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Site-directed mutation modified lytic polysaccharide monooxygenase as well as construction method and application thereof

A polysaccharide monooxygenase and mutant technology, applied in the field of genetic engineering, can solve the problem of less application and achieve the effect of improving enzyme activity

Active Publication Date: 2019-12-10
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology is still rarely used in the transformation of LPMOs, especially for fungal LPMOs, there are few literature reports on site-directed mutation transformation

Method used

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  • Site-directed mutation modified lytic polysaccharide monooxygenase as well as construction method and application thereof
  • Site-directed mutation modified lytic polysaccharide monooxygenase as well as construction method and application thereof
  • Site-directed mutation modified lytic polysaccharide monooxygenase as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Preparation of the lytic polysaccharide monooxygenase mutant described in Example 1

[0030] (1) Construction of recombinant plasmid pPIC9K-MtLPMO9A: a recombinant vector encoding the gene of wild-type Myceliophthora thermophila lytic polysaccharide monooxygenase (shown in amino acid sequence SEQ ID No.3).

[0031] Firstly, according to the original amino acid sequence (GenBank: AKO82493.1), the codon-optimized synthetic gene was used as a yeast expression host to obtain the optimized lytic polysaccharide monooxygenase coding gene (nucleotide sequence such as SEQ ID No.4), The gene encoding the lytic polysaccharide monooxygenase and the pPIC9K vector were double-digested with BamHI and EcoRI enzymes respectively, and then recovered separately, and the recovered coding gene fragment was connected to the pPIC9K vector with ligase to obtain the recombinant vector pPIC9K -MtLPMO9A, transfer the recombinant vector pPIC9K-MtLPMO9A to the cloning host E.coli Jm109, verify whet...

Embodiment 2

[0036] Example 2 Expression and Enzyme Activity Determination of Mutant Gene Engineering Bacteria Containing Cracking Polysaccharide Monooxygenase of the Present Invention

[0037] (1) Expression of genetically engineered bacteria:

[0038]Place the screened genetically engineered bacteria in 5 mL of YPD medium and culture at 30°C for 24 hours, then transfer 1 mL to 50 mL of glycerol-containing BMGY medium and culture at 30°C for 16-18 hours, then centrifuge at 4000r / min for 10 minutes to collect the cells, and use BMMY medium Cells were further washed. Finally, the cells were resuspended in BMMY, and 100% methanol was added every 24 hours to a final concentration of 0.5% to induce the expression of the target protein. After 6 days of induced expression, the supernatant of the fermentation broth was collected by centrifugation at 12,000 r / min for 30 minutes, which was the crude enzyme solution, and filtered through an aqueous membrane with a pore size of 0.22 μm. use Ni 2+ ...

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Abstract

The invention discloses site-directed mutation modified lytic polysaccharide monooxygenase as well as a construction method and application thereof, and belongs to the field of gene engineering. The activity of the site-directed mutation modified lytic polysaccharide monooxygenase provides a degrading function in degradation of crystalline polysaccharide of cellulose. A lytic polysaccharide monooxygenase mutant provided by the invention is formed by mutating Arg into Leu in the 34th site on the basis of the amino acid sequence of lytic polysaccharide monooxygenase of wild type myceliophthora thermophila, wherein the optimum temperature of the mutant enzyme and the wild enzyme is 85 DEG C; the specific activity of the mutant enzyme is significantly increased by about two times compared withthat of the wild enzyme; and when pH is more than 5.5, the specific activity of the mutant enzyme is significantly about two times higher than that of the wild enzyme. When microcrystalline celluloseserves as a substrate, the content of glucose generated through synergistic reaction of cellulase and the mutant enzyme is significantly increased by 48.5 percent compared with that in the method ofdegrading the substrate singly by the celluase, so it is observed that the mutant enzyme significantly improves the enzyme activity and the economic cost of industrial application can be reduced.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a site-directed mutation method for preparing lytic polysaccharide monooxygenase mutants. Background technique [0002] Although my country has become the world's second largest economy, it still faces challenges in energy, resources and the environment. Petroleum resources are facing depletion, and it is urgent to seek renewable resources for alternative energy sources. Bioethanol is a renewable and carbon-neutral bio-liquid fuel that can alleviate the problem of energy shortage. Therefore, the extraction of ethanol from biomass has always been the focus of government support. First-generation biofuels derived from starch or sugar are already a well-established technology, but their reliance on food resources, combined with a lack of sufficient resources to meet future energy demands, meant the search for alternatives. Therefore, obtaining second-generation biof...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P19/02C12R1/84
CPCC12N9/0083C12P19/02C12Y114/99
Inventor 刘夫锋郭宵安亚静柴成程路福平
Owner TIANJIN UNIV OF SCI & TECH
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