Dextran sucrase mutant as well as preparation method and application thereof

A technology of dextran and sucrase, applied in the field of bioengineering, can solve the problem that prebiotics are difficult to meet the demand, and achieve the effect of concentrated molecular weight distribution

Inactive Publication Date: 2022-05-31
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have shown that prebiotics play an important role in regulating the structure of intestinal flora, improving intestinal microecology, regulating lipid metabolism, lowering cholesterol, and enhancing immunity; Flavoring agents, leavening agents, stabilizers, etc. have shown superior physical properties and are widely used in food, feed, cosmetics, and medicine. tons is still difficult to meet the market demand

Method used

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  • Dextran sucrase mutant as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1 Construction of dextran sucrase mutant

[0023] Using the genome from Leuconostoc citri NRRL B-1299 as a template, primers for the mutant were designed, and a plasmid containing the mutant gene was amplified by PCR. After the PCR amplification products were checked and determined by 1% agarose gel electrophoresis, a suitable enzyme digestion system was designed, and DpnI was added to remove the original template with methylation. After digestion for 4 hours, the digested product was purified by PCR, and the purified product was confirmed by nucleic acid electrophoresis. The obtained purified product was mixed with Escherichia coli DH5α competently, placed on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and quickly placed on ice for 5 minutes. Add the competent cells obtained in the previous step to 1 mL of LB medium, activate at 37°C and 180rpm for 50min, take 100uL of the competent cells after activation and spread evenly on the LB solid mediu...

Embodiment 2

[0024] Example 2 Expression of Dextran Sucrase Mutants

[0025] The plasmid of the dextran sucrase mutant was digested and linearized by DNA restriction endonuclease EcorI at 37°C, and the digested product was confirmed by nucleic acid electrophoresis after digestion for 5 hours. Then the PCR product was purified, and finally eluted with sterile water, and the purified product was transformed into Escherichia coli BL21 (DE3), and the transformation solution was spread on an LB plate and incubated at 37°C for 12 hours, and the single colony that grew out was expression strain. Pick a single colony and inoculate it into 5 mL of LB medium for 12 hours at 37°C and 200 rpm, and then inoculate it into 200 mL of LB medium for expansion. When the OD600 of the bacterial solution grows to 0.6-0.8, add 0.1 mMIPTG to induce culture for 24 hours.

Embodiment 3

[0026] Example 3 Dextransucrase Mutant Expression Enzyme Activity Determination

[0027] Ultrasonic crush the bacterial liquid containing the dextran sucrase mutant, centrifuge to collect the supernatant after crushing, take 1 mL and 2.5 mL of the enzyme activity reaction system for measuring enzyme activity, mix and react for 30 min, add quantitative DNS reagent after the reaction, boil for 5 min, use The spectrophotometer measures at a wavelength of 540nm, and substitutes into the fructose standard curve to calculate the fructose content of the reaction and determine the enzyme activity.

[0028] The enzyme activity reaction system is: sodium acetate 1.46 g / L, calcium chloride 0.05 g / L, sucrose 120 g / L, and the pH is adjusted to 5.5 with 20% hydrochloric acid.

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Abstract

The invention discloses a dextransucrase mutant and a method for preparing prebiotics by using maltose as a receptor substrate. The sequence diagram of the dextransucrase is shown in the figure, the optimal catalysis pH is 5.5, and the temperature is 30 DEG C. The mutant dextransucrase is used for catalyzing dextran, maltose is used as a receptor substrate to directly obtain a product, the method has the advantages of being easy to operate, low in energy consumption and high in efficiency, and prebiotics produced through catalysis of the dextransucrase have huge market application potential in the aspects of food, medicine and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a dextran sucrase mutant and its preparation method and application. Background technique [0002] With the rapid development of society and economy, people's pace of life is constantly accelerating. Unhealthy living habits such as irregular diet and staying up late make more and more people in a sub-health state. Health problems have increasingly become the research focus of scholars at home and abroad. Studies have shown that prebiotics play an important role in regulating the structure of intestinal flora, improving intestinal microecology, regulating lipid metabolism, lowering cholesterol, and enhancing immunity; Flavoring agents, leavening agents, stabilizers, etc. have shown superior physical properties and are widely used in food, feed, cosmetics, and medicine. tons is still difficult to meet market demand. Except for a few oligosaccharides such as soy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/08A61K38/45C12R1/19
CPCC12N9/1051C12N15/70C12P19/08A61K38/00C12Y204/01005Y02A50/30
Inventor 马江锋姜岷孙涛蒋志明吴昊
Owner NANJING UNIV OF TECH
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