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Continuous endo-cellulase mutant and application thereof

A technology of endo-cellulase and mutants, applied in the field of genetic engineering, can solve the problem of unclear catalytic mechanism of crystalline cellulose and achieve the effect of improving enzyme activity

Active Publication Date: 2022-03-29
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few types of persistent endocellulase, and the catalytic mechanism for crystalline cellulose is still unclear, so improving its catalytic efficiency is still a huge challenge.

Method used

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  • Continuous endo-cellulase mutant and application thereof
  • Continuous endo-cellulase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Determination of key amino acid residues in the framework of the processive endocellulase EG5C-1 enzyme activity (1) Homology modeling: BLAST analysis was performed according to the NCBI and PDB databases, and the catalytic structure of BsCel5A derived from B.subtilis 168 was used The crystal structure of the domain (PDB ID: 3PZT) was used as a template, and its sequence was 99% similar to the original enzyme EG5C-1 amino acid sequence SEQ ID NO: 1. The three-dimensional structure of EG5C-1 was constructed using the MODELER program of Discovery Studio 3.5 (DS 3.5), and the final quality analysis of the constructed model was carried out using the PROCHECK and Profile-3D programs. The visualization and analysis of the model were performed using Discovery Studio 2018Client and MOE2019.01 software.

[0029](2) Molecular docking: extract the cellohexaose structure from the endoglucanase GH48 (PDB ID: 5CVY) of B. pumilus, and use Autodock 4.2 software to dock it to ...

Embodiment 2

[0031] Example 2 Construction of the Processive Endocellulase EG5C-1 Mutant

[0032] The key amino acid residues determined in Example 1 were mutated to alanine, and the method disclosed by BinWu (Biotechnology for Biofuels, 2018, 11:20) was used to construct a recombinant plasmid containing SEQ ID NO: 2, which was represented by SEQ ID NO: 2 The sequence shown is a template. Refer to Vazyme biological products and operation manuals, and use the corresponding mutation primers designed as follows to amplify the site-directed mutation sequence from the whole plasmid. The primers used are as follows:

[0033]

[0034]

[0035] The PCR reaction system is as follows:

[0036]

[0037] The PCR program was set as follows:

[0038] 95℃, 3min;

[0039] 95℃, 15s; 60℃, 15s; 72℃, 8min; 30 cycles;

[0040] 72°C, 10min;

[0041] 4°C, Hold.

[0042] After the amplification of the whole plasmid was completed, 2 μL of the PCR product was taken for nucleic acid electrophoresis ver...

Embodiment 3

[0064] Example 3 Screening procedure for optimal mutants of persistent endocellulase

[0065] With the 23 mutants obtained in embodiment two, taking carboxymethylcellulose sodium CMC and microcrystalline cellulose Avicel as the substrate measurement enzyme activity change, the alanine mutation of key amino acid is to CMC (A) and Avicel (B) ) The results of the relative hydrolysis activity of the substrate are as follows image 3 shown. image 3 It was shown that the activities of most residues were significantly decreased for CMC and Avicel substrates after substitution of alanine. Among them, mutations of aromatic amino acids and polar amino acid residues are particularly significant. The D70, T177 and S235 sites have little effect on the alanine mutation, therefore, the four sites D70, T177, A234 and S235 were selected as the hotspot residues for further modification.

[0066] Referring to the method in Example 2, site-directed saturation mutation was performed on the fou...

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Abstract

The invention relates to a persistent endo-cellulase mutant with remarkably improved enzyme activity and application thereof. The mutant comprises 70th and / or 235th amino acid mutation of an amino acid sequence, and the amino acid sequence of the endo-cellulase is as shown in SEQ ID NO: 1. A single-point mutation technology is adopted, based on homologous modeling and molecular docking methods, key amino acids in an enzyme activity framework are selected to optimize the molecular structure of the persistent endo-cellulase, and the enzyme activity is improved. Compared with a wild type enzyme, the content of reducing sugar generated by degrading filter paper by the continuous endo-cellulase mutant is greatly increased, and the continuous endo-cellulase mutant has important significance on efficient degradation of a cellulose substrate and reduction of the production cost.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a persistent endo-cellulase mutant with improved enzyme activity and application thereof. Background technique [0002] Cellulose is the most widely distributed and abundant renewable resource on the earth, and its efficient biotransformation is of great significance to the sustainable development of human society. The complete degradation of cellulose requires the synergistic action of a series of enzymes such as endoglucanase, exoglucanase and β-glucosidase. The problem of too high is still an important bottleneck restricting the commercialization of cellulose conversion technology. [0003] Processive endoglucanase (Processive endoglucanase) is a newly discovered bifunctional cellulolytic enzyme, which not only has the action characteristics of an endoenzyme, but also has the sustained catalytic degradation ability of an exoenzyme, and can efficiently degrade C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/14C12R1/19
CPCC12N9/2437C12N15/70C12P19/14C12Y302/01004
Inventor 吴斌吕科旻高振童心陈杰王子露张辉马江锋何冰芳
Owner NANJING UNIV OF TECH
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