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

5-hydroxymethylfurfural oxidase gene HMFO and codase thereof as well as application

A hydroxymethylfurfural oxidase and gene technology, applied in the direction of oxidoreductase, application, genetic engineering, etc., can solve the problem of not being able to complete the three-step oxidation of HMF at the same time, and achieve the effect of short catalytic reaction time and high activity

Active Publication Date: 2018-06-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
View PDF2 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Galactose oxidase can selectively oxidize HMF into DFF, and xanthine oxidase oxidizes HMF into HMFCA (Qin Y-Z, Li Y-M, Zong M-H, et al. Enzyme-catalyzed selective oxidation of 5-hydroxymethylfurfural (HMF) and separation of HMF and 2,5-diformylfuran using deep eutectic solvents[J].Green Chem.,2015,17:3718-3722.), these enzymes have high selectivity, but they cannot complete the three-step oxidation of HMF to FDCA at the same time, and the reaction pH mostly neutral

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
  • 5-hydroxymethylfurfural oxidase gene HMFO and codase thereof as well as application
  • 5-hydroxymethylfurfural oxidase gene HMFO and codase thereof as well as application
  • 5-hydroxymethylfurfural oxidase gene HMFO and codase thereof as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Implementation example 1: 5-hydroxymethylfurfural oxidase gene sequence analysis

[0032] The sequencing results were analyzed by Basic Local Alignment Search Tool (BLAST) in the GenBank database, and Vector NTI Suite 8.0 software was used for multiple sequence alignment to analyze sequence information.

[0033] The obtained 5-hydroxymethyl furfural oxidase gene (named HMFO) has a coding region of 1632 bp in length, and its nucleotide sequence is shown in SEQ ID NO 1. HMFO encodes 543 amino acids and a stop codon. Its amino acid sequence is shown in SEQ ID NO 2. The theoretical molecular weight of the protein is 58686 Da, and the predicted isoelectric point is 5.96. Among the amino acids, 152 are charged, accounting for 34.45%. 65, accounting for 13.17%, 52 basic amino acids, accounting for 12.98%, polar amino acids, 102, accounting for 19.07%, and hydrophobic amino acids, 205, accounting for 36.44%. There are 58 in Ala, 56 in Gly, and 26 in Glu.

[0034] (1) SEQ ID No.1 inf...

Embodiment 2

[0056] Example 2 Cloning of the full-length gene of 5-hydroxymethylfurfural oxidase HMFO

[0057] The genomic DNA of Pseudomonas nitroreduced bacteria was extracted according to the operation steps of the bacterial genomic DNA extraction kit (TaKaRa company). After performing multiple sequence alignment analysis on the 5-hydroxymethylfurfural oxidase gene sequence, primers F: 5'-AGTCCATATGACCACTCCTCGCTATGAC-3' and R: 5'-ATATAAGCTTGGCTCCGCCCAGAATG-3' were designed. The genomic DNA of Pseudomonas nitroreducing strain was used as a template for PCR amplification. The PCR reaction conditions were: 94°C for 4min, 1 cycle; 98°C for 10s, 68°C for 5s, each cycle reduced by 0.2°C, 72°C for 1min 45s, 35 cycles; 72°C for 10min, 1 cycle. The PCR product was detected by 1% agarose gel electrophoresis, and the product size was 1596 bp. Gel recovery kit (purchased from Axygen company) to purify PCR products.

Embodiment 3

[0058] Example 3 Construction of recombinant plasmid PET21a-HMFO

[0059] The purified PCR product and the empty vector pET21a were digested with restriction enzymes Nde Ⅰ and Hid Ⅲ, respectively. The digested product was purified by the gel recovery kit. Under the action of T4 DNA ligase, the purified digested product was subjected to After ligation, the ligation product is transformed into E. coli Top10 competent cells, and then coated on 100μg / ml ampicillin LB (yeast powder 5g / L, tryptone 10g / L, sodium chloride 10g / L, agar powder 15g / L) solid On the medium, incubate at 37°C for 12 hours. Take a single clone into a liquid LB medium containing 100 μg / ml ampicillin for culture, and extract the plasmid. The recombinant plasmid was digested with restriction enzymes Nde Ⅰ and Hid Ⅲ, and the digested products were detected by 1% agarose gel electrophoresis. The digested products with the correct band size were obtained. The constructed recombinant plasmid was initially proved to be ...

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 provides pseudomonas nitroreducens 5-hydroxymethylfurfural oxidase and a preparation method thereof. Particularly, the DNA sequence of 5-hydroxymethylfurfural oxidase is cloned to plasmid, and recombinant plasmid is integrated into host bacteria to obtain a gene engineering strain capable of heterologous expression of the oxidase. The prepared 5-hydroxymethylfurfural oxidase subjected to heterologous expression of the strain can oxidize the 5-hydroxymethylfurfural oxidase and convert the 5-hydroxymethylfurfural oxidase into 2,5-furandicarboxylic acid, furan-2,5-dicarbaldehyde and5-formyl-2-furancarboxylic acid. The invention provides a technological base for preparing furan bulk chemicals by oxidizing 5-hydroxymethylfurfural through bioanalysis.

Description

Technical field [0001] The invention relates to a gene sequence of 5-hydroxymethyl furfural oxidase in nitro-reducing pseudomonas and its preparation method and application. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the 5-hydroxymethyl furfural. The 5-hydroxymethylfurfural oxidase of the present invention is mainly used in the field of preparing bulk chemicals by biological methods, and is specifically used in the field of preparing furan bulk chemicals by oxidation of 5-hydroxymethyl furfural. technical background [0002] With the increasing consumption of fossil resources and the emission of a large amount of greenhouse gases, the development and utilization of renewable energy and renewable resources have attracted widespread attention worldwide. Biomass is the only renewable carbon resource. Converting biomass into platform compounds and bulk chemicals is an important development direction for biomass conversion and ut...

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): C12N15/53C12N9/04C12N15/70C12N15/81C12P17/04C12R1/38C12R1/19C12R1/84
CPCC12N9/0006C12N15/70C12N15/815C12N2800/101C12N2800/102C12P17/04C12Y101/03
Inventor 尹恒吴树丽刘启顺谭海东王文霞
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