Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Lytic polysaccharide monooxygenase LPMO M1 encoding gene and enzyme thereof, and preparation method and application

A technology of LPMOM1 and monooxygenase, which is applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of low degradation efficiency in substrate crystallization area, limited efficient utilization of biomass such as cellulose, and high cost question

Inactive Publication Date: 2017-05-31
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the traditional cellulase systems belong to the glycoside hydrolase family, their degradation efficiency to the crystallization region of the substrate is low and the cost is high, which makes it difficult for the traditional cellulase systems to meet the needs of industrial applications and limits the development of cellulose. efficient use of biomass

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Lytic polysaccharide monooxygenase LPMO M1 encoding gene and enzyme thereof, and preparation method and application
  • Lytic polysaccharide monooxygenase LPMO M1 encoding gene and enzyme thereof, and preparation method and application
  • Lytic polysaccharide monooxygenase LPMO M1 encoding gene and enzyme thereof, and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Polysaccharide cleavage monooxygenase full-length gene cloning

[0031] Extract the RNA of Magnaporthegrisea 70-15 from the blast fungus strain Magnaporthegrisea 70-15 according to the operation steps of the fungal RNA extraction kit (Shanghai Shenggong, SK8659), and synthesize cDNA according to the operation steps of the first cDNA synthesis kit (CodeNO.: 6210A) of TaKaRa Biological Company . Primers were designed according to the nucleotide sequence of the target gene, upstream primer 5'-CTGCCCAGCCGGCGATGGCCCACTACAACTTCGAGTCCCTC-3', downstream primer: 5'-TTGTTAGCAGCCGGATCTCAGTAGTGTGCGGAGCGGCGGC-3'. PCR amplification was performed using the synthesized cDNA as a template. The PCR reaction conditions were: pre-denaturation at 98°C for 3min, denaturation at 98°C for 10s, annealing at 55°C for 5s, extension at 72°C for 2min, a total of 30 cycles, and extension at 72°C for 5min. After the PCR product was analyzed by agarose gel electrophoresis, the target fragm...

Embodiment 2

[0032] Example 2 Polysaccharide Cleavage Monooxygenase Gene Sequence Analysis

[0033]The sequencing results were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, and the Vector NTI Suite 8.0 software was used for multiple sequence alignments to analyze their homology. The domains of the sequences were analyzed using the Simple Modular Architecture Research Tool (SMART) online tool.

[0034] The coding region of the obtained polysaccharide cleavage monooxygenase gene (named lpmo M1) is 819 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. Lpmo M1 encodes 271 amino acids and 1 stop codon. Its amino acid sequence is shown in SEQ ID NO 2. The theoretical molecular weight of the protein is 28.85kDa, and the predicted isoelectric point is 7.66. SMART analysis showed that the 1-21 positions in the amino acid sequence of LPMO M1 were signal peptides. The domain characteristics of LPMOM1 are more similar to those of AA9 family members...

Embodiment 3

[0039] Recombinant expression of embodiment 3 lpmo M1 gene in escherichia coli

[0040] Using the genomic DNA of Magnaporthe grisea cDNA as a template, use the designed upstream primer (5'-CTGCCCAGCCGGCGATGGCCCACTACAACTTCGAGTCCCTC-3') and downstream primer (5'-TTGTTAGCAGCCGGATCTCAGTAGTGTGCGGAGCGGCGGC-3') to amplify the coding polysaccharide cleavage single plus The gene sequence of the mature protein of the oxygenase (excluding the signal peptide gene). The PCR amplification product and the expression vector pET22b (Novagen, USA) were ligated by unlimited cloning (RF cloning), and the ligated product was transformed into Escherichia coli TOP10 competent cells, and spread on Luria-Bertani medium containing 100 μg / mL ampicillin On a solid plate, culture at 37°C for 12-16 hours, pick a single clone, and use the upstream and downstream primers for colony PCR verification, and the result is an amplified product of the correct size; insert the correct single clone into a liquid cont...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses a lytic polysaccharide monooxygenase (LPMO) M1 encoding gene coming from magnaporthe grisea, and a preparation method and an application thereof. The lytic polysaccharide monooxygenase LPMO M1 is sourced from the magnaporthegrisea 70-15, which is a pathogenic bacterium of paddy rice. The invention also provides a method of producing the novel LPMO, wherein by means of genetic engineering technical methods, the gene of the novel LPMO is cloned onto an escherichia coli expression vector to obtain an escherichia coli recombination strain that can heterologously express the enzyme. The LPMO M1 prepared through the heterologous expression by the strain can degrade PASC (microcrystalline cellulose processed by phosphoric acid) to generate cello-oligosaccharide and cello-oligosaccharide acid. The LPMO M1 can be widely applied in the fields of chemical engineering, agriculture, feed additives, medicines and the like, and has huge production potential and economic value.

Description

technical field [0001] The invention relates to a gene sequence of a polysaccharide cleavage monooxygenase, a preparation method and application thereof. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the polysaccharide cleavage monooxygenase. The polysaccharide cleavage monooxygenase LPMO M1 of the invention can be widely used in the fields of agriculture, food, feed addition, medicine and the like. Background technique [0002] With energy demand growing, reserves dwindling, and the global warming effect intensifying, the search for renewable energy alternatives to fossil fuels has become urgent. In addition to clean energy such as solar energy and wind energy, abundant biomass energy has become the focus of attention. Lignocellulose is an extremely widely distributed and abundant biomass resource in nature. The global annual cellulose production is as high as about 8×10 13 kg, rational use of such resources will be of ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12P19/04C12P7/58C12P7/10
CPCY02E50/10
Inventor 尹恒张鑫谭海东王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com