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Immobilized glucosyltransferase, preparation method thereof and method for catalytic production of rebaudioside D

A technology of glucosyl and transferase, applied in the field of bioengineering, can solve the problems of difficult product separation, the enzyme cannot be reused, etc., and achieves the effects of good reusability and reduced inactivation rate.

Pending Publication Date: 2020-03-10
TIANJIN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention overcomes the deficiency that the enzyme existing in the prior art cannot be reused and is not easy to be separated from the product, and provides an immobilized glucosyltransferase

Method used

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  • Immobilized glucosyltransferase, preparation method thereof and method for catalytic production of rebaudioside D
  • Immobilized glucosyltransferase, preparation method thereof and method for catalytic production of rebaudioside D

Examples

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Effect test

Embodiment 1

[0025] Example 1 Construction of Recombinant Plasmid pET28a-UGT1 and Induced Expression and Purification of Glucosyltransferase UGT1

[0026] Preparation of glucosyltransferase UGT1: The NCBI database was used to screen the glucosyltransferase gene sequence from rice, and the codon optimization of the gene with the accession number AK121682 in the Gene Bank database was performed. The codon optimization work was completed by Gen Script. Synthesize the nucleotide sequence shown in SEQ ID NO.1 (secretly expressing the gene sequence of glucose transferase UGT1) by artificially synthesizing double-stranded DNA molecules in vitro, and design the NcoI that is inserted into the pET-28a vector (commodity) and XhoI restriction site, the plasmid pET28a-UGT1 was obtained.

[0027] The recombinant plasmid pET28a-UGT1 was transformed into Escherichia coli BL21 strain to obtain the engineered strain E.coli BL21-pET-28a(+)-UGT1 expressing glucosyltransferase UGT1, and a single colony carryin...

Embodiment 2

[0031] A preparation method for immobilized glucosyltransferase, comprising the following steps:

[0032] (1) be that the acetic acid aqueous solution of 2% is solvent with volume concentration, preparation concentration is the chitosan solution of 40g / L, described chitosan solution is dripped in the NaOH aqueous solution of 2.5M, leave standstill 3 hours, prepare Form into pellets with a diameter of 0.5-3.5 mm, rinse the pellets with water until no NaOH remains, and obtain chitosan pellets;

[0033] (2) According to the ratio of 1g:7mL, chitosan pellets are mixed with glutaraldehyde aqueous solution of 5% volume concentration, cross-linked and activated at room temperature for 3 hours, and washed with water to obtain glutaraldehyde-crosslinked chitosan pellets. ball;

[0034] (3) According to the ratio of 1g:7mL, mix the chitosan pellets cross-linked by glutaraldehyde with the aqueous solution of glucosyltransferase UGT1 at a concentration of 30 μg / mL, react at 20° C. for 20...

Embodiment 3

[0036] A preparation method for immobilized glucosyltransferase, comprising the following steps:

[0037] (1) With the volumetric concentration of 1% acetic acid aqueous solution as a solvent, prepare a chitosan solution with a concentration of 20g / L, drop the chitosan solution into a 1M NaOH aqueous solution, and leave it for 2 hours to prepare For pellets with a diameter of 0.5-3.5mm, wash the pellets with water until no NaOH remains, and obtain chitosan pellets;

[0038] (2) According to the ratio of 1g:2mL, chitosan pellets were mixed with glutaraldehyde aqueous solution with a volume concentration of 0.5%, cross-linked and activated at room temperature for 5 hours, and washed with water to obtain glutaraldehyde-crosslinked chitosan pellets. ball;

[0039] (3) According to the ratio of 1g:5mL, mix the chitosan pellets cross-linked by glutaraldehyde with the aqueous solution of glucosyltransferase UGT1 at a concentration of 6 μg / mL, react at 4°C for 25h under stirring, and...

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Abstract

The invention discloses immobilized glucosyltransferase, a preparation method thereof and a method for catalytic production of rebaudioside D. The preparation method comprises the following steps: (1)preparing a chitosan solution, dropwisely adding the chitosan solution into a NaOH aqueous solution, standing the solution, preparing pellets, and washing the pellets to obtain chitosan pellets, (2)mixing the chitosan pellets and a glutaraldehyde aqueous solution, cross-linking and activating the mixed solution and carrying out cleaning to obtain glutaraldehyde cross-linked chitosan pellets and(3) uniformly mixing the glutaraldehyde cross-linked chitosan pellets and a glucosyltransferase aqueous solution, carrying out a reacting process, filtering the product and cleaning the product with water to obtain immobilized glucosyltransferase. The immobilized glucosyltransferase is easy to separate from a reaction product, so that the separation and purification of the product and the recoveryof the enzyme are facilitated. Recycling is realized, after the immobilized enzyme is used for the third time, the enzyme activity is gradually increased and the RD yield is gradually increased alongwith the increasing of the repeated operation times of the immobilized enzyme, and after the immobilized enzyme is repeatedly used for 8 times, the enzyme activity of the immobilized enzyme is gradually reduced and the RD yield is reduced, so that the reusability is better.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an immobilized glucosyltransferase, a preparation method and a method for catalyzing the production of rebaudioside D. Background technique [0002] At present, diabetic patients in the world account for about 9% of the total population over the age of 18, and the number of obese patients exceeds 2 billion. These people need to strictly control their sugar intake in their diet. However, most of the non-caloric sugar substitutes currently on the market have health risks and are not suitable for long-term use. Therefore, the market needs new and safe sugar substitute sweeteners. Stevioside is a class of stevioside compounds extracted and separated from the leaves of Stevia rebaudiana. Because of its high sweetness, low calorie, and non-toxicity, it has been certified as a safe food additive by the US Food and Drug Administration. Recognized by more and more nati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12P19/56C12P19/18
CPCC12N11/10C12N9/1051C12Y204/01C12P19/56C12P19/18
Inventor 宋浩刘伟刘文斌马媛媛洪解放汪振洋来庆英
Owner TIANJIN UNIV
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