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High-purity steviol glycosides

A technology of steviol glycosides and steviol, which is applied in the direction of sugar derivatives, sugar derivatives, glycosyltransferases, etc., and can solve problems such as not being suitable for commercial use

Pending Publication Date: 2021-08-06
PURECIRCLE USA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although methods are known for the preparation of steviol glycosides from Stevia rebaudiana, many of these methods are not suitable for commercial use

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0484] Protein sequences of engineered enzymes for use in biocatalytic methods

[0485] SEQ ID 1 :

[0486] >SuSy_At, variant PM1-54-2-E05 (engineered sucrose synthase; source of WT gene: Arabidopsis)

[0487]

[0488] SEQ ID 2. :

[0489] > UGTS12, variant 0234 (engineered glucosyltransferase; source of WT gene: tomato)

[0490]

[0491] SEQ ID 3 :

[0492] > UGT76G1, variant 0042 (engineered glucosyltransferase; source of WT gene: Stevia)

[0493]

Embodiment 2

[0495] Expression and formulation of the SuSy_At variant of SEQ ID 1

[0496] The gene encoding the SuSy_At variant of SEQ ID 1 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0497] Cells were cultured at 37°C in ZYM505 medium (F. William Studier, "Protein Expression and Purification", Vol. 41, 2005, pp. 207-234) supplemented with kanamycin (50 mg / l). Gene expression was induced by IPTG (0.2 mM) in log phase and performed at 30° C. and 200 rpm for 16-18 hours.

[0498] by centrifugation ( 20 min, 4°C) to harvest the cells and wash with cell lysis buffer (100mM Tris-HCl, pH 7.0; 2mM MgCl 2 , DNA nuclease 20U / mL, lysozyme 0.5mg / mL) resuspended to an optical density of 200 (measured at 600nm (OD 600 )). Cells were then disrupted by sonication, and crude extracts were separated from cell debris by centrifugation (18000 xg, 40 min, 4°C). Sterilize the superna...

Embodiment 3

[0501] Expression and formulation of the UGTS12 variant of SEQ ID 2

[0502] The gene encoding the UGTS12 variant of SEQ ID 2 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0503] Cells were cultured at 37°C in ZYM505 medium (F. William Studier, "Protein Expression and Purification", Vol. 41, 2005, pp. 207-234) supplemented with kanamycin (50 mg / l). Gene expression was induced by IPTG (0.1 mM) in log phase and carried out at 30°C and 200 rpm for 16-18 hours.

[0504] Cells were harvested by centrifugation (3220×g, 20 min, 4°C) and washed with cell lysis buffer (100 mM Tris-HCl, pH 7.0; 2 mM MgCl 2 , DNA nuclease 20U / mL, lysozyme 0.5mg / mL) resuspended to an optical density of 200 (measured at 600nm (OD 600 )). Cells were then disrupted by sonication, and crude extracts were separated from cell debris by centrifugation (18000 xg, 40 min, 4°C). Sterilize the ...

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Abstract

Methods of preparing highly purified steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 2a and / or SvG7 are described. The methods include utilizing enzyme preparations and recombinant microorganisms for converting various staring compositions to target steviol glycosides. The highly purified steviol glycosides are useful as non-caloric sweetener, flavor enhancer, sweetness enhancer, and foaming suppressor in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

Description

[0001] sequence listing [0002] The text file titled "39227_80PROV_Sequence_Listing_ST25.txt," created on November 27, 2018, having 15 kilobytes of data and filed concurrently with this document, is hereby incorporated by reference in its entirety into this application. technical field [0003] The present invention relates to a method for preparing steviol glycoside-containing compositions, including highly purified steviol glycoside compositions. Background technique [0004] High-intensity sweeteners have sweetness levels many times higher than those of sucrose. They are essentially non-caloric and are often used in weight loss and reduced calorie products, including food and beverages. High-intensity sweeteners do not elicit a glycemic response, making them suitable for products aimed at diabetics and others interested in controlling their carbohydrate intake. [0005] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a per...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H1/00C07H15/256C12P19/56
CPCC07H15/256C12P19/56C07H1/00C12N9/1051A23L27/00C12Y204/01C12Y204/01013A23L27/88A23L29/30A23L27/36A23L2/56A23L2/60A23V2002/00B01D19/0495
Inventor 阿韦季克·马尔科西亚周绍银海尔·尼扎姆本纳威克里斯蒂娜·查汗穆罕默德·阿夫扎尔本哈西姆沙拉瓦南·A·L·拉曼达赫
Owner PURECIRCLE USA
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