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Tannase AfTan2.0 mutant and application thereof

A technology of tannase and mutants, applied in the field of molecular biology, can solve problems such as changes in fermentation temperature, influence of tannase catalytic activity, poor thermal stability of tannase, etc., and achieve the effect of improving thermal stability

Active Publication Date: 2021-11-12
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the thermal stability of the current tannase is poor, and in the actual production process, it is often faced with a large change in the fermentation temperature, which greatly affects the catalytic activity of the existing tannase

Method used

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  • Tannase AfTan2.0 mutant and application thereof
  • Tannase AfTan2.0 mutant and application thereof
  • Tannase AfTan2.0 mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The mutation strategy of embodiment 1 tannase AfTan2.0

[0020] The three-dimensional structure model of AfTan2.0 was constructed using the protein online modeling software SWISS-MODEL (https: / / swissmodel.expasy.org / interactive), and the crystal structure of Aspergillus niger tannase (PDB: 7k4o) was also selected as a template. The model constructed by SWISS-MODEL was optimized using the online tool GalaxyRefine (http: / / galaxy.seoklab.org / cgi-bin / submit.cgi?type=REFINE). Use PROCHECK to detect the quality of the model before and after optimization, and select the optimal model for molecular dynamics simulation. At a temperature of 400K, a 20ns molecular dynamics simulation of the protein was carried out using the software NAMD2.12 and 100 frames of structure were extracted from the simulated trajectory. FoldX4.0 software was used to perform scanning mutation operation on each frame of protein structure extracted from the molecular dynamics simulation trajectory, and th...

Embodiment 2

[0022] The mutant construction of embodiment 2 tannase AfTan2.0

[0023] The primers used to construct the mutants for the thermal stability study of AfTan2.0 are shown in Table 1, and the mutant primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The method of mutant construction adopts conventional methods.

[0024] Table 1 Mutant primers for thermostability studies

[0025]

[0026]

[0027] Note: The underlined mark is the mutation site

Embodiment 3

[0028] Example 3 SDS-PAGE analysis and activity identification of tannase thermostable mutants

[0029] SDS-PAGE analysis was carried out on the thermostable mutants of tannase. All 20 mutants were expressed in the recombinant strain of Pichia pastoris GS115, and the results were as follows: figure 1 shown. The apparent molecular weight of each mutant is between 75-100kDa, which is the same as that of AfTan2.0. When the tannase activity of each mutant was determined, it was found that the mutant R230L lost the tannase activity, and the remaining 19 mutants T163L, T176L, T176F, Q211L, A222V, R230F, Q234L, S242L, C255P, I295N, I295K, T412L, T437K, T437R, A459L, G475E, G475P, V476E and S477L still had tannase enzyme activity.

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Abstract

The invention discloses a tannase AfTan2.0 gene mutant and application thereof. The mutants are G475E, T437K and A222V. According to the invention, 20 single-point mutants are designed on the basis of molecular dynamics simulation by applying a calculation program FoldX; activity identification shows that the mutant R212L loses the tannase activity; and the mutants G475E, T437K and A222V which have an obvious improvement effect on the thermal stability of tannase are screened out by measuring the residual enzyme activity of the residual 19 mutants under the treatment conditions of 50 DEG C and 60 DEG C.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a tannase AfTan2.0 mutant derived from a thermophilic fungus and an application thereof. Background technique [0002] Tannase (Tannase EC 3.1.1.20), also known as tannase, is a kind of tannin hydrolase, which can hydrolyze the ester bond and decapsyl carboxyl bond in tannin to generate glucose, gallic acid and their corresponding alcohols class of compounds. Tannase is widely distributed in nature, such as animals, plants and microorganisms. At present, tannase used in production mainly comes from microorganisms of the genus Aspergillus. The U.S. Food and Drug Administration (FDA) has recognized tannase as a safe food additive, and it has been used to clarify liquids in tea, beverages, and brewing projects. In addition, it is also used in industrial fields such as chemical industry, medicine and feed. [0003] At present, the production cost of tannase l...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/81C12N1/19C12R1/84
CPCC12N9/18C12N15/815C12Y301/0102Y02A50/30
Inventor 卢海强谷新晰陈伟谷子林陈宝江
Owner HEBEI AGRICULTURAL UNIV.
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