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Construction method of in-vitro artificial scaffold protein mediated trehalose multienzyme complex

A complex and scaffolding technology, applied in the biological field, can solve the problems that have not been expanded to non-cellulase, and achieve the effects of reducing production costs, reducing waste, improving utilization and catalytic efficiency

Pending Publication Date: 2021-03-30
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, the specific interaction of cohesin-dockerin is mostly applied to the degradation of cellulose, and has not been extended to other fields other than cellulase

Method used

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  • Construction method of in-vitro artificial scaffold protein mediated trehalose multienzyme complex
  • Construction method of in-vitro artificial scaffold protein mediated trehalose multienzyme complex
  • Construction method of in-vitro artificial scaffold protein mediated trehalose multienzyme complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Construction of recombinant strain WB800n-ScafCCR

[0152] (1) Clone the ScafCCR gene fragment

[0153] Using the ScafCCR bacterial liquid synthesized by Shanghai Sangong Company as a template, design primers for PCR amplification of the Rfcoh-Ctcoh-CBM-Cccoh gene fragment: using the Bacillus subtilis WB800n genome as a template, design primers for PCR amplification of the gene fragment of the P43 promoter, The gene fragment of phoD signal peptide, the gel electrophoresis picture is as follows Figure 5 shown;

[0154] The PCR primers of the ScafCCR protein gene sequence are as follows:

[0155] ScafCCR-F:5'-GGGGCCTTTGAAGTAATGACAACAACAGGCGGC-3'SEQ ID NO.10;

[0156] ScafCCR-R: 5'-CGACTCTAGAGGATCCTTAATGATGGTGATGATGATGTTGTGTGC-3' SEQ ID NO.11.

[0157] The PCR reaction system is as follows:

[0158] 2×Phanta Max Master Mix 25 μL, 10 μmol / L upstream primer ScafCCR-F 2.5 μL, 10 μmol / L downstream primer ScafCCR-R 2.5 μL, template 2.5 μL, use ddH 2 O supplemented to 50 ...

Embodiment 2

[0198] Construction of recombinant strain WB800n-P43-phoD-treY-Ccdoc

[0199] (1) Clone the treY and Ccdoc gene fragments of the maltooligosaccharyl trehalose synthase (MTSase) gene fragment

[0200] Using the genome of Bacillus subtilis WB800n as a template, primers were designed to amplify the gene fragment of the P43 promoter and the gene fragment of the phoD signal peptide by PCR, and the genome of Sulfolobus acidocaldarius ATCC 33909 was used as a template to amplify maltooligosaccharides by PCR Based on the trehalose synthase (MTSase) treY gene fragment, using the Sase2-Ccdoc bacterial liquid synthesized by Shanghai Sangong Company as a template, primers were designed for PCR amplification of the docking protein Ccdoc gene fragment, and the gel electrophoresis diagram is as follows Figure 5 shown.

[0201] The PCR primers of the P43 promoter gene sequence are as follows:

[0202] P43-F: 5'-AGTGAATTCGAGCTCAGCTTCGTGCATGCAGGCCGG-3'SEQ ID NO.6;

[0203] P43-R: 5'-TCAAAAC...

Embodiment 3

[0251] Construction of recombinant strain WB800n-P43-phoD-treZ-Ctdoc

[0252] (1) Clone the maltooligosaccharide-based trehalose hydrolase (MTHase) gene fragment treZ and Ctdoc gene fragment

[0253] Using the Escherichia coli strain P43-phoD-MTHase ​​genome constructed in our laboratory as a template, primers were designed to amplify the gene sequence of phoD-maltooligosaccharide-based trehalose (MTHase) treZ by PCR. Template, design primers for PCR amplification of the docking protein Ctdoc gene fragment, the gel electrophoresis picture is as follows Figure 5 shown;

[0254] The PCR primers of the phoD-treZ gene sequence are as follows:

[0255] phoD-Hase-F:5'-GAATTAATAACAGAAGCTTATGGCATACGACAGTCGTTTTGATG-3'SEQ ID NO.17;

[0256] phoD-Hase-R: 5'-TGCCCGGAACTTTATACGTTTCTAATTGATATACCCCAACACCT-3' SEQ ID NO. 18.

[0257] The PCR reaction system is as follows:

[0258] 2×Phanta Max Master Mix 25 μL, 10 μmol / L upstream primer phoD-Hase-F 2.5 μL, 10 μmol / L downstream primer pho...

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Abstract

The invention relates to a construction method of an in-vitro artificial scaffold protein mediated trehalose multienzyme complex. The construction method mainly comprises the following steps: constructing a self-assembled scaffold protein module recombinant bacterium WB800n-ScafCCR; constructing a recombinant bacterium WB800n-P43-phoD-treYC-cdoc of a self-assembled catalytic module; constructing arecombinant bacterium WB800n-P43-phoD-treZ-Ctdoc of a self-assembled catalytic module; constructing a recombinant bacterium WB800n-P43-phoD-cgt-Rfdoc of a self-assembled catalytic module; performingsecretory expression on the recombinant bacteria, performing self-assembly in vitro, and obtaining a recombinant trehalose multi-enzyme complex; the efficiency of preparing trehalose by catalyzing thetrehalose multi-enzyme complex is higher than the catalytic efficiency of mixed free enzymes; the cellulose microspheres can be used for producing high-quality trehalose after being immobilized.

Description

technical field [0001] The invention relates to a construction method of an in vitro artificial scaffold protein-mediated trehalose multienzyme complex, belonging to the field of biotechnology. Background technique [0002] Trehalose is a non-reducing disaccharide ubiquitous in nature. It is connected by glucose residues through α-1,1 glycosidic bonds. It is an excellent natural desiccant and preservative. polysaccharides. Under harsh environmental conditions such as high temperature, high cold, high osmotic pressure and dehydration, a unique protective film can be formed on the cell surface to effectively protect protein molecules from denaturation and inactivation, thereby maintaining the life process and biological characteristics of living organisms. This unique functional property makes trehalose an excellent active protectant for protein drugs, enzymes, vaccines and other biological products. The special biological properties of trehalose make it widely used in the f...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/66C12N15/62C12N1/21C12N11/18C12P19/24C12P19/18C12P19/14C12P19/12C12R1/125
CPCC12N15/75C12N15/52C12N9/90C12N9/2408C12N9/1051C12N11/18C12P19/24C12P19/18C12P19/14C12P19/12C12Y504/99015C12Y302/01141C07K2319/735C07K2319/02C12N11/12C12Y204/01C12N15/03
Inventor 王腾飞张欣怡刘洪玲王瑞明
Owner QILU UNIV OF TECH
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