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5-Hydroxymethylfurfural oxidase gene hmfo and its encoding enzyme and application

A technology of hydroxymethyl furfural oxidase and hydroxymethyl furfural, which is applied in the directions 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 achieves a short catalytic reaction time and high activity. Effect

Active Publication Date: 2021-04-13
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • 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

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  • 5-Hydroxymethylfurfural oxidase gene hmfo and its encoding enzyme and application
  • 5-Hydroxymethylfurfural oxidase gene hmfo and its encoding enzyme and application
  • 5-Hydroxymethylfurfural oxidase gene hmfo and its encoding enzyme and application

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Implementation Example 1: 5-Hydroxymethylfurfural Oxidase Gene Sequence Analysis

[0033] The results of the sequencing 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 alignment and sequence information analysis.

[0034] The coding region of the obtained 5-hydroxymethylfurfural oxidase gene (named HMFO) is 1632 bp long, 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 58686Da, and the predicted isoelectric point is 5.96. There are 152 charged amino acids, accounting for 34.45%. 65, accounting for 13.17%, 52 basic amino acids, accounting for 12.98%, 102 polar amino acids, accounting for 19.07%, 205 hydrophobic amino acids, accounting for 36.44%. Ala has 58, Gly has 56, and Glu has 26.

[0035] (1) Information ...

Embodiment 2

[0057] Example 2 Cloning of the full-length gene of 5-hydroxymethylfurfural oxidase HMFO

[0058] The genomic DNA of Pseudomonas nitroreductans was extracted according to the operation steps of the bacterial genomic DNA extraction kit (TaKaRa Company). After multiple sequence alignment analysis of 5-hydroxymethylfurfural oxidase gene sequence, primers F: 5'-AGTCCATATGACCACTCCTCGCTATGAC-3' and R: 5'-ATATAAGCTTGGCTCCGCCCAGAATG-3' were designed. PCR amplification was performed using the genomic DNA of Pseudomonas nitroreductors as a template. The PCR reaction conditions were as follows: 94°C for 4 min, 1 cycle; 98°C for 10 s, 68°C for 5 s, each cycle lowered by 0.2°C, 72°C for 1 min 45 s, 35 cycles; 72°C for 10 min, 1 cycle. The PCR product was detected by 1% agarose gel electrophoresis, and the product size was 1596bp. Gel recovery kit (purchased from Axygen) was used to purify PCR products.

Embodiment 3

[0059] The construction of embodiment 3 recombinant plasmid PET21a-HMFO

[0060] The purified PCR product and the empty vector pET21a were double-digested with restriction endonucleases Nde Ⅰ and Hid Ⅲ respectively, and the digested products were purified by the gel recovery kit. Under the action of T4 DNA ligase, the purified digested products were Ligation, after the ligation product is transformed into Escherichia coli Top10 competent cells, spread it on a solid containing 100 μg / ml ampicillin LB (yeast powder 5g / L, tryptone 10g / L, sodium chloride 10g / L, agar powder 15g / L) culture medium at 37°C for 12 hours. Take a single clone and insert it into liquid LB medium containing 100 μg / ml ampicillin for culture, and extract the plasmid. The recombinant plasmids were double-digested with restriction endonucleases Nde Ⅰ and Hid Ⅲ, and the digested products were detected by 1% agarose gel electrophoresis, and the digested products with the correct band size were obtained, which p...

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Abstract

The invention provides a kind of nitroreducing Pseudomonas (Pseudomonas nitroreducens) 5-hydroxymethylfurfural oxidase and a preparation method thereof, specifically cloning the DNA sequence of the 5-hydroxymethylfurfural oxidase into a plasmid, And the recombinant plasmid is integrated into the host bacteria to obtain a genetically engineered strain that can express the enzyme heterologously, and the 5-hydroxymethylfurfural oxidase prepared by the heterologous expression of the strain can oxidize 5-hydroxymethylfurfural into 2 ,5‑furandicarboxylic acid, 2,5‑furandicarbaldehyde and 5‑formyl‑2‑furandicarboxylic acid. This patent provides a technical basis for the preparation of furan bulk chemicals by biologically oxidizing 5‑hydroxymethylfurfural.

Description

technical field [0001] The invention relates to a gene sequence of 5-hydroxymethylfurfural oxidase in Pseudomonas nitroreductors, a preparation method and application thereof. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the 5-hydroxymethylfurfural. The 5-hydroxymethylfurfural oxidase of the 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-hydroxymethylfurfural. [0002] technical background [0003] 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 has attracted widespread attention worldwide. Biomass is the only renewable carbon resource, and the conversion of biomass into platform compounds and bulk chemicals is an important development direction for biomass co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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