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A lytic polysaccharide monooxygenase modified by site-directed mutation and its construction method and application

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: 2021-06-08
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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  • A lytic polysaccharide monooxygenase modified by site-directed mutation and its construction method and application
  • A lytic polysaccharide monooxygenase modified by site-directed mutation and its construction method and application
  • A lytic polysaccharide monooxygenase modified by site-directed mutation and its construction method and application

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 a lytic polysaccharide monooxygenase modified by site-directed mutation, its construction method and application, and belongs to the field of genetic engineering. The enzyme activity provides a degradative function in the degradation of crystalline polysaccharides of cellulose. The lytic polysaccharide monooxygenase mutant of the present invention is based on the amino acid sequence of the lytic polysaccharide monooxygenase of wild-type Myceliophthora thermophila, and the 34th position is mutated from Arg to Leu. The optimal temperature of the wild enzyme and the wild enzyme is both 85°C, and the specific activity of the mutant enzyme is about 2 times higher than that of the wild enzyme, and when the pH is greater than 5.5, the specific activity of the mutant enzyme is about 2 times higher than that of the wild enzyme. When microcrystalline cellulose was used as the substrate, the glucose content produced by the synergistic reaction of cellulase and mutant enzyme was significantly increased by 48.5% compared with the substrate degraded by cellulase alone. It can be seen that the mutant enzyme significantly improved the enzyme activity, which can Lower economic costs for industrial applications.

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 Patents(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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