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Fusion protein for cellulose accessibility measurement and application thereof

A fusion protein and cellulose technology, which is applied in the direction of fusion with spectrum/fluorescence detection, measurement devices, enzymes, etc., can solve the problems of large measurement deviation, poor repeatability, time-consuming and laborious measurement process, etc., to improve accuracy and avoid changes Effect

Inactive Publication Date: 2017-02-22
TSINGHUA INNOVATION CENT IN DONGGUAN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing technology for measuring cellulose accessibility has the problems that the measured material needs to be dried, the measurement deviation is large, the measurement process is time-consuming and laborious, and the repeatability is poor.

Method used

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  • Fusion protein for cellulose accessibility measurement and application thereof
  • Fusion protein for cellulose accessibility measurement and application thereof
  • Fusion protein for cellulose accessibility measurement and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Construction of recombinant plasmid pET28a-GFP2-CBM in Escherichia coli

[0036] According to the sequences of GFP gene and CBM gene in Genebank, two gene sequences of GFP and CBM fusion protein were designed and synthesized on the pUC57-simple vector. According to the gene sequence of the fusion protein GFP2-CBM and the characteristics of the multiple cloning site on the Escherichia coli expression vector pET28a, the following two primers were designed and synthesized:

[0037] GFP_Fe: 5'-GACgaattcATGCGTAAAGGCGAAGAGCTGTTC-3'

[0038] CBM_Rh:5'- GACaagcttTTACAAGCACTGAGAGTAGTAAGG-3'

[0039] EcoRI and HindIII restriction sites were designed at the two ends of GFP_Fe and CBM_Rh, respectively, and are indicated in lowercase fonts. Using GFP_Fe, CBM_Rh primers, using the recombinant plasmid pUC57-simple-GFP2-CBM constructed in our laboratory as a template, the GFP2-CBM gene was amplified by PCR. PCR conditions: pre-denaturation at 95°C for 5 minutes; then 30 cycles of de...

Embodiment 2

[0042] Expression of fusion protein GFP2-CBM

[0043] The recombinant plasmid pET28a-GFP2-CBM was transformed into Escherichia coli expression strain BL21, and the bacterial solution was spread on LB plates containing 50 μg / ml Kanamycin for resistance screening, single clones were picked for PCR detection, and positive clones were selected.

[0044] Pick the recombinant Escherichia coli expression strain BL21-pET28a-GFP2-CBM, inoculate it in 5 ml LB medium, and cultivate it overnight at 37°C on a shaker at 200 rpm. Then take 1 ml of the bacterial liquid and transfer it to 100 ml of LB medium, and culture it on a shaker at 37°C and 200 rpm to OD 600 0.6, add IPTG to a final concentration of 1 mM, and culture at 30°C for 6 h on a shaker at 200 rpm. The bacteria were collected by centrifugation, supernatant was obtained by ultrasonic crushing, purified by Ni-NTA medium column, and dialyzed to remove salt. The final protein yield was 100 mg / L bacterial liquid. After SDS-PAGE ele...

Embodiment 3

[0046] Example 3 Recognition of pure cellulose by fusion protein

[0047] The fusion protein GFP-GFP-CBM was obtained according to the steps described in Examples 1 and 2. And use similar steps to obtain the fluorescent protein GFP-GFP without CBM. Two proteins, GFP-GFP-CBM and GFP-GFP, were respectively adsorbed on filter paper and microcrystalline cellulose as substrates at 37 °C and 200 rpm for 2 h, and the adsorbed filter paper cellulose was observed under a fluorescence microscope (attached figure 1 ) and microcrystalline cellulose (with figure 2 ), it can be found that due to the recognition of CBM on cellulose, the GFP-GFP-CBM fusion protein can be effectively adsorbed on the cellulose, while the GFP-GFP fusion protein does not have this function, so it is not obvious under the fluorescence microscope fluorescence phenomenon.

[0048] Result analysis: The results of this example show that the fusion protein provided by the present invention can obtain visual analysi...

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Abstract

The invention provides a fusion protein for cellulose accessibility measurement and an application thereof. According to the invention, through fusion of recognition functional components of fungus cellulase to a cellulose substrate with a fluorescent protein, the accessibility of cellulose to the fungus cellulase is accurately measured. The fusion protein provided by the invention has a molecular size similar to the molecular size of a key component, namely exoglucanase, of the fungus cellulase, can specifically recognize the cellulose substrate, can obtain fluorescence signals for visual analysis and quantitative measurement, can be used for measuring the accessibility of a substrate under the water-containing state, and avoids needing the step of drying of the substrate in a traditional method for analyzing a pore structure of a solid substance, thereby being able to obtain a more accurate measurement result.

Description

technical field [0001] The invention relates to the field of biochemical industry, in particular to a fusion protein used for measuring the accessibility of cellulose and its application. Background technique [0002] Due to the increasingly serious shortage of fossil resources and the increasingly serious environmental pollution, the production of bioenergy and industrial chemicals from a wide range of sources and renewable lignocellulose has attracted widespread attention from all over the world. Among them, the biotransformation of lignocellulose with biotechnology as the core has become one of the key technologies in bioenergy development, biomass resource processing and green chemical process, and related research has also become a hot spot and difficulty in current industrial microbial technology. However, plants have formed a complex lignocellulose structure to defend against the attack of animals and microorganisms during the long-term evolution process, and the vari...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70G01N21/64
CPCG01N21/6428C12N9/2437C12N15/62C12N15/70C12Y302/01091C07K2319/60Y02E50/10
Inventor 赵雪冰李恬欧先金刘德华
Owner TSINGHUA INNOVATION CENT IN DONGGUAN
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