell wall of Saccharomyces cerevisiae using a-lectin as the carrier protein , and prepared as a whole-cell catalyst to catalyze the hydrolysis of naringin (Bioresource Technol, 2012, 123:144-149). The author tried to display the rhA gene derived from Aspergillus niger A. niger on the surface of Saccharomyces cerevisiae cells, but the protein expression level was low. After analysis, it may be that some factors have an inhibitory effect on gene expression, such as codon usage preference etc., ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/81C12N9/24C12R1/865
CPCC12N9/2402C12N15/81C12Y302/0104
Inventor 王俊王方芹张凡何殊
Owner JIANGSU UNIV OF SCI & TECH
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