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Novel tannase and application thereof

A kind of tannase, a new type of technology, applied in the field of genetic engineering

Inactive Publication Date: 2012-10-10
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no studies have reported using metagenomic technology to screen tannase genes from the environment

Method used

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  • Novel tannase and application thereof
  • Novel tannase and application thereof
  • Novel tannase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Establishment of metagenomic library and acquisition of positive clones, gene cloning and expression

[0034] 1. Genomic DNA extraction

[0035] (1) Get 4g of seabed mud sample and put it into a 50ml sterile centrifuge tube;

[0036] (2) Add 13.5ml DNA extraction solution buffer, shake at 37°C, 220r / min for 30min;

[0037] (3) Add 1.5ml 20% SDS;

[0038] (4) 65°C water bath for 2 hours, during which time the centrifuge tube was gently inverted several times every 20 minutes, and mixed;

[0039] (5) Centrifuge at 6000×g for 10 minutes at 4°C;

[0040] (6) Take the supernatant, add an equal volume of chloroform, and gently invert several times up and down;

[0041] (7) Centrifuge at 16000×g for 10 minutes at 4°C;

[0042] (8) Repeat (6), (7) steps twice;

[0043](9) Take the supernatant, add 0.6v of isopropanol, mix gently, and place at room temperature for 1-2h;

[0044] (10) Centrifuge at 16000×g for 20 minutes at 4°C;

[0045] (11) Wash twice with 75%...

Embodiment 2

[0067] The enzymatic property research of embodiment 2 recombinant tannase Tan410

[0068] Take 200 μL of crude enzyme solution, add 200 μL of 15 mM propyl gallate, 37 ° C, water bath for 5 min, then add 200 μ L of methanol tannin (0.667%), 37 ° C for 5 min, add 200 μ L of KOH (0.5 M), 37 ° C for 5 min At the same time, the inactivated crude enzyme solution was used as a control, and the light absorption value was measured at 520nm. The greater the absorption value, the higher the enzyme activity.

[0069] 1. Optimum reaction temperature and thermal stability of recombinant tannase Tan410

[0070] Under the condition of reaction temperature of 20-60° C., the enzyme activity was measured according to the above method, and the optimum reaction temperature was obtained (recorded as 100% when the enzyme activity was the highest). The enzyme was incubated at 25°C, 35°C, 45°C, 50°C, and 55°C for 0.25h, 0.5h, 1h, 2h, 4h, and 12h respectively, and the remaining enzyme activity was me...

Embodiment 3

[0080] Example 3 Effect of recombinant tannase Tan410 on degradation of propyl gallate, tannic acid, epicatechin gallate, epigallocatechin ester, chlorogenic acid, rosmarinic acid and ethyl ferulic acid HPLC analysis

[0081] Take 6 2ml centrifuge tubes, add 200 μL of enzyme solution, and then add 0.5 mg / mL of 200 μL methyl gallate, tannic acid, epicatechin gallate, epigallocatechin gallate, Chlorogenic acid, rosmarinic acid and ethyl ferulic acid were placed at 30°C for 40 minutes and then analyzed by HPLC.

[0082] The chromatographic conditions are: Diamonsil C18 chromatographic column (250×4.6mm, 5μm), column temperature 30°C, the ultraviolet detection wavelength of gallic acid is 260nm, and the ultraviolet detection wavelength of ferulic acid and caffeic acid is 320nm. The injection volume was 0.5 μL, and the peak area was quantified by the external standard method.

[0083] Mobile phase A: acetonitrile B: pH 3.5 phosphate buffered saline solution, A:B=60:40, flow rate ...

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Abstract

The invention discloses a novel tannase. An amino acid sequence of the tannase is shown as SEQ ID NO.3. The invention further discloses a cDNA of the novel tannase, and a nucleotide sequence of the cDNA is shown as SEQ ID NO.1. The invention further discloses an expression vector containing the novel tannase cDNA, recombinant tannase, a preparation method of the recombinant tannase and degradation application of the recombinant tannase by taking propyl gallate, tannic acid, epicatechin gallate, epigallocatechin-3-gallate, chlorogenic acid, rosmarinic acid and ethyl ferulate as substrates. The novel tannase and recombinant tannase have soluble expression in an escherichia coli expression system with high efficiency, and the recombinant tannase has efficient degradation effect on tannic acid, epicatechin gallate, epigallocatechin-3-gallate, chlorogenic acid, rosmarinic acid and ethyl ferulate.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a novel tannase gene isolated and screened from a seabed mud metagenomic library, heterologous expression and application thereof. Background technique [0002] In the production process of tea beverages, the high-temperature extract of tea leaves will become turbid after cooling, and produce flocculent precipitates (tea cheese), which seriously affects the taste and aroma of tea beverages. The formation process of tea cheese is as follows: when the temperature is high, the tea polyphenols and their oxides, caffeine, protein and lipids and other substances in the tea extract exist in a free state; as the temperature decreases, the polyphenols ( Especially theaflavin, thearubigin and gallate, etc.) are combined with the ketone amino group of caffeine and the peptide group of protein to form a complex, which gradually aggregates and precipitates. During the productio...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/10C12N15/63C12N15/70
Inventor 刘玉焕姚健范新炯
Owner SUN YAT SEN UNIV
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