Method for validating influence of disulfide bond on thermal stability of xylanase

A xylanase and thermal stabilization technology, which is applied in the field of bioengineering, can solve problems such as unseen thermal stability analysis, and achieve the effect of accurate and reliable experimental basis.

Inactive Publication Date: 2013-03-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no literature or patent report on the thermal stability analysis of the N-terminal disulfide bond on the thermostable hybrid xylanase AEx11A of the 11 family

Method used

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  • Method for validating influence of disulfide bond on thermal stability of xylanase
  • Method for validating influence of disulfide bond on thermal stability of xylanase
  • Method for validating influence of disulfide bond on thermal stability of xylanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Homology modeling to simulate the spatial structure of AEx11A:

[0024] Homologous comparison was performed in the PDB database, and the primary structure homology with AEx11A was found to be higher, which were derived from Penicillium funiculosum (1TE1), E.coli (2VUL) and Hypocrea jecorina (1ENX) Crystal structure of family 11 xylanases as a template for homology modeling. Homology modeling of AEx11A was performed using SALIGN (http: / / salilab.org / DBAli / ?page=tools_&action=f_salign) and MODELLER 9.9 (http: / / salilab.org / modeller / ) programs. Through the homology modeling of the AEx11A structure and its alignment with the AoXyn11A structure, it was found that a disulfide bond (Cys 5 -Cys 32 ). The cysteine ​​at position 5 was mutated to threonine (C5T) by site-directed mutagenesis, and the disulfide bond was removed to explore its effect on the thermal stability of AEx11A. The genes before and after the mutation were AEx11A and AEx11A C5T .

Embodiment 2

[0025] Example 2 Mutant gene AEx11A C5T and construction of expression plasmids

[0026] Design primers based on pPIC9K-AEx11A (this experiment is preserved)

[0027] F1: 5′- GAATTC AACGCTCAAACTactCTTACCTCT-3′, containing EcoR I restriction site and mutation point Thr 5

[0028] R1: 5'- GCGGCCGCT CAATAAACAGTGATAATAGCAG-3′, with Not I restriction site

[0029]The pPIC9K-AEx11A stored in our laboratory was used as a template, and F1 and R1 were used as primers for PCR reaction. 94°C for 2min; 30 cycles, 94°C for 30s, 55°C for 30s, 72°C for 12s; 72°C for 10min. The PCR product was analyzed by 1% agarose gel electrophoresis, and the target band was recovered by tapping the gel and ligated with pUCm-T; transformed into JM109, and sent to Shanghai Sangon for correct sequencing of pUCm-T-AEx11A after enzyme digestion and identification C5T Both the pPIC9K plasmid and the pPIC9K plasmid were digested with EcoR I and Not I, and the recovered digested products were ligated unde...

Embodiment 3

[0030] Embodiment 3GS115 / AEx11A C5T Construction, expression, product purification and activity determination of

[0031] pPIC9K-AEx11A with Sal I C5T Perform linearization, perform electrotransformation and screening according to the Pichia expression manual, and obtain high-copy Pichia recombinant GS115 / AEx11A C5T ; The engineered bacteria were induced with 1.0% methanol for 72 hours, and the mutant xylanase activity in the fermentation broth was basically consistent with that of AEx11A as measured by DNS method. The supernatant after centrifugation of the fermentation broth is the mutant AEx11A C5T The crude enzyme solution was concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10kDa, and then purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. After purification, a single band was detected by SDS-PAGE, And it shows that the molecular weight of the mutant xylanase is basically the s...

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Abstract

The invention proposes a method for heat-resistant analysis on hybrid xylanase AEx11A from 11 family of Aspergillus oryzae. According to the invention, a disulfide bond (cys5-cys32) is introduced at the N end of AEx11A by homologous modeling of an AEx11A structure and comparison between the AEx11A structure and an AoXyn11A structure; 5-site cysteine is subjected to mutation to form threonine (C5T) by a site-directed mutagenesis method, and the disulfide bond is removed to discuss the effect of the disulfide bond on the thermal stability of the AEx11A. The result shows that Topt of the mutant enzyme (AEx11AC5T) is reduced to 60 DEG C from 75 DEG C before mutation, and t1/270 and t1/280 are respectively shortened to 3.0 minutes and 1.0 minute from 197 minutes and 25 minutes before mutation, thus the N-end disulfide bond has a certain influence on the 11 family of xylanase, and a theoretical basis can be established for clarification of a thermal stability mechanism of the 11 family of xylanase and transformation of heat-stable protein engineering.

Description

technical field [0001] The present invention relates to the homology modeling of the AEx11A structure derived from the 11 family thermostable heterozygous xylanase and its comparison analysis with the AoXyn11A structure, the mutant xylanase AEx11A C5T The construction of engineering bacteria and the high-efficiency expression, purification and activity determination methods of mutant xylanase belong to the technical field of bioengineering. Background technique [0002] Xylanases (EC 3.2.1.8) are an important class of industrial enzymes. It mainly acts on the main chain of xylan, randomly cuts the xylosidic bonds inside xylan, and the hydrolyzed products are mainly xylooligosaccharides with different degrees of polymerization and a small amount of xylose, which is the most critical xylan degrading enzyme enzymes. In recent years, due to the potential industrial application and economic value of xylanase in the feed industry, food processing and brewing industries, especial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/81C12N1/19C12R1/84
Inventor 邬敏辰余涛殷欣李剑芳
Owner JIANGNAN UNIV
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