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Glycosyltransferase StUGT capable of catalyzing rebaudioside A to generate rebaudioside D

A technology of glycosyltransferase and glycosylation reaction, applied in the field of bioengineering, can solve problems such as poor catalytic efficiency, and achieve the effect of high catalytic efficiency

Active Publication Date: 2021-05-07
SINOCHEM HEALTH IND DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problem of poor catalytic efficiency of the enzyme solution capable of catalyzing rebaudioside A to generate rebaudioside D in the prior art, the present invention provides a novel recombinant strain containing the gene StUGT encoding a glycosyltransferase, and Induced expression of the recombinant bacteria, the obtained crude enzyme solution or purified enzyme protein, the crude enzyme solution or purified enzyme protein can be used to efficiently catalyze rebaudioside A to generate rebaudioside D

Method used

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  • Glycosyltransferase StUGT capable of catalyzing rebaudioside A to generate rebaudioside D
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  • Glycosyltransferase StUGT capable of catalyzing rebaudioside A to generate rebaudioside D

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Acquisition of Glycosyltransferase StUGT Gene and Construction of Recombinant Strain

[0068] The potato (Solanum tuberosum) glycosyltransferase amino acid sequence (GenBank: XP_006367681.1; SEQ ID NO: 2) and nucleic acid sequence (GenBank: XM_006367619.1; SEQ ID NO: 1) were downloaded from GenBank. The corresponding sequence is shown in Table 1, and the nucleic acid sequence is codon-optimized by GenScript Company, and the optimized synthetic gene is connected to the vector pET28a(+) through the restriction site Nco1 and Xho1 to obtain the plasmid pET28a(+)-StUGT, For plasmid map see figure 1 .

[0069] The obtained plasmid pET28a-StUGT was transformed into E.coli BL21 competent cells, using LB (1% peptone, 0.5% yeast powder, 1% NaCl, 1.6% agar powder) solid plate containing 50 μg / ml kanamycin. After screening, the screened monoclonal transformants were identified by colony PCR to obtain a recombinant strain E.coli BL21 (pET28a-StUGT).

[0070] The above recombinant...

Embodiment 2

[0076] Induced expression of recombinant strain and preparation of crude enzyme solution

[0077] Taking the recombinant strain BL21(DE3)(pET28a-StUGT) as an example, the expression mode of the glycosyltransferase gene StUGT in E. coli is illustrated.

[0078] Strain BL21(DE3) (pET28a-StUGT) was grown to OD in LB liquid medium (1% peptone, 0.5% yeast powder, 0.5% NaCl) containing 50 μg / ml kanamycin at 37°C, 220 rpm 600 In 0.4-1.2, isopropyl-β-D-thiogalactoside (IPTG) was added to make the final concentration of the bacterial solution 0.1-1 mM, and expression was induced at 18°C-30°C for 5-17h.

[0079] The culture of induced expression was centrifuged (12000rpm, 4°C, 10min), the supernatant was discarded, and the cell pellet was collected. The collected cells were washed once with 10 mM PBS (pH 7.2) to remove the remaining medium on the cells; the cells were resuspended with 10 mM PBS (pH 7.2) at a ratio of 1 / 20 of the original cell volume, The cells were disrupted by ultras...

Embodiment 3

[0082] StUGT catalyzes the glycosylation of rebaudioside A to recitidine D

[0083] Using Rebaudioside A as a substrate, StUGT catalyzes the reaction system to generate Rebaudioside D as follows: Add RebA with a final concentration of 1-100 g / L, and uridine diphosphate glucose (UDPG) with a final concentration of 1-3 mM. ) with a final concentration of 3 mM Mg 2+ (magnesium chloride), add the crude enzyme liquid of the recombinant bacteria StUGT obtained in Example 2.

[0084] After the glycosylation reaction system is prepared, the reaction is allowed to stand at 18-45°C for 6-48h. After the reaction was completed, 500 μL of 60% (v / v) acetonitrile was added, and after shaking and mixing, centrifuged at 12,000 rpm for 10 min at room temperature, and the supernatant was passed through a 0.2 μm organic membrane and then analyzed by HPLC. HPLC used a Luna C18 reverse-phase bonded silica gel separation column (4.6mm×250mm, 5μm), the mobile phase was acetonitrile: sodium phosphat...

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Abstract

The invention belongs to the field of bioengineering and provides use of a nucleotide sequence for coding glycosyltransferase StUGT in preparing a recombinant protein capable of catalyzing rebaudioside A to generate rebaudioside D. The nucleotide sequence is as follows: a) a nucleotide sequence as shown in SEQ ID NO.1; or a nucleotide sequence which is different from the nucleotide sequence in a) and capable of encoding an amino acid sequence shown in SEQ ID NO.2. After a gene of the glycosyltransferase StUGT is connected to an expression vector, the gene is transferred into a host cell to obtain a recombinant bacterium. The recombinant protein takes UDPG as a glycosyl donor, the substrate rebaudioside A is catalyzed to generate rebaudioside D, and high catalytic efficiency can be achieved.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a glycosyltransferase and a gene encoding the glycosyltransferase, and the application of a recombinant strain in the production of glycoside compounds. Background technique [0002] Rebaudioside D (RebD) is a natural non-calorie sweetener extracted from stevia, which is 350 times sweeter than sucrose. Compared to most other steviol glycosides (SGS), it has a slightly better mouthfeel and a much shorter after-bitterness. However, pure rebaudioside D has poor water solubility, and the content of rebaudioside D in leaves is very low, only about 0.5% in stevia. The extraction method from stevia leaves cannot meet the market demand for rebaudioside D Therefore, the use of biocatalysis to obtain sufficient rebaudioside D has attracted extensive attention. [0003] Glycosyltransferases are a large class of enzymes widely found in nature, which can catalyze the linking of activated sugar...

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
CPCC12N9/1048C12N15/70C12P19/56
Inventor 马媛媛汪振洋来庆英宋浩魏晓珍
Owner SINOCHEM HEALTH IND DEV CO LTD
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