Polysaccharide cracking monooxygenase LPMO 9D coding gene and enzyme and preparation and application thereof

A technology of monooxygenase and coding gene, which is applied in application, genetic engineering, oxidoreductase, etc., and can solve the problems of low degradation efficiency, easy loss of vitality, and high cost

Active Publication Date: 2019-06-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the conversion of cellulose in industry needs to go through two processes of pretreatment and saccharification. The whole process mainly uses chemical and enzymatic methods to degrade cellulose. The application of enzymatic methods in the saccharification process is mainly through The glycoside hydrolase enzyme system composed of glycoside hydrolase an

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
  • Polysaccharide cracking monooxygenase LPMO 9D coding gene and enzyme and preparation and application thereof
  • Polysaccharide cracking monooxygenase LPMO 9D coding gene and enzyme and preparation and application thereof
  • Polysaccharide cracking monooxygenase LPMO 9D coding gene and enzyme and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Polysaccharide cleavage monooxygenase full-length gene cloning

[0030] Extract the RNA of the Myceliophthora thermophila strain Thermothelomyces thermophila ATCC 42464 according to the operation steps of the fungal RNA extraction kit (Shanghai Shenggong, SK8659), and follow the operation of the cDNA first-strand synthesis kit (Code NO.: 6210A) of TaKaRa Biological Company Steps to synthesize cDNA. Primers were designed according to the nucleotide sequence of the target gene, upstream primer 5'-GACC1TCGAGAAAAGACACTACATTCTTTCAGCAGCTG-3', downstream primer: 5'-CTACAAGCACTGGCTGTAGTAGTC-3'. Using the synthesized cDNA as a template, PCR amplification was performed using the PrimerStar HS DNA polymerase kit (Code No.: R010A) from TaKaRa Biological Company. The PCR reaction conditions were: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 1 min, a total of 30 cycles, and extension at 72°C for 5 min. ...

Embodiment 2

[0031]Example 2 Polysaccharide Cleavage Monooxygenase Gene Sequence Analysis

[0032] The sequencing results were analyzed using Basic Local Alignment Search Tool (BLAST) in the GenBank database, DNAMAN software was used for multiple sequence alignment, and VectorNTI 8.0 was used to analyze the sequence information. The coding region of the obtained polysaccharide cleavage monooxygenase gene (named lpmo 9D) is 912 bp long, and its nucleotide sequence is shown in SEQ ID NO.1. lpmo 9D encodes 303 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 31.53kDa, and its predicted isoelectric point is 6.16. SignalP analysis showed that the 1-20 positions in the amino acid sequence of LPMO 9D were signal peptides. The domain characteristics of LPMO 9D are more similar to members of the AA9 family, which indicates that LPMO 9D is a new member of the AA9 family. Using the SWISS-MODEL homology modeling server ...

Embodiment 3

[0054] Recombinant expression of embodiment 3lpmo 9D gene in Pichia pastoris

[0055] Using the genomic DNA of Myceliophthora thermophila cDNA as a template, use the designed upstream primer (5'-GACC1TCGAGAAAAGACACTACATCTTTCAGCAGCTG-3', downstream primer: 5'-CTACAAGCACTGGCTGTAGTAGTC-3'.) to amplify the polysaccharide cleavage code according to the procedure in Example 1 The gene sequence of the mature protein of the monooxygenase (excluding the signal peptide gene). The PCR amplification product and the expression vector pPICZαA (Novagen, USA) were ligated by T4 DNA ligase, and the ligated product was transformed into Escherichia coli TOP10 competent cells, and spread on the solid of low-salt Luria-Bertani medium containing 100 μg / mL bleomycin On the 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...

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
Theoretical molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention discloses a method for preparing a polysaccharide cracking monooxygenase LPMO 9D coding gene derived from myceliophthora thermophila and an enzyme thereof, and an application thereof. The polysaccharide cracking monooxygenase LPMO 9D of the present invention is derived from Thermomyxomyces thermophila ATCC 42464. The invention also provides a method for preparing the novel polysaccharide cracking monooxygenase, which is characterized in that the gene of the new polysaccharide cracking monooxygenase is cloned into a Pichia pastoris expression vector by using a genetic engineeringmethod to obtain a recombinant strain of Pichia pastoris capable of performing heterogenous expressing of the enzyme, and the polysaccharide cracking monooxygenase LPMO 9D prepared by heterologous expression of the strain can degrade PASC (phosphorylated microcrystalline cellulose) to produce cellooligosaccharide and cellooligosaccharide. The polysaccharide cracking monooxygenase LPMO 9D providedby the invention can be widely used in the fields of chemical industry, agriculture, feed addition and medicine, and has great production potential and economic value.

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 also provides the recombinant plasmid and recombinant genetic engineering strain of the polysaccharide cleavage monooxygenase. The polysaccharide cleavage monooxygenase LPMO 9D of the invention can be widely used in the fields of agriculture, food, feed addition, medicine and the like. Background technique [0002] With the rapid economic development and rapid population growth, traditional fossil energy such as coal, oil and other non-renewable resources can no longer meet the economic development needs of the world today. In addition to clean energy such as solar energy and wind energy, lignocellulose is the most abundant renewable resource in the world, and the development and production of biomass fuels and chemicals using it as a raw material has attracted widespread attention from various count...

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
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12P19/04C12P19/14C12P7/10
CPCY02E50/10
Inventor 尹恒鞠酒于作琛周海川王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap