Carbohydrate binding module CBM6B protein capable of specifically recognizing xanthan gum side chains and application

A carbohydrate and protein technology, applied in the biological field, can solve problems that have not yet been tried, and achieve the effect of improving affinity and catalytic ability, and improving enzyme activity.

Pending Publication Date: 2021-03-12
李宪臻
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no one has attempted the fusion expression of CBM and carbohydrate-active enzymes to efficiently hydrolyze xanthan gum

Method used

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  • Carbohydrate binding module CBM6B protein capable of specifically recognizing xanthan gum side chains and application
  • Carbohydrate binding module CBM6B protein capable of specifically recognizing xanthan gum side chains and application
  • Carbohydrate binding module CBM6B protein capable of specifically recognizing xanthan gum side chains and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of carbohydrate binding module CBM6B expression vector

[0041] A. Extraction of Microbacterium sp.XT11 genomic DNA (CTAB / NaCl method)

[0042] Microbacterium sp.XT11 (the xanthan gum-degrading bacterium XT11 screened from the soil by our laboratory has the number AB2016011 of the China Center for Type Culture Collection, and the accession number of the genome sequence in the DDBJ / EMBL / GenBank database is CP013859).

[0043]Collect 10 mL of fresh Microbacterium XT11 seed liquid by centrifugation at 4°C, and resuspend and wash the cells with pure water. The cells were lysed with 20 μL of lysozyme at a concentration of 10 mg / mL, added with 1 mL of 1×TE, mixed gently, and reacted at 37 °C for 1 h. After the reaction, the cells were transferred to a new 2 mL imported centrifuge tube, 10 μL of 10 mg / mL proteinase K and 60 μL of 10% SDS were added in sequence, mixed well, and reacted at 37°C for 1 h.

[0044] Add 100 μL of 5M NaCl and 80 μL of CTAB / Na...

Embodiment 2

[0050] The construction of the fusion expression vector of embodiment 2 carbohydrate activity enzyme CtCel8A and CBM6B

[0051] A high-activity cellulase was selected as the object of transformation, and it was fused with the substrate binding domain CBM6B to construct a new type of xanthan endonuclease.

[0052] A. Using PCR technology to amplify the coding gene of endocellulase CtCel8A

[0053] The codon-optimized sequence of the high-activity endocellulase CtCel8A from Clostridium thermocellum (genebank: CP016502.1) synthesized by the whole gene is shown in SEQ ID NO.5. The specific reaction system and reaction conditions of PCR are as follows: 150ng pUC57-CtCel8, 30 μL 5×PrimeSTAR Buffer, 15 μL dimethyl sulfoxide (DMSO), 30 μM each of upstream and downstream primers (upstream primer sequence such as SEQ ID NO.6, downstream primer sequence As shown in SEQ ID NO.7), 12 μl of 2.5mM dNTPs, 3.75U PrimeSTAR DNA polymerase, and 150 μL of pure water. The reaction parameters were...

Embodiment 3

[0057] The construction of the fusion expression vector of embodiment 3 carbohydrate activity enzyme MiXen and CBM6B

[0058] Select the catalytic domain sequence MiXen-CD of the xanthan endonuclease MiXen from the xanthan gum degrading bacteria Microbacterium sp.XT11, see SEQ ID NO.8 (genebank: LX1-1GL001095), and express it in fusion with the substrate binding domain CBM6B , to construct a novel xanthan endonuclease.

[0059] A. Using PCR technology to amplify the coding gene of carbohydrate binding module CBM6B

[0060] The specific reaction system and reaction conditions of PCR are as follows: 150ng pET28a-CBM6B, 30 μL 5×PrimeSTARBuffer, 15 μL dimethyl sulfoxide (DMSO), 30 μM each of the upstream and downstream primers (the sequence of the upstream primer is as SEQ ID NO.9, and the sequence of the downstream primer is as SEQ ID NO.9). ID NO.10), 12μl 2.5mM dNTPs, 3.75U PrimeSTAR DNA polymerase, and 150μL of pure water. The reaction parameters were: pre-denaturation at 95...

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Abstract

The invention discloses a carbohydrate binding module CBM6B protein capable of specifically recognizing xanthan gum side chains and application, and belongs to the technical field of biology. According to the invention, a CBM6B gene is cloned from Microbacterium sp. XT11 by using a modern molecular biological means, and then a carbohydrate active enzyme and a fusion expression vector are constructed, and the carbohydrate active enzyme and the fusion expression vector are transformed into Escherichia coli to carry out high-level expression on the fusion enzyme. The CBM6B can improve the affinity and catalytic ability of recombinase and substrate xanthan gum, and lays a foundation for efficiently and accurately cutting xanthan gum and preparing xanthan gum oligosaccharide.

Description

technical field [0001] The invention relates to a carbohydrate binding module CBM6B protein that specifically recognizes the side chain of xanthan gum and its application, belonging to the field of biotechnology. Background technique [0002] As the first mass-produced biopolymer since 1969, xanthan gum has many excellent properties and is widely used. Its degradation product, xanthan gum oligosaccharide, has been proved to be a new type of functional oligosaccharide integrating antibacterial, antioxidative, antitumor and other activities, and has broad application prospects in food, medicine and other fields. Therefore, it is imminent to find a method that can efficiently and accurately degrade xanthan gum. At present, the degradation of xanthan gum is mainly realized by physical, chemical and biological methods. For the preparation of oligosaccharides, the physical method requires high equipment and consumes a lot of energy, making it difficult to achieve large-scale ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N15/62C12N1/21C12P19/00C12P19/04C12R1/19
CPCC12N9/88C12Y402/02012C12N15/70C12P19/00C12P19/04C07K2319/00
Inventor 杨帆王雪妍李宪臻
Owner 李宪臻
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