Cloning, expression and application of alpha-L-rhamnosidase gene

A rhamnosidase and gene technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low yield, high cost, difficulty in separation and purification of α-L-rhamnosidase, etc., and achieve the effect of reducing production costs

Inactive Publication Date: 2016-12-07
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But, directly produce α-L-rhamnosidase by original bacterial strain fermentation, not only yield is not high, and often exist the β-D-glucosidase simi...

Method used

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  • Cloning, expression and application of alpha-L-rhamnosidase gene
  • Cloning, expression and application of alpha-L-rhamnosidase gene
  • Cloning, expression and application of alpha-L-rhamnosidase gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, clone expression of gene

[0036] 1. Main reagents

[0037] DNA polymerase, T4 DNA ligase, DNA marker, cloning vector pMD-18T, restriction endonucleases (Bln Ⅰ and Not Ⅰ), competent cell preparation kits were purchased from Takara Company; PCR product recovery kit, gel recovery Kits, rapid plasmid extraction kits, etc. were purchased from Shanghai Sangong; RNA extraction kits, cDNA reverse transcription kits were purchased from Beijing Quanshijin. RCR primers were synthesized by Beijing Liuhe Huada Gene Company; IPTG, ampicillin, and sodium dodecyl sulfate (SDS) were purchased from Shanghai Sangong; other routine reagents were chemically analytically pure.

[0038] 2. Plasmids and strains

[0039] Aspergillus tubingensis, Escherichia coli DH5α, Pichia pastoris GS115 and pPIC9K are all conventional biological materials.

[0040] 3. Method

[0041] 3.1 Aspergillus tubingensis was inoculated on the activation medium, cultured at 28°C for 2 days, transferr...

Embodiment 2

[0053] Example 2. Enzymatic properties of recombinant α-L-rhamnosidase

[0054] 2.1 The influence of pH on recombinant α-L-rhamnosidase

[0055] Na at pH 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 at a concentration of 0.05 mol / L 2 HPO 4 -C 4 h 2 o 7 Enzyme activity was measured in the reaction system, and the highest enzyme activity was taken as 100%, and the rest were compared with it, and the relative enzyme activity was plotted against the pH to obtain the enzyme reaction pH impact on enzyme activity; Glycosidase was mixed with 400 μL of the above-mentioned different pH buffers, and then placed at 4°C for 24 hours to measure the remaining enzyme activity, and the relative enzyme activity was plotted against the pH to obtain the pH stable range of the enzyme. The results are as follows figure 2 shown.

[0056] 2.2 Effect of temperature on recombinant α-L-rhamnosidase

[0057] The enzyme activity was measured at 30, 40, 50, 60, 70, and 80°C respectively, and the enzyme activity...

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Abstract

The invention discloses cloning, expression and application of an alpha-L-rhamnosidase gene. A coding gene is obtained from aspergillus tubingensis and is expressed in pichia pastoris GS115; specifically, total RNA (Ribonucleic Acid) of the aspergillus tubingensis is used as a template and cDNA (complementary Deoxyribonucleic Acid) of alpha-L-rhamnosidase is cloned through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and RACE (Rapid Amplification of cDNA Ends) technologies; pPIC9k is used as an expression vector and is subjected to secreting expression in the pichia pastoris GS115. The alpha-L-rhamnosidase can be used for efficiently hydrolyzing naringin; when the naringin is used as a substrate, Km and Vmax values respectively are 1.27mu.mol/mL and 3445mu.M min<-1>; the thermal stability at 60 DEG C is good and the alpha-L-rhamnosidase gene has a good application prospect in food and biopharmaceutical industries.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and enzyme engineering, in particular to the cloning, expression and application of an α-L-rhamnosidase gene. Background technique [0002] α-L-rhamnosidase (α-L-rhamnosidase) is a glycoside hydrolase that can specifically hydrolyze L-rhamnose at the end of many substances, such as flavonoids, polysaccharides, steroids and glycolipids. α-L-rhamnosidase is produced in some animal and plant tissues, fungi, and bacteria. α-L-rhamnosidase has important application value in the fields of food and medicine. The main functions are: [0003] (1) Debittering effect: Too much bitter substances such as naringin in citrus fruit juices hinders its market development. In order to reduce the content of naringin in fruit juices to improve the taste of fruit juices, people use The enzyme acts on the flavonoid bitter substance to remove the terminal L-rhamnose, thereby greatly reducing the bitterness...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/81C12N1/19C12N15/66C12P19/60C12P19/14C12P19/02C12G1/022A23L2/84A23L5/20C12R1/84
CPCC12N9/2402A23L2/84C12G1/0203C12N15/66C12N15/815C12N2800/102C12P19/02C12P19/14C12P19/60C12Y302/0104
Inventor 李利君高庭倪辉杨远帆王耸朱艳冰杜希萍黄高凌肖安风陈艳红
Owner JIMEI UNIV
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