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Biosensing elements based on specific binding of cbm to cellulose

A technology of biosensing and sensing elements, applied in the fields of biological enzyme genetic engineering and biosensing

Active Publication Date: 2022-05-27
BIOLOGY INST OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Utilizing the specific affinity adsorption properties of CBM for cellulose to construct fusion enzymes, and using nanocellulose membrane as the immobilization carrier of fusion enzyme molecules, the transformation strategy to improve the stability of enzyme electrode sensors has hardly been reported at home and abroad.

Method used

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  • Biosensing elements based on specific binding of cbm to cellulose
  • Biosensing elements based on specific binding of cbm to cellulose
  • Biosensing elements based on specific binding of cbm to cellulose

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1, the fusion expression of CBM and GOD

[0027] CBM1, CBM2, CBM3, CBM5, and CBM10 families with binding specificity to cellulose were selected, and the amino acid frequencies and functions of different CBMs were analyzed using structural bioinformatics analysis tools (SWISS-MODEL, ClustalX, VMD and PyMO1 software). Statistical analysis was carried out on the information such as the architecture sequence spectrum, and the CBM2 in Thermobifida fusca was screened out for the construction of the hybrid enzyme.

[0028]Based on the analysis of the structural characteristics of CBM2 and GOD, the fusion enzyme molecule linker sequences ((GGGGS)2, (EAAAK)3) were selected from the LinkerDB database, and the endo-β-xylanase (EM_PRO:Z81013. 1) The natural linker sequence connecting the catalytic domain and the CBM2 domain (LGGDSSGGGPGEPGGPGGPGEPGGPGGPGEPGGPGDGT); the fusion protein structure with different linker connections or no linker connection is modeled by the FP...

Embodiment 3

[0047] Example 3. Morphology and composition detection of GOD-NL-CBM2 / cellulose membrane

[0048] Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS) and Infrared Spectroscopy (FTIR) were used to analyze the morphological and compositional characteristics of cellulose membrane (a mixture of nitrocellulose and cellulose acetate) without or with glucose oxidase. The results show that the three-dimensional structures formed by cellulose nanofibers have pores of different micron and nanometer sizes, and the nanofibers with a diameter of about 200 nm are also porous ( image 3 a1). When GOD reacted with the cellulose membrane, the diameter of the nanofibers increased, which may be due to the swelling effect of the buffer, but the pores in the nanofibers were still obvious and the size did not change significantly ( image 3 b1). In contrast, when GOD-NL-CBM2 interacted with the cellulose membrane, a clear morphological change could be observed, and the surfac...

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Abstract

The invention utilizes the specific affinity adsorption properties of CBM to cellulose to construct a fusion protein, the fusion protein includes glucose oxidase and CBM, and then uses the nanocellulose membrane as an immobilized carrier of the fusion protein to prepare a biosensing element, and the obtained The biosensing element can exhibit good catalytic properties and detection stability.

Description

technical field [0001] The invention relates to the linking of an oxidoreductase gene and a carbohydrate binding module (CBM) gene through a linker gene to construct an oxidoreductase-linker-CBM fusion enzyme, and using the fusion enzyme to prepare a nanocellulose carrier membrane specific binding A biosensing element belongs to the field of biological enzyme genetic engineering and biosensing technology. Background technique [0002] Oxidoreductase is a key detection sensitive element in biosensors. It is effectively immobilized on the surface of the carrier and can maintain its active state for a long time and efficiently. It is an important guarantee for the high stability and high sensitivity of biosensors. The immobilization of enzyme molecular elements on the electrode surface usually uses physical adsorption or covalent bonding to randomly immobilize enzyme molecules on a carrier membrane with an area of ​​less than one square centimeter. However, the biologically ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/81C07K17/12G01N27/327
CPCC12N9/0006C12Y101/03004C07K14/00C12N15/815C07K17/12G01N27/3277G01N27/3278C07K2319/00G01N33/0098C12N9/2437
Inventor 公维丽马耀宏蔡雷王丙莲孟庆军刘庆艾郑岚杨艳杨俊慧李一苇史建国韩庆晔
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI