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Cellulosome with improved catalytic activity and assembly method and application thereof

A technology for improving catalytic activity and cellulosomes, applied in chemical instruments and methods, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problem of low catalytic activity of artificial cellulosomes, and achieve synergistic catalytic effect, The effect of reducing consumption and production cost and good application prospect

Pending Publication Date: 2021-03-19
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on displaying cellulosomes on the cell surface of Saccharomyces cerevisiae has achieved certain research results, but the catalytic activity of artificial cellulosomes is still not high.

Method used

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  • Cellulosome with improved catalytic activity and assembly method and application thereof
  • Cellulosome with improved catalytic activity and assembly method and application thereof
  • Cellulosome with improved catalytic activity and assembly method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Fusion of Lignocellulose Degrading Enzyme Gene and Docking Protein Gene

[0037] The coding genes of docking proteins Doc1 and Doc2 were derived from R. flavefaciens (GenBank: WP_009985128) and R. flavefaciens (GenBank: 5M2O-B), respectively, and the nucleotide sequences were optimized according to the codon preference of E. coli, Doc1 and Doc2 The nucleotide sequences of Doc1 and Doc2 are shown in SEQ ID NO.1 and SEQ ID NO.2, and the amino acid sequences of Doc1 and Doc2 are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0038] The coding genes of Doc1 and Doc2 were respectively fused to the C genes of xylanase AExynM (GenBank: HQ724284.1) and glucanase EG1 (GenBank: AF329732) with high catalytic activity preserved in the laboratory by overlapping PCR. At the end, the primer sequences are shown in Table 1, and the specific steps are as follows:

[0039] Use the pET-32a(+)-AExynM preserved in this experiment as a template, and use Xyn-F and Xyn-R as primers for the first roun...

Embodiment 2

[0044] Induced expression of fusion genes

[0045] Correctly sequenced Escherichia coli recombinants pET-32a(+)-AExynM-Doc1 and pET-32a(+)-EG1-Doc2 and BL21-pET-32a(+)-AExynM and BL21-pET-32a preserved in the laboratory (+)-EG1 was subjected to IPTG induction culture respectively: inoculate the strains in LB medium respectively, cultivate overnight as the seed medium, inoculate 1% into 30mL LB medium, and cultivate at 37°C and 200rpm until the cell OD 600 =0.6~0.8, add inducer IPTG to a final concentration of 0.8mmol / L, induce culture at 28°C and 200rpm for 4~6h.

[0046] Take 30mL of the above induced culture, centrifuge at 8,000rpm for 10min, collect the bacteria, and use Na 2 HPO 4 - Citrate buffer (pH 5.5) containing 10mM CaCl 2 , wash the precipitate, and ultrasonically disrupt the cells in an ice bath, centrifuge at 15,000 rpm for 10 minutes, collect the supernatant, which is the crude enzyme solution, and perform enzyme activity determination and SDS-PAGE detection....

Embodiment 3

[0051] Cohesin display on the surface of Saccharomyces cerevisiae

[0052] 1) Construction of recombinant expression vector pYD1-Coh1-Coh2

[0053] The coding genes of cohesion proteins Coh1 and Coh2 are derived from R. flavefaciens (GenBank: AM262974) and R. flavefaciens (GenBank: 5M2O-A) respectively, and the specific sequences are obtained by artificial synthesis, and a connecting peptide is inserted between the two GGGGSGGGGSGGGGS, the nucleotide and amino acid sequences of Coh1-Coh2 are shown in SEQ ID NO.5 and SEQ ID NO.10, and this sequence is inserted between the EcoRI and XhoI sites of the surface display plasmid pYD1 to obtain pYD1-Coh1- Coh2.

[0054] 2) Surface display of cohesin Coh1-Coh2

[0055] The lithium acetate chemical method was used to transform pYD1-Coh1-Coh2 into competent cells of Saccharomyces cerevisiaeEBY100, and the yeast recombinants were screened on a tryptophan selection plate, and the genomic DNA of the recombinants was extracted. Using this...

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Abstract

The invention discloses a cellulosome with improved catalytic activity and an assembly method and application thereof, and belongs to the technical field of biology. According to the assembly method,xylanase AExynM-Doc1 containing a docking domain and glucanase EG1-Doc2 containing a docking domain are expressed heterologously by using escherichia coli; fibronectin Coh1-Coh2 are surface-displayedby using saccharomyces cerevisiae, to obtain a recombinant yeast EBY100 / Coh1-Coh2; and the recombinant proteins AExynM-Doc1 and EG1-Doc2 are mixed with the recombinant yeast EBY100 / Coh1-Coh2, and arerespectively bound with the Coh1 and the Coh2 to finish in-vitro assembly of the cellulosome. According to the invention, the xylanase and glucanase with high catalytic activity are combined in scaffold protein of the cellosome, so that the synergistic catalytic action between the two enzymes is greatly improved, and lignocellulose can be more effectively degraded.

Description

technical field [0001] The invention relates to a cellulosome with improved catalytic activity and its assembly method and application, belonging to the field of biotechnology. Background technique [0002] Lignocellulose accounts for about 50% of the total biomass on the earth, but most of it is burned or discarded, which not only causes environmental pollution, but also wastes resources. Using modern biotechnology to convert it into bioethanol or chemical raw materials can not only free human beings from dependence on fossil fuels, but also reduce environmental pollution and promote sustainable development of society. The key to bioconversion of lignocellulose is to degrade cellulose or hemicellulose into fermentable sugars. In traditional processes, it is usually completed by the synergy of a variety of commercial enzymes, and the cost of enzyme preparations can account for production costs. 20%, which seriously hinders the development of lignocellulose bioconversion. T...

Claims

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Application Information

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IPC IPC(8): C12N11/18C12N11/16C12N9/42C12N9/24C07K19/00C12N15/81C12P19/14C12P19/00C12R1/865
CPCC12N11/18C12N11/16C12N9/2482C12N9/2437C07K14/195C12N15/81C12P19/14C12P19/00C12Y302/01008C12Y302/01004C07K2319/01
Inventor 张慧敏姜惠沈子亮杨章平邬敏辰毛永江李明勋陈志孙雨佳徐天乐
Owner YANGZHOU UNIV
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