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Recombinant cellulase with primary product cellotetrose and building method and application thereof

A cellotetraose and cellulase technology, applied in the field of enzyme engineering, can solve the problems of high production cost and complicated purification process, and achieve the effects of high concentration, high purity and high enzyme activity

Active Publication Date: 2019-08-02
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current production of cellotetraose uses chemical synthesis methods, and the purification process is complicated, making its production cost very expensive

Method used

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  • Recombinant cellulase with primary product cellotetrose and building method and application thereof
  • Recombinant cellulase with primary product cellotetrose and building method and application thereof
  • Recombinant cellulase with primary product cellotetrose and building method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Obtaining the cellulase that continuously produces and accumulates cellotetraose includes the following steps:

[0047] 1) Homology modeling and molecular docking: Using the three-dimensional structures of Cel9G (PDB: 1GA2) in Clostridium cellulolyticum and Cel9A (PDB: 4TF4) in Thermobifida fusca as templates, use Discovery Studio 4.0 Construct the structural model of the target sequence; then use Pymol 1.7 (http: / / www.pymol.org) to use the cellulose single chain (the cellulose single chain is a cellulose chain greater than 6 glucose units, which can fill the active center of the model) Molecular docking was carried out between the cellulose single chain and the structure model of the above obtained enzyme molecule, and the enzyme with the same structural domain as CcCel9A was obtained.

[0048] And in the obtained model, the active center of the enzyme molecule and the amino acid site where the CBM region binds to the substrate are marked.

[0049] 2) Selection of mutat...

Embodiment 2

[0107] Example 2: Enzymatic hydrolysis of amorphous cellulose by point mutants

[0108] After expressing and purifying the point mutant protein obtained from the above construction, it was added to the cellulose substrate for the production of cellotetraose, and the wild type was also used as a control. The reaction system was as follows: Add to 10g / L amorphous cellulose Enzyme with a final concentration of 1 μM and a total volume of 5 mL were incubated at 60° C. for 48 hours. After the protein was inactivated, the supernatant was collected by centrifugation for detection of product spectrum.

[0109] Among them, the product map analysis of point mutants: add 3 μM of the above-mentioned purified enzyme solution obtained with different point mutations to 50 μl of 1% RAC solution, then add PC buffer to a total volume of 100 μl, and place at 60° C. for reaction. Samples were taken at 5 minutes, 10 minutes, 20 minutes, 40 minutes, and 120 minutes, and the volume of each sample was...

Embodiment 3

[0112] Example 3: Point mutant proteins participate in the compounding of cellulase preparations

[0113]After expressing and purifying the constructed point mutant protein H372A / Y415A, it was compounded with Novozymes cellulase preparation Cellic CTec2, and mixed in 10g / L corn stover after gas explosion treatment at a molar ratio of 1:5 Add the above compounded enzyme at a final concentration of 5 μM to a total volume of 5 mL, and incubate at 60° C. for 48 h. Take the sample without point mutant protein H372A / R374A as a control, and take the same treatment as the experimental group. After inactivating the protein, the supernatant was centrifuged to detect the product. The results showed that the yields of cellooligosaccharides in the experimental group were 30% and 40% respectively compared with the control group. Sugar production increased by 10%. It can be seen that mixing the enzymes obtained in the above examples with existing enzymes for degradation can produce a syner...

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Abstract

The invention belongs to the field of enzyme engineering, and particularly relates to recombinant cellulase with a primary product cellotetrose and a building method and application thereof. A targetside, which adjusts and controls mutation of the primary product cellotetrose, of persistent endo-cellulase CcCe19A is one or more of H123, H372, R374 and Y415. The endo-cellulase can be fast expressed in escherichia coli, and is high in enzyme activity and stability so that the endo-cellulase and other enzymes can be compounded for cellulase hydrolysis. The cellotetrose generated by means of a cellulase substrate is high in concentration and purity, and can be used as a standard substance for scientific research.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, in particular to a recombinant cellulase whose main product is cellotetraose and its construction method and application. Background technique [0002] Cellotetrose, the chemical structure is (β-D-Glc-[1→4]) 3 -D-Glc, composed of 4 glucose units connected by β-1,4 glycosidic bonds, is mainly used for standards. Since lignocellulose is the most abundant renewable biomass resource on the earth, the research on cellulase has been unabated for many years. Cellooligosaccharides are often necessary in the study of cellulase, especially in the study of the enzyme's minimal substrate and product profiles. In addition, cellotetraose can promote the proliferation of bifidobacteria in the intestinal tract of humans and animals, and is beneficial to intestinal peristalsis, but it will not be absorbed by the intestinal tract. The sweetness of cellotetraose is about 30% to 40% of that of sucrose, so it can...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12P19/14C12N15/70C12N1/21C12N15/11C12R1/19
CPCC12N9/2437C12P19/14C12N15/70
Inventor 李福利张坤迪王禄山
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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