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Glycosyltransferase UGTZJ1 mutant and application thereof

A glycosyltransferase and mutant technology, applied in the field of genetic engineering, can solve the problems of high enzyme production cost, large amount of recombinant bacteria, energy consumption and high cost of preparation, so as to simplify the process, increase the yield and avoid separation and purification Effect

Active Publication Date: 2020-08-11
广东广业清怡食品科技股份有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of recombinant cells in the reaction system requires the addition of toluene or other cell permeabilizers; or the use of recombinant cell lyophilized powder, and the preparation of lyophilized powder consumes energy and costs high
Although the process is optimized in patent CN201410019981.4, the addition ratio of glycosyltransferase and sucrose synthase needs to be adjusted, the amount of recombinant bacteria is large, and the cost of enzyme production is high

Method used

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  • Glycosyltransferase UGTZJ1 mutant and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The construction of embodiment 1 glycosyltransferase UGTZJ1 mutant

[0035] The amino acid sequence of the glycosyltransferase UGTZJ1 mutant provided in this example is shown in any one of SEQ ID NO.2-4.

[0036] Genes encoding the above mutants.

[0037]The preparation method of the above-mentioned glycosyltransferase UGTZJ1 mutant, the 48th asparagine in the amino acid sequence of the glycosyltransferase UGTZJ1 shown in SEQ ID NO.1 is mutated into threonine or glycine, or the 75th glycine Mutated to histidine.

[0038] It can also be a carrier or cell line carrying the above-mentioned genes and genetically engineered bacteria.

[0039] The genetically engineered bacterium uses Escherichia coli as a host and simultaneously expresses the sucrose synthase gene and the above-mentioned glycosyltransferase UGTZJ1 mutant.

[0040] The sucrose synthase gene of the present invention can be a gene with sucrose synthase activity from any source, such as the sucrose synthase S...

Embodiment 2

[0056] Induced expression of embodiment 2 genetically engineered bacteria and preparation of crude enzyme solution

[0057] The different genetically engineered bacteria that 4 strains appearing in embodiment 1 respectively contain wild-type UGTZJ1 gene and mutant gene thereof are respectively inoculated into 5mL LB liquid culture medium (LB (g / L): peptone 10 , sodium chloride 10, and yeast extract 5) in a 50-mL shaking tube, cultured on a shaker at 37° C. for 8 hours at a constant temperature of 200 rpm. Then insert the culture solution into a 500mL shake flask containing 100mL induction medium TB (TB (g / L): yeast powder 25, tryptone 15, sodium chloride 10, glucose 2, lactose 3) according to 4% inoculum Incubate at 200rpm, 37°C for 2h, until OD 600 When it reaches about 0.2, turn to 16°C to induce 24h and centrifuge to collect the bacteria. Sonicate the bacteria, centrifuge and take the supernatant as a crude enzyme solution, and store it in a 4°C refrigerator for later use...

Embodiment 3

[0062] The influence of embodiment 3 temperature on catalytic reaction

[0063] The crude enzyme solution obtained after the induced expression of the genetically engineered bacteria E.coli BL21(DE3) / pET28a-UGTZJ1(N48T)-StSUS1 with the highest yield obtained in Example 2 was used for the reaction. In a 50mL Erlenmeyer flask, 10mL of the reaction mixture contained 20g / L rebaudioside A, 60g / L sucrose, 50mM sodium phosphate buffer at pH 7.0, 3mg / mL crude enzyme solution, and reacted at 20°C, 30°C, Carried out at 37°C, 42°C, 50°C and 200rpm. The reaction was started by adding crude enzyme solution, and after 16 hours of reaction, samples were taken and heated at 95°C for 5 minutes to inactivate the enzyme, and the supernatant was collected by centrifugation. The content of rebaudioside D in the reaction solution at different reaction temperatures was measured.

[0064] The content of rebaudioside D in the reaction solution under the different reaction temperatures of table 2

[...

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Abstract

The invention discloses a glycosyltransferase UGTZJ1 mutant. The amino acid sequence of the mutant is shown as any one of SEQ ID NO.2-4, and a gene for encoding the mutant and a preparation method ofthe mutant are also disclosed. The invention also discloses a vector, a cell and a genetically engineered bacterium containing the mutant encoding gene. The invention also discloses applications of the glycosyltransferase UGTZJ1 mutant, the gene for encoding the mutant, the vector and a cell line comprising the mutant encoding gene, particularly the genetically engineered bacterium containing theglycosyltransferase UGTZJ1 mutant, and the like in the aspect of catalyzing rebaudioside A to generate rebaudioside D. The yield of the rebaudioside D, which is generated by catalyzing the rebaudioside A through the genetically engineered bacterium containing the mutant, is increased by about 4 times compared with that of a wild type, and the yield of the rebaudioside D can reach 47.56 g / L under the optimal reaction condition.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a glycosyltransferase UGTZJ1 mutant and application thereof. Background technique [0002] With the increasing awareness of people's health concerns, consumers gradually reduce the intake of sucrose, and the food industry is committed to the development of natural sweeteners with low calorie value and high sweetness. Stevioside is a class of steviol glycosides extracted and separated from the leaves of Stevia rebaudiana. Because of its advantages of high sweetness, low calorie, non-toxicity, high temperature resistance, acid and alkali resistance and good water solubility, it has been favored by the scientific community and industry. It has received extensive attention in many fields such as the world, and has been recognized as a new generation of sweeteners. The leaves of Stevia rebaudiana contain various natural sweet glycosides such as stevioside, reba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/56C12R1/19
CPCC12N9/1048C12P19/56
Inventor 韩诗蕾王三永李春荣曾伟山叶鹏菁吕琪严明魏淼陈圣章志林陈晶晶
Owner 广东广业清怡食品科技股份有限公司
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