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Heat-resistant dextran sucrase mutant as well as a preparation method and application thereof

A technology of dextran and mutants, applied in the fields of genetic engineering and molecular enzyme engineering, can solve the problems of thermal stability and loss of vitality, and achieve the effects of no bacteria growth, high solubility and strong thermal stability

Active Publication Date: 2021-08-03
广西产研院生物制造技术研究所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2017, Frenchman Marion Claverie et al. (M.Claverie et al.2017.) developed a dextran sucrase mutant DSR-M2Δ, which was obtained by intercepting the dextran sucrase 164-1433 interval of Leuconostoccitreum NRRL B-1299 bacteria Peptides composed of amino acids, the mutant enzyme can convert sucrose to directly generate low molecular weight dextran, the reaction temperature of the mutant enzyme is 30°C, and the thermal stability of the enzyme needs to be improved, but its research results have proved that through molecular enzyme engineering technology Improvement of dextran sucrase is an effective means to prepare low and micro molecular weight dextran
Wang Chao et al. (C.Wang et al.2017.) performed molecular splicing on the dextran sucrase of Leuconostoc mesenteroides 0326, and expressed or truncated the peptides in different intervals of the peptide chain. The mutant enzyme catalyzes sucrose to produce low molecular weight or micromolecular weight dextran products or oligosaccharide products, but the optimal temperature of the mutant is between 25 and 30°C, and its activity is almost completely lost when the temperature reaches 40°C

Method used

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  • Heat-resistant dextran sucrase mutant as well as a preparation method and application thereof
  • Heat-resistant dextran sucrase mutant as well as a preparation method and application thereof
  • Heat-resistant dextran sucrase mutant as well as a preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Synthesis of dextran sucrase mutant dsrVΔ gene and construction of recombinant bacteria

[0017] The inventors carried out homologous comparison and analysis on the dextransucrase DSR from sources such as Leuconostoc citreum NRRL B-1299, Leuconostoc mesenteroides 0326 and other thermophilic bacteria, specifically Through sequence comparison, homology modeling, structure comparison and optimization, molecular design and molecular cutting, the peptide segment that retains the amino acid composition of the 37-1430 interval of the above-mentioned DsrV protein is mainly intercepted, and the dextran sucrase is finally obtained after structural optimization design The amino acid sequence of the mutant DsrVΔ is shown in SEQ ID NO.1. The inventor then followed the amino acid sequence of the mutant DsrVΔ, while considering the need for high-efficiency expression and purification of its protein, and according to the principle of codon preference in Escherichia coli, the...

Embodiment 2

[0019] Example 2. Study on Induced Expression and Enzymatic Properties of Dextran Sucrase Mutant DsrVΔ

[0020] On a plate coated with recombinant Escherichia coli BL21(DE3) / pET-22b-dsrVΔ, pick a single colony and place it in 5ml LB (containing 100μg / mL ampicillin, Amp) liquid medium, and cultivate overnight at 37°C and 220rpm. Inoculate 2% of the inoculum from 5 mL of LB bacteria liquid into a 500 mL shaker flask filled with 200 mL of LB (containing 100 μg / mL Amp) liquid medium. 600 When the concentration is 0.5-0.7, add IPTG to a final concentration of 1mmol / L, add 5mL sterile Tris-HCl solution (pH 6.4, 100mmol / L) at the same time, shake and cultivate in a shaker at 30°C and 220rpm for 4h-8h . Take 200 mL of the culture medium after induction culture for 5 h, collect the cells by centrifugation, add 5 mL of cell-breaking buffer (20 mM sodium acetate buffer, pH 5.4, 20 mM CaCl 2 , 0.2g / L sodium amide), added Triton X-100 to a final concentration of 0.5%, after ultrasonic di...

Embodiment 3

[0026] Example 3. Experiments on the preparation of dextran by converting sucrose with dextran sucrase mutant DsrVΔ

[0027] According to the method of Example 2 of the present invention, a sufficient amount of dextran sucrase mutant crude enzyme solution was prepared, and the experiment of converting sucrose to dextran was carried out. The inventor adds 2000mL of sodium acetate buffer solution (20mmol / L sodium acetate, pH 5.7, 25mmol / L CaCl 2 ), stirring at a speed of 100rpm and starting to raise the temperature of the kettle body, adding sucrose at the same time, the total amount of sucrose added is 600g, after the sucrose is fully dissolved by stirring, the temperature of the reactor is controlled at about 63°C by water bath circulation, and then adding dextran Sucrase mutant crude enzyme solution 1000U, catalyzed reaction under the condition of 63 ℃ temperature, 100rpm for 15 hours, during the reaction, take samples every 3 hours to detect the molecular weight of dextran (...

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Abstract

The invention relates to a heat-resistant dextran sucrase mutant as well as a preparation method and application thereof, and is characterized in that the mutant is a new mutant obtained by intercepting an amino acid peptide fragment in an interval 37-1430 of Leuconostoc citreum dextran sucrase DsrV and carrying out structure optimization, the optimum temperature of the mutant is 63 DEG C, and the mutant has good heat stability under a high-temperature condition of 58-68 DEG C. The mutant can catalyze cane sugar to generate micro-molecular-weight dextran, and the catalytic process has the characteristics of low viscosity of conversion liquid, no growth of infectious microbes, low energy consumption and simplicity in operation. The mutant is suitable for industrial application, and is more favorable for reducing the production cost of the micro-molecular-weight dextran.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and molecular enzyme engineering, and specifically relates to a heat-resistant dextran sucrase mutant and a preparation method thereof, as well as the application of the mutant in one-step catalysis of sucrose to generate micromolecular-weight dextran and dextran derivatives. Background technique [0002] Dextran (Dextran), also known as dextran, dextran, dextran, and polyglucose, is catalyzed by dextran sucrase (Dextransucrase, DSR, EC2.4.5.1), which converts D- The glucose group is transferred to the receptor molecule, forming a high molecular polysaccharide polymer connected by several glucose groups, accompanied by the generation of fructose. The main chain of dextran is connected by α-D-1,6 glycosidic bonds, and the side chain branch points are connected by α-D-1,3 and α-D-1,2. Plasma-applied microbial polysaccharides. Due to its loose and soft texture, dextran has many advanta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/18C12P19/08
CPCC12N9/1051C12N15/70C12P19/18C12P19/08C12Y204/01005C12N2800/22Y02A50/30
Inventor 韦旭钦彭小玉吴睿阮恒李广曹志强王则奋胡雪玲李略韦志明吴建璋
Owner 广西产研院生物制造技术研究所有限公司
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