Glycosylated 5-hydroxymethyl furfural oxidase and preparation thereof

A hydroxymethylfurfural oxidase, glycosylation technology, applied in the directions of oxidoreductase, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low expression, no active inclusion bodies, etc., to avoid toxins The effect of producing, improving purity, and increasing yield

Inactive Publication Date: 2017-04-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme is mainly expressed in Escherichia coli at present, and it faces two problems: the expression level is very low, and the 5-hydroxymethylfurfural oxidase produced per liter of fermentation broth is less than 100 mg, and it is easy to form inactive inclusion bodies

Method used

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  • Glycosylated 5-hydroxymethyl furfural oxidase and preparation thereof
  • Glycosylated 5-hydroxymethyl furfural oxidase and preparation thereof
  • Glycosylated 5-hydroxymethyl furfural oxidase and preparation thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. With the 5-hydroxymethylfurfural oxidase gene (see the sequence table SEQ ID NO: 1 for the sequence), after NadeI and XhoI double-digestion (the gene is on the pUC18 plasmid, the plasmid is double-digested, plasmid 36ul, NdeI and 3ul each of XhoI, plus 5ul of 10x H buffer. Enzyme digestion reaction conditions, 37°C, 3h). Then, the cleaved gene fragment was recovered by 1% agarose electrophoresis (using a fragment recovery kit, Axygen Company, Cat. No. AP-GX-50). The purified fragment was ligated to the PET28a carrier with double enzyme digestion under the same conditions, and ligated overnight at 16°C with T4 ligase (the composition of each material in the reaction system: 8ul plasmid, 1ul reaction buffer, 1ul T4 ligase). Transform Escherichia coli Top10 competent that was frozen in our laboratory. Take 5ul of the connection solution and add it to 100ul competent cells, mix on ice for 10 minutes, place in a 42°C water bath for 45 seconds, and place on ice for 2 minu...

Embodiment 2

[0039] The optimization of gene codon mainly considers the following factors:

[0040] 1. The expression of foreign genes in Pichia pastoris is also closely related to the selection of gene codons. The codon usage bias of Pichia pastoris has important implications for the regulation of HMFO gene expression from translation. Pichia pastoris codon preference, by analyzing the synonymous codon usage of 30 protein-coding genes of Pichia pastoris and calculating the codon usage of the yeast, the high-expression superior codons of Pichia pastoris were determined.

[0041] 2. To balance GC content, genes with high AT content often cannot be efficiently transcribed due to premature termination. Early termination is a species-specific phenomenon. GC content should be controlled between 40-60%.

[0042]3. mRNA secondary structure, stable mRNA secondary structure and molecules near the 5' end also have an important impact on gene expression. Using the open reading frame upstream of t...

Embodiment 3

[0047] 1. The 5-hydroxymethylfurfural oxidase gene is codon-optimized (see the sequence listing SEQ ID NO: 1 for the sequence), after double digestion with restriction endonucleases XhoI and XbaI (the gene is on the pUC18 plasmid, and the plasmid enzyme Cut into components, plasmid 36ul, XbaI and XhoI 3ul each, add 10x H buffer 5ul. Enzyme digestion reaction conditions, 37°C, 3h). Then, the cleaved gene fragment was recovered by 1% agarose electrophoresis (using a fragment recovery kit, Axygen Company, Cat. No. AP-GX-50). The purified fragment was ligated to the pPICZa-A vector with double enzyme digestion under the same conditions, and ligated overnight at 16°C by T4 ligase (the composition of each material in the reaction system: 8ul plasmid, 1ul reaction buffer, 1ul T4 ligase). Transform Escherichia coli Top10 competent that was frozen in our laboratory. Take 5 ul of the connection solution and add it to 100 ul of competent cells Escherichia coli TOP10, mix on ice for 10 m...

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Abstract

The invention relates to the field of eukaryotic expression of industrial enzymes, and particularly relates to a method of producing glycosylated 5-hydroxymethyl furfural oxidase (HMFO) by means of pichia pastoris. According to the codon preference of the pichia pastoris, the gene sequence of the HMFO is optimized; the optimized sequence is connected to a pPICZa-A carrier to convert pichia pastoris X-33. Pichia pastoris having high-copy HMFO gene is then screened, thereby achieving high-effective secretion expression of the HMFO in the pichia pastoris. In addition, the purity of the expressed protein is high and purification steps are simple. Meanwhile, the protein, when being subjected to galactosylated modification, is higher in activity than other proteins in the prior art. The preparation process is low in energy consumption and is high in efficiency and has simple and green production process. The glycosylated HMFO has excellent industrial application prospect.

Description

technical field [0001] The invention relates to the field of preparing industrialized enzymes by a genetic engineering method, in particular, Pichia pastoris is used to efficiently secrete and express glycosylated modified HMFO. Background technique [0002] HMFO is a biological enzyme that can convert hydroxymethylfurfural (HMF) into 2,5 furandicarboxylic acid (FDCA). However, the enzyme is mainly expressed in Escherichia coli at present, and it faces two problems: the expression level is very low, and the 5-hydroxymethylfurfural oxidase produced per liter of fermentation broth is less than 100 mg, and it is easy to form inactive inclusion bodies. (Reference Dijkman, W.P., and Fraaije, M.W.(2014). Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688. Applied and environmental microbiology 80, 1082-1090.). [0003] If the protein is modified, such as post-translational glycosylation, its stability and activity can be improve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/81C12R1/84
CPCC12N9/0006C12Y101/03
Inventor 尹恒谭海东刘启顺吴树丽王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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