Heat-resistant N-acetylglucosamine deacetylase and encoding gene and application thereof

A technology of deacetylase and acetylamino, which is applied to heat-resistant N-acetylglucosamine deacetylase and its encoding gene and application field, can solve the problems of low catalytic conversion rate, limited application of enzymatic deacetylation and the like, and achieves good performance. Application value, good enzymatic properties, good heat resistance

Active Publication Date: 2019-11-22
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to enzyme stability, catalytic activity, cost and other reasons, there is currently no commercially available N-acetylglucosamine deacetylase specifically for the preparation of glucosamine, and the industry has not yet adopted enzymatic deacetylation to prepare glucosamine on a large scale.
Most of the currently disclosed N-acetylglucosamine deacetylases are room temperature enzymes, and the catalytic conversion rate is not high, which limits the application of enzymatic deacetylation
[0006] Currently, there are few reports on specific deacetylases that can be applied to the deacetylation reaction of N-acetylglucosamine, and only a small amount of deacetylases derived from microorganisms have similar activities. Acetylase and its coding gene

Method used

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  • Heat-resistant N-acetylglucosamine deacetylase and encoding gene and application thereof
  • Heat-resistant N-acetylglucosamine deacetylase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, the acquisition of gene TcDac and the construction of expression vector

[0062] According to the above method, the candidate genes were screened from the gene database.

[0063] A candidate gene shown in positions 1-798 of SEQ ID NO.1 is named gene TcDac, and the protein encoded by the gene is named protein TcDac, and its amino acid sequence is shown in SEQ ID NO.2.

[0064] According to the DNA sequence information shown in SEQ ID NO.1, the target gene is artificially synthesized.

[0065] The upstream primer TcDac-up1 (5'-ATGTTTGAGGATGTGAACG-3') and the downstream primer TcDac-down1 (5'-AATCAGCTCCGCGAAG-3',) were designed, and the target DNA fragment was amplified by PCR. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and 30 cycles; finally, extension at 72°C for 10 min. PCR products were recovered by 1% agarose gel electropho...

Embodiment 2

[0071] Embodiment 2, expression and purification of recombinant N-acetylglucosamine deacetylase (TcDac)

[0072] The present invention also relates to the construction of a recombinant host cell for expressing TcDac, which comprises the nucleotide encoding TcDac described in the present invention. A host cell can be any cell useful in the recombinant production of proteins of the present invention, such as prokaryotic or eukaryotic cells.

[0073] The prokaryotic host cell can be any gram-positive or gram-negative bacterium, including but not limited to: Bacillus, Clostridium, Lactobacillus, Streptomyces, Staphylococcus, Escherichia coli, Pseudomonas, Paenibacillus.

[0074] Eukaryotic host cells can be mammalian, insect, plant or fungal cells, including but not limited to: filamentous fungi (Aspergillus, Mucor, Rhizopus, Penicillium, etc.), yeasts (Pichia pastoris, Candida spp. yeast, etc.).

[0075] In one embodiment, the expression host cell selected in the present inven...

Embodiment 3

[0079] Example 3, Construction of Pichia pastoris expression system of N-acetylglucosamine deacetylase (TcDac) and high-density fermentation to obtain recombinant protein

[0080] 1. Yeast expression system construction

[0081] Design the upstream primer TcDac-EcoRI (5'-ATTCCG GAATTC ATGTTTGAGGATGTGAACG-3', the underline indicates the EcoRI restriction site) and the downstream primer TcDac-NotI (5'-ATAAGAAT GCGGCCGCTTAAATCAGCTCCGCGAAG-3', the underline shows the NotI restriction site), and using the DNA fragment obtained in the above-mentioned Example 1 as a template, PCR amplifies the amino acid sequence encoding the mature protein. The amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and 30 cycles; finally, extension at 72°C for 5 min. The product was recovered by 1% agarose gel electrophoresis and digested with EcoRI and NotI. The digested product was ligated with...

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Abstract

The invention provides a heat-resistant N-acetylglucosamine deacetylase and an encoding gene and application thereof. The N-acetylglucosamine deacetylase is a) a protein encoded by the amino acid sequence shown in SEQ ID NO.2, or b) a fusion protein obtained by connecting an N-terminal and / or C-terminal of the protein shown in SEQ ID NO.2 and a tag, or c) a protein, with same functions, obtained by performing substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence shown in SEQ ID NO.2. The invention further relates to application of the protein in the preparation of glucosamine. The invention further relates to a biological material related to the heat-resistant N-acetylglucosamine deacetylase. The deacetylase can catalyze the deacetylation reaction of N-acetylglucosamine, and is applied to industrial preparation of glucosamine, the reaction conditions are mild, the energy consumption is less, no contaminant is generated, and the deacetylase has better industrial application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a heat-resistant N-acetylglucosamine deacetylase and its coding gene and application. Background technique [0002] Glucosamine (C 6 h 13 NO 5 ), also known as glucosamine, glucosamine or glucosamine, is an amino monosaccharide formed after the hydroxyl group at the 2-position of glucose is replaced by an amino group. Glucosamine is one of the most abundant monosaccharides in nature, and it mostly exists in articular cartilage, connective tissue and cell membranes in the human body. Glucosamine is also an important precursor in the glycosylation reaction of biological proteins or lipids, an important nutrient for the formation of chondrocytes, and a natural tissue component of healthy articular cartilage. [0003] Glucosamine is an important functional food as well as an adjuvant therapy drug, which mainly relieves pain, stiffness and swelling caused by arthritis; rel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/70C12N15/81C12N1/21C12N1/19C12P19/26C12R1/19C12R1/84
CPCC12N9/80C12N15/70C12N15/815C12P19/26C12Y305/01033
Inventor 赵黎明秦臻马佳菲邱勇隽
Owner EAST CHINA UNIV OF SCI & TECH
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