Medium and low temperature neutral tannase TanXZ7 and gene and application thereof

A tannase and neutral technology, applied in the field of genetic engineering, can solve problems such as slow progress, and achieve the effect of low acid-base low temperature adaptability and high activity

Active Publication Date: 2014-08-27
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microbial sources of tannase have a long history of research, but the progress is slow. At present, 58 strains of fungi / bacteria that can produce tannase have been reported, 25 tannases have been studied, and only 3 tannases have been realized. Heterologous expression, 1 completed structure analysis

Method used

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  • Medium and low temperature neutral tannase TanXZ7 and gene and application thereof
  • Medium and low temperature neutral tannase TanXZ7 and gene and application thereof
  • Medium and low temperature neutral tannase TanXZ7 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Cloning of embodiment 1 Thielavia subthermophila XZ7 tannase coding gene tanXZ7

[0049] The strain Thielavia subthermophila XZ7 was collected from the root sand of vegetation in the desert area of ​​Ningxia Autonomous Region. It can grow normally at 45°C, but cannot grow at 20°C. It is a typical thermophilic fungus.

[0050] Extraction of Thielavia subthermophila XZ7 genomic DNA:

[0051]Filter the mycelium cultured in liquid for 3 days into a mortar with sterile filter paper, add liquid nitrogen to make it pre-cooled, put the mycelia in the mortar and grind it into a white powder, transfer the white powder to a 50mL centrifuge Add 22.5mL of CTAB solution preheated at 65°C (add 2% mercaptoethanol before use) and 2.5mL of 20% SDS solution to the tube, lyse in a water bath at 65°C for 2 hours, invert and mix once every 10min, and centrifuge at 12,000rpm at 4°C 15min. The supernatant was extracted in chloroform / isoamyl alcohol (24:1) to remove impurities, and then 0.6 t...

Embodiment 2

[0060] The preparation of embodiment 2 recombinant tannase

[0061] The expression vector pPIC9 was subjected to double digestion (SpeI and NotI), and the gene tanXZ7 encoding tannase was double-digested (SpeI and NotI) at the same time, and the gene fragment encoding tannase was cut out and connected to the expression vector pPIC9 to obtain a shuttle containing The recombinant plasmid pPIC-tanXZ7 of Thielavia subthermophila XZ7 tannase gene tanXZ7 was transformed into Pichia pastoris GS115 to obtain recombinant Pichia pastoris strain GS115 / tanXZ7.

[0062] The GS115 strain containing the recombinant plasmid was inoculated in 400mL of BMGY culture medium, shaken at 250rpm at 30°C for 48h, and then collected by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 72 hours of induction, the supernatant was collected by centrifugation. Determination of tannase activity. The expression level of recombinant tannase was 10.52U / mL. SDS-PAGE ...

Embodiment 3

[0064] The activity analysis of embodiment 3 recombinant tannase

[0065] Rotanin Chromogenic Method: The specific method is as follows: at pH 5.0 and 40°C, a 500 μL reaction system includes 100 μL of an appropriate diluted enzyme solution, 400 μL of propyl gallate solution (1.25 mmol / L, pH 5.0), After reacting for 10 minutes, 300 μL of methanolic rhodanine solution (50 mmol / L) was added to terminate the reaction, and 200 μL of KOH (0.5 M) was added for color development after 5 minutes. After cooling to room temperature, the OD value was measured at 520 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of gallic acid per minute under given conditions.

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Abstract

The invention relates to the field of genetic engineering, in particular to medium and low temperature neutral tannase TanXZ7 and a gene and application of the medium and low temperature neutral tannase TanXZ7. The amino acid base sequence is shown in SEQ ID NO.1. The optimum pH of the medium and low temperature neutral tannase TanXZ7 is 6.0, and stability of the medium and low temperature neutral tannase TanXZ7 is better with pH ranging from 3.0 to 8.0; the optimum temperature is 40 DEG C, the medium and low temperature neutral tannase TanXZ7 has high enzyme activity at the temperature ranging from 25 DEG C to 60 DEG C, and stability of the medium and low temperature neutral tannase TanXZ7 is good at the temperature of 40 DEG C; due to the medium and low temperature neutral characteristic, the medium and low temperature neutral tannase TanXZ7 can be applied to food industrial production.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a medium-low temperature neutral tannase TanXZ7 and its gene and application. Background technique [0002] Tannins are widely distributed in plants, including roots, wood, bark, fruits and leaves. As the main secondary metabolite in plants, the content of tannin is only lower than that of cellulose, hemicellulose and lignin. According to its chemical structure, it can be divided into hydrolyzable citric acid tannins and ellagitannins and relatively stable condensation tannins composed of polyflavanol polyphenols or proanthocyanidins (ie, hydroxyflavanol monomers). quality tannins. Tannins contain a large number of benzene rings and phenolic hydroxyl groups, which are easy to anneal with metals to form larger polymers, and can combine with polysaccharides and proteins to produce astringent and sedimentation effects. [0003] In terms of food and animal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/81C12N1/19A23K1/165A23L2/70C12R1/84A23K20/189
CPCC12N9/18C12N15/815C12Y301/0102
Inventor 姚斌马锐张三燕石鹏君黄火清柏映国罗会颖王亚茹杨培龙孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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