Polysaccharide cleavage monooxygenase encoding gene and enzyme thereof, and preparation method and applications

A technology of monooxygenase and polysaccharide, which is applied in polysaccharide cleavage monooxygenase coding gene and enzyme preparation and application field, can solve the problems such as no literature or patent report of gene and amino acid sequence, and achieve efficient degradation effect

Pending Publication Date: 2019-07-02
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are only three reports on LPMOs derived from Myceliophthora thermophila
In 2015, MtLPMO9A was found to have oxidative activity on cellulose and xylan, as well as a synergistic effect on cellulase, but there is no related mechanism research
[0006] The gene and amino acid sequence described in this patent have not been reported in literature or patents

Method used

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  • Polysaccharide cleavage monooxygenase encoding gene and enzyme thereof, and preparation method and applications
  • Polysaccharide cleavage monooxygenase encoding gene and enzyme thereof, and preparation method and applications
  • Polysaccharide cleavage monooxygenase encoding gene and enzyme thereof, and preparation method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of polysaccharide cleavage monooxygenase MtLPMO9E eukaryotic expression strain

[0062] After performing multiple sequence alignment analysis on the polysaccharide cleavage monooxygenase gene sequence in The National Center for Biotechnology Information (NCBI) database, the polysaccharide cleavage monooxygenase of Myceliophthora thermophila was prepared to be synthesized. The coding region of the target gene MtLPMO9E in the Myceliophthora thermophila genome is 741bp long, its nucleotide sequence is shown in SEQ ID NO1, encoding 246 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 2, and its theoretical molecular weight It is 26.04KDa. The MtLPMO9E signal peptide is predicted to be 1-17 amino acids, the molecular weight is predicted to be 24.29KDa after removing the signal peptide, and the predicted isoelectric point is 6.03. The amino acid encoded by MtLPMO9E contains an AA9 (GH61) family domain. Through codon optimiza...

Embodiment 2

[0064] Example 2 Heterologous expression and purification of polysaccharide cleavage monooxygenase MtLPMO9E in Pichia pastoris

[0065] The polysaccharide cleavage monooxygenase MtLPMO9E was induced and expressed according to the method of Pichia pastoris expression manual. Polyacrylamide gel electrophoresis was used to detect the expression of polysaccharide cleavage monooxygenase LPMO9E, and the results were as follows: figure 2 shown. The BIO-PAD protein purification system was used to purify the target protein according to the isoelectric point of the protein. The purified polysaccharide cleavage monooxygenase LPMO9E showed a single band on the electrophoresis gel, and its position was consistent with the predicted molecular weight.

Embodiment 3

[0066] Example 3 Analysis of substrate binding activity of polysaccharide cleavage monooxygenase MtLPMO9E

[0067] Use affinity incubation and SDS-PAGE techniques to study the binding ability of MtLPMO9E to insoluble substrates: MtLPMO9E was incubated with the pretreated and washed substrate for 24 hours under buffer conditions, centrifuged to obtain the supernatant and precipitate, and the precipitate needed to be washed twice. SDS-PAGE was used to detect the proportion of MtLPMO9E protein in the upper layer and the precipitate to determine its binding ability, and it was found that: MtLPMO9E can bind to cellulose substrates (see image 3 ), MtLPMO9E has no binding activity with chitin substrates (see Figure 4 ).

[0068] Using isothermal calorimetry (ITC) to study the binding ability of MtLPMO9E to soluble substrates: MtLPMO9E and xyloglucan were equilibrated overnight in the same buffer, and titrated according to the operating procedure of the isothermal calorimeter. The ...

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Abstract

The present invention discloses a polysaccharide cleavage monooxygenase encoding gene and an enzyme thereof derived from myceliophthora thermophila, and a preparation method and applications. A genetic engineering technical method is used to clone the gene of the polysaccharide cleavage monooxygenase into pichia pastoris expression vectors to obtain a pichia pastoris recombinant strain capable ofheterologously expressing the enzyme. The polysaccharide cleavage monooxygenase heterologously expressed by the strain can efficiently degrade cellulose (oxidative cleavage of beta-1,4-glycosidic bonds). The provided polysaccharide cleavage monooxygenase can be widely applied to the fields of energy, agriculture, food, feed additives, medicines, etc.

Description

technical field [0001] The invention relates to a gene sequence of polysaccharide cleavage monooxygenase and the preparation method and application of the enzyme. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the polysaccharide cleavage monooxygenase and its application in polysaccharide degradation. The polysaccharide cleavage monooxygenase provided by the invention can be widely used in the fields of energy, agriculture, food, feed addition, medicine and the like. Background technique [0002] Plant cell walls are mainly composed of polysaccharides and lignin. In the dry weight composition of plant cell walls, cellulose, hemicellulose and lignin account for 20-50%, 15-35%, and 10-30%, respectively. Among them, cellulose is the most widely distributed and most abundant biomass source in nature. The effective use of cellulose can provide a large number of chemicals and energy products for human society. There are many ways to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/80C12N1/19C12P19/14C12R1/84
CPCC12N9/0083C12N15/815C12P19/14
Inventor 尹恒于作琛周海川鞠酒
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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