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Fusion enzyme for directional synthesis of dextran as well as construction method and application thereof

A dextran and construction method technology, applied in the field of directional synthesis of low-molecular-weight dextran fusion enzyme construction, can solve the problems of uniformity, restriction of catalytic efficiency, and difficulty in controlling the distance between enzyme molecules, and achieve high conversion rate and simple separation and purification.

Pending Publication Date: 2021-10-29
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, in the process of double-enzyme synergistic catalysis, the enzyme is in a free state, and the distance between enzyme molecules is difficult to control, which restricts its catalytic efficiency; and the oligosaccharides prepared under the existing conditions are all mixed sugars with a polymerization degree of 3-10 , it is very difficult to prepare oligosaccharides with a single degree of polymerization or highly concentrated
It is difficult to obtain a large amount of dextran with a uniform molecular weight, especially below 5000Da, through the synergy of two enzymes

Method used

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  • Fusion enzyme for directional synthesis of dextran as well as construction method and application thereof
  • Fusion enzyme for directional synthesis of dextran as well as construction method and application thereof
  • Fusion enzyme for directional synthesis of dextran as well as construction method and application thereof

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Embodiment 1

[0064] A method for constructing a fusion enzyme Escherichia coli capable of directional synthesis of dextran, using P473S / P856S double mutant heat-stable dextran sucrase gene and streptococcus-derived dextranase gene as templates, using (EAAAK) 1 Homologous recombination of two enzyme genes in order to connect peptides and transformation into BL21(DE3) Escherichia coli to obtain a fusion enzyme capable of directional synthesis of dextran Escherichia coli BL21(DE3) / dex-YG-G-a1dex genetic engineering bacteria. The other two fusion enzyme genetic engineering bacteria and construction methods are consistent with BL21(DE3) / dex-YG-G-a1dex genetic engineering bacteria. The BL21(DE3) / dex-YG-G-a1dex genetically engineered bacterium is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), and the classification is named Directed Synthesis Low-molecular-weight dextran ...

Embodiment 2

[0085] Expression of Fusion Enzyme Escherichia coli BL21(DE3) / dex-YG-G-a1dex Genetic Engineering Bacteria Capable of Directly Synthesizing Dextran

[0086] The genetically engineered bacterium BL21(DE3) / dex-YG-G-a1dex that embodiment 1 obtains is inoculated in the LB culture medium that contains 40~60 μ g / ml kanamycin by the inoculum size of 0.5%, rotating speed 250r / min, Cultivate at 37°C for 16 hours; draw 2mL from the above culture solution and add it to 200mL of medium A, culture on a shaker at 37°C, when the enriched culture solution is diluted 10 times with distilled water, the OD 600 Add 500μL IPTG to induce enzyme production at 0.20~0.24 o’clock. After inducing fermentation at 25℃ for 3.5~4 hours, carry out crushing and centrifugation, and perform high-performance liquid chromatography analysis on the samples taken. The protein molecular weight of the expressed fusion enzyme is about 265KDa, consistent with the predicted value. Wherein, each liter of the A medium cont...

Embodiment 3

[0088] The fermentation enzyme production of the fusion enzyme E. coli engineering bacteria BL21(DE3) / dex-YG-G-a1dex genetically engineered bacteria capable of directional synthesis of dextran specifically includes the following steps:

[0089] Inoculate the genetically engineered bacteria BL21(DE3) / dex-YG-G-a1dex into the LB medium containing 40-60 μg / ml kanamycin at an inoculum size of 0.5% by volume fraction, at a speed of 250 r / min, at 37° C. Cultivate under low temperature for 16 hours; draw 2mL from the above culture solution and add it to 200mL A medium, place it on a shaker at 37°C for culture, when the enriched culture solution is diluted 10 times with distilled water, the OD 600 At 0.20 to 0.24, add 500 μL IPTG to induce enzyme production, keep at 25°C to induce fermentation for 3.5 to 4 hours, and centrifuge the bacterial suspension after induced fermentation at 4°C at 8000r / min for 15min, and a centrifuge tube corresponds to Add a bottle of bacterial suspension, th...

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Abstract

The invention discloses a construction method and application of a fusion enzyme Escherichia coli engineering bacterium capable of directionally synthesizing low molecular weight dextran, a P473S / P856S double-mutation heat-stable dextransucrase gene and a streptococcus-derived dextranase gene are used as templates, the two enzyme genes are homologously recombined in catalytic order and transformed into BL21(DE3) E. coli by using (EAAAK)n(n= 0, 1, 2) as connecting peptide, the fusion enzyme Escherichia coli BL21 (DE3) / dex-YG-nG-a1dex (n = 0, 1, 2) genetically engineered bacterium capable of directionally synthesizing dextran with different low molecular weight is obtained. The dextran synthesized by the dex-YG-nG-a1dex (n = 0, 1, 2) genetically engineered bacterium constructed by the invention is concentrated in molecular weight, the dex-YG-nG-a1dex (n = 0, 1, 2) genetically engineered bacterium constructed by the invention can directly convert most sucrose into dextran with different low molecular weights by controlling conditions and connecting peptides, and the weight-average molecular weight of the dextran can be directly obtained and is 1000-2000Da, 5000Da and 10000Da.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion enzyme construction method for directional synthesis of low-molecular-weight dextran and its application. Background technique [0002] Dextran and its derivatives have been widely used in food systems, such as thickeners, stabilizers, prebiotics, and improving the quality of wheat bread. In addition, dextran and its derivatives are also used in the pharmaceutical industry (as a carrier, blood volume expander, antithrombotic, anticoagulant), and in the field of biotechnology (molecular sieves). Because human α-amylase can only slowly hydrolyze the α(1,6) branch of glycogen, and can quickly hydrolyze the α(1,4) branch of starch and glycogen. Dextran-induced allergic reactions (DIARs) appear to be related to the chemical structure. High molecular weight dextran and / or the ratio of non-α(1,6) linkages in dextran are associated with a higher incidence of allergic reactions. T...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/62C12N15/70C12P19/08C12R1/19
CPCC12N9/1051C12N9/2454C12N15/70C12P19/08C12Y204/01005C12Y302/01011C07K2319/00
Inventor 张洪斌张宇馨杨静文胡雪芹
Owner HEFEI UNIV OF TECH
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