Fructosamine desaccharase carrier, transgenic cell line and genetically engineered bacterium for expressing fructosamine desaccharase, and application of fructosamine desaccharase

The technology of fructosamine decarboxylation enzyme and glycosaminoglycan decarboxylation enzyme is applied in the field of biological enzymatic production, and can solve the problems of high cost, low yield, complicated refining process and the like

Pending Publication Date: 2021-11-02
WUHAN BAIANHUIJI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the yield is low, the cost is high,

Method used

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  • Fructosamine desaccharase carrier, transgenic cell line and genetically engineered bacterium for expressing fructosamine desaccharase, and application of fructosamine desaccharase
  • Fructosamine desaccharase carrier, transgenic cell line and genetically engineered bacterium for expressing fructosamine desaccharase, and application of fructosamine desaccharase
  • Fructosamine desaccharase carrier, transgenic cell line and genetically engineered bacterium for expressing fructosamine desaccharase, and application of fructosamine desaccharase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Acquisition of fructokinase and fructosamine desugarase genes

[0053] Firstly, the fructokinase (Fructokinase) and fructosamine desugarase (Fructosamine deglycase) genes were searched through the NCBI (National Center for Biotechnology Information) database. The fructokinase (GenBank: ESD97983.1) and the fructosamine desugarase (GenBank: AOR99552.1) from Escherichia coli (Escherichia coli 908658) and Bacillus subtilis (GenBank: AOR99552.1) were obtained through gene mining and delivered to the biological company Perform gene synthesis. Wherein the synthesized gene sequence is introduced into SnaBI and NotI restriction enzyme cutting sites (without signal peptide), the restriction enzyme cutting sites are protective bases, and the effective sequence is the sequence after the restriction enzyme cutting sites. And the sequence is stored in the plasmid pMD-19T to form the recombinant plasmid pMD-19T-FRK1 or pMD-19T-FrIB.

[0054] Transform the synthesized gene sequence, ...

Embodiment 2

[0057] Construction of prokaryotic expression vectors of fructokinase and fructosamine desugarase genes, recombinant expression and protein expression

[0058] 1. Construction of prokaryotic expression vector

[0059] (1) Primer design: design primers starting from the mature peptide sequence following the signal peptide.

[0060] Fructosamine desugarase primers:

[0061] Forward primer:

[0062] 5'- TACGTA AATATATTGTAATATCAGATTACGT-3' (SEQ ID NO.5)

[0063] Reverse primer:

[0064] 5'- GCGGCCGCG TTATATAACATTATAGTCTAATGCA-3' (SEQ ID NO. 6)

[0065] The underline is the enzyme cutting site, and the enzymes used are SnaBI and NotⅠ

[0066] Fructose kinase primer:

[0067] Forward primer:

[0068] 5'- TACGTA GAGAACACCGGTATTGGTGCGTCGC-3' (SEQ ID NO. 7)

[0069] Reverse primer:

[0070] 5'- GCGGCCGCG CTCTTGTGGCCATAACCACGCAGCG-3' (SEQ ID NO.8)

[0071] The underline is the enzyme cutting site, and the enzymes used are SnaBI and NotⅠ

[0072] (2) PCR reaction, usin...

Embodiment 3

[0086] Purification of fructokinase and fructosamine desugarase proteins

[0087] Purification of recombinant enzyme and its analysis by SDS-PAGE gel electrophoresis

[0088] For protein purification, refer to GE Healthcare guidelines, and for SDS-PAGE analysis, follow the "Molecular Cloning Experiment Guide" (Third Edition), the gel concentration used is 12.5%, and the loading volume is 5-25 μL. Proteins were stained with Coomassie brilliant blue R-250.

[0089] Among them, native-SDS-PAGE experimental steps:

[0090]A. Add 5-10 μl sample buffer [0.1mol / L Tris-HCl (Tris-HCl), pH 6.8; 2% SDS (weight: volume), 10% glycerol ( volume: volume), 0.01% bromophenol blue (weight: volume)] placed in a water bath at 37°C for 5-10 min, and then separated by electrophoresis. Note: When extracting samples, the purpose of not adding mercaptoethanol to the sample extract is to moderately denature proteases during electrophoresis, so that the activity of these proteases can be restored aft...

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Abstract

The invention relates to a fructosamine decarboxylase carrier, a transgenic cell line and a genetically engineered bacterium for expressing fructosamine decarboxylase and application of fructosamine desaccharase, and belongs to the technical field of biological enzyme method production. The invention provides a method for catalyzing transfer of an amino group from an amino donor compound to an amino receptor compound, which comprises the following steps of: utilizing an enzyme (fructosamine desaccharase), or an expression vector or a cloning vector for expressing the enzyme, or a transgenic cell line for expressing the enzyme, or a genetically engineered bacterium for expressing the enzyme; and the catalytic amino group is transferred from the amino donor compound to the amino acceptor compound. The method provided by the invention has the advantages of sufficient substrate source, no limitation of raw materials, mild preparation conditions, small environmental pollution, high reaction specificity, few impurities in the system, easiness in downstream separation and purification, and low production cost.

Description

technical field [0001] The invention relates to the technical field of biological enzymatic production, in particular to the application of a fructosamine desugarase carrier, a transgenic cell line expressing the fructosamine desugarase, genetically engineered bacteria and the fructosamine desugarase. Background technique [0002] Glucosamine (GlcN), that is, 2-amino-2-deoxy-D-glucose. Glucosamine has important physiological functions to the human body, and is widely used in food, health products, medicines and other fields. In clinical medicine, it can be used as a substance for treating bone and joint diseases, and can also be used as a drug for treating rheumatoid arthritis. In food It can be used as an additive in food for infants and young children. With the further improvement of the quality of life and the intensification of the aging population, the global demand for glucosamine-based medicines, food and nutritional health products will continue to increase. [000...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/70C12N1/21C12P7/40C12P19/26C12R1/19
CPCC12N9/78C12N15/70C12P19/26C12P7/40
Inventor 王怀英
Owner WUHAN BAIANHUIJI BIOTECHNOLOGY CO LTD
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