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Alternaria alpha-L-rhamnosidase gene and application thereof

A technology of rhamnosidase and gene, applied in the field of genetic engineering

Inactive Publication Date: 2012-01-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on the properties of α-L-rhamnosidase mainly involves the hydrolysis function, and there are few research reports on its application in glycoside synthesis

Method used

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  • Alternaria alpha-L-rhamnosidase gene and application thereof
  • Alternaria alpha-L-rhamnosidase gene and application thereof
  • Alternaria alpha-L-rhamnosidase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Extraction of Alternaria sp.L1 total RNA

[0029] Inoculate Alternaria sp.L1, strain preservation number CGMCC No.3688, into 150ml of enzyme-producing fermentation broth, culture at 28°C for 32h, and wash the cells three times with DEPC-treated ultrapure water , filter paper to collect the bacteria, and liquid nitrogen to grind and break the wall. Take 100mg of bacterial cells, add 1ml Takara RNAiso Plus, mix well, let stand at 15-30°C for 5min, then add 200μL chloroform, shake for 15s, let stand at room temperature for 5min, centrifuge at 12000 rpm, 4°C for 30min, and remove the supernatant Transfer to a new tube, add an equal volume of isopropanol, mix upside down, let stand at 15-30°C for 10min, centrifuge at 2000 rpm, 4°C for 10min, remove the supernatant; add 1mL of 75% ethanol to wash the precipitate, 7500 Centrifuge at 4°C for 5 minutes to remove ethanol, and dry for 5 minutes; dissolve the precipitate in a certain amount of DEPC-treated sterilized ul...

Embodiment 2

[0031] Embodiment 2: PCR amplification of Alternaria L1α-L-rhamnosidase gene partial cDNA sequence

[0032] The cDNA sequences of α-L-rhamnosidase genes of different strains were retrieved from GenBank, and a pair of degenerate PCR primers (N-TER and G1-R) were designed:

[0033] oligo-dT: 5′-TTTTTTTTTTTTTTTTTT-3′SEQ ID NO.6

[0034] N-TER: 5′-ACCCCHTACTCNCARTACATCYTCGC-3′SEQ ID NO.7

[0035] G1-R: 5′-GGRCCNGYRGACCANCCRTG-3′SEQ ID NO.8

[0036] Using the extracted Alternaria total RNA as a template, use the above-mentioned oligo-dT primers to carry out reverse transcription to synthesize the first strand of cDNA. The total reaction volume is 20 μL. A total of 12 μL of ultrapure water for the enzyme was pre-denatured at 65°C for 5 minutes, and placed on ice; then, 4 μL of 5× cDNA synthesis buffer, 1 μL of RNase inhibitor (20 U / μL), 2 μL of 10 mM dNTP, and reverse transcriptase ( 200U / μL) 1 μL, mix well, extend at 42°C for 60 minutes, and terminate the reaction at 70°C for 5 ...

Embodiment 3

[0037] Embodiment 3: Amplification of Alternaria L1α-L-rhamnosidase gene cDNA 3' end sequence

[0038] According to the sequence of SEQ ID No.3, in addition to N-TER, a nested PCR sense primer (F2) was designed, and the 3' end fragment of α-L-rhamnosidase gene cDNA was synthesized by 3'RACE method. The primer sequences are as follows:

[0039] 3'AP: 5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'SEQ ID NO.9

[0040] First-round PCR primers

[0041] N-TER: 5′-ACCCCHTACTCNCARTACATCYTCGC-3′SEQ ID NO.10

[0042] 3'AUAP: 5'-GGCCACGCGTCGACTAGTAC-3'SEQ ID NO.11

[0043] Second round of PCR primers

[0044] F2: 5'-CTGGCTTCGCTTCGGTATGGG-3' SEQ ID NO.12

[0045] 3'AUAP: 5'-GGCCACGCGTCGACTAGTAC-3'SEQ ID NO.13

[0046] Using the extracted Alternaria total RNA as a template, the first strand of cDNA was synthesized by reverse transcription using the above-mentioned 3'AP primer. Using the first strand of cDNA as a template, N-TER and 3'AUAP primers were used for the first round of PCR ampl...

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Abstract

The invention relates to an alpha-L-rhamnosidase gene, especially relates to an alpha-L-rhamnosidase gene from alternaria and an application thereof, and belongs to the technical field of gene engineering. An alpha-L-rhamnosidase gene has a nucleotide sequence as shown in SEQ ID No. 1. An alpha-L-rhamnosidase coded by the alpha-L-rhamnosidase gene has an amino acid sequence as shown in SEQ ID No. 2. The alpha-L-rhamnosidase gene of the invention can be cloned on a Ppic9K expression vector; pichia yeast GS115 is transformed; expression is induced by methanol; and the enzyme yield of fermentation broth is 426 mg / L. The recombinase of the alpha-L-rhamnosidase gene expressed in pichia yeast can efficiently hydrolyze the main bitter substance of naringin in citrus fruit juice, can synthesize transfructosylation products by using rhamnose as a donor and using various substances as receptors, and has important potential application value in food, pharmacy and chemical industry.

Description

field of invention [0001] The invention relates to an α-L-rhamnosidase gene, in particular to an α-L-rhamnosidase gene derived from Alternaria sp. and its application, belonging to the technical field of genetic engineering. technical background [0002] α-L-rhamnosidase (α-L-rhamnosidase, EC 3.2.1.40) widely exists in animals, plants and microorganisms, and can hydrolyze the α-L-rhamnosidic bonds in polysaccharides and other glycosides. It has important application value in the field of medicine and chemical industry. α-L-rhamnosidase can hydrolyze naringin, the main bitter substance in citrus fruit juices, greatly reduce bitterness and improve the taste of juice; it can also hydrolyze a large number of bonded aromatic substances in wine, release aglycone, and generate free Aromatic substances that add aroma and flavor to wine. In addition, α-L-rhamnosidase is also widely used in the structural identification of compounds and the improvement of the activity of anticancer ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/63C12N1/19C12N15/81C12P19/14A23L2/84C12R1/84
Inventor 肖敏刘倩卢丽丽
Owner SHANDONG UNIV
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