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High-purity steviol glycosides

A technology of steviol glycoside and steviobioside, which is applied in sugar derivatives, enzymes, organic chemistry, etc., and can solve problems such as inapplicability

Pending Publication Date: 2021-03-16
PURECIRCLE USA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although methods for preparing steviol glycosides from Stevia rebaudiana are known, many of these methods are not commercially available

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0729] Protein sequences of engineered enzymes used in biocatalytic methods

[0730] SEQ ID 1:

[0731] >SuSy_At, variant PM1-54-2-E05 (engineered sucrose synthase; WT gene source: Arabidopsis)

[0732] MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELVNLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYLEM...

Embodiment 2

[0740] Expression and formulation of the SuSy_At variant of SEQ ID 1

[0741]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.

[0742] Cells were cultured at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / l). IPTG (0.2 mM) induced gene expression in log phase and at 30°C and 200 rpm for 16-18 hours.

[0743] Cells were harvested by centrifugation (3220xg, 20min, 4°C) and washed with cell lysis buffer (100mM Tris-HClpH7.0; 2mM MgCl 2 , DNA nuclease 20U / mL, lysozyme 0.5mg / mL) resuspended to an optical density of 200 (at 600nm (OD 600 ) measured at ). Cells were then disrupted by sonication and the crude extract was separated from cell debris by centrifugation (18000 xg for 40 min, 4°C). The supernatant was sterilized by filtration...

Embodiment 3

[0746] Expression and formulation of the UGTS12 variant of SEQ ID 2

[0747] 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.

[0748] Cells were cultured at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / l). Gene expression was induced in log phase with IPTG (0.1 mM) at 30°C and 200 rpm for 16-18 hours.

[0749] Cells were harvested by centrifugation (3220xg, 20min, 4°C) and washed 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 the crude extract was separated from cell debris by centrifugation (18000 xg for 40 min, 4°C). The supernatant was sterilized by filtrati...

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Abstract

Methods of preparing highly purified steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudiosideB, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, 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 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 21, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and / or SvG7 are described. The methods include utilizing enzyme preparations and recombinant microorganisms for converting various staring compositions to target steviol glycosides. Highly purified steviol glycosides are useful as non-caloric sweeteners, flavor enhancers, sweetness enhancers and foam inhibitors in edible and chewable compositions such as any beverage, confectionery, bakery, biscuits and chewing gums.

Description

technical field [0001] The present invention relates to methods for preparing steviol glycoside-containing compositions, including highly purified steviol glycoside compositions. [0002] Background of the invention [0003] High intensity sweeteners have a sweetness level that is many times greater than the sweetness level of sucrose. They are primarily non-caloric and are often used in meals and reduced-calorie products, including food and beverages. High-intensity sweeteners do not elicit an insulin-elevating response, making them suitable for products targeting diabetes and others beneficial in controlling their sugar intake. [0004] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub in the family Asteraceae (Compositae) native to certain regions of South America. Their structure is characterized by the monobasic steviol, distinguished by the presence of carbohydrate residues at the C13 and C19 positions. Th...

Claims

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Application Information

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IPC IPC(8): A23L27/30A23L2/60C07H15/256
CPCA23L2/60C07H15/256A23L27/36C12P19/56A23L27/88A23L27/30C12N9/1051C12N9/1048
Inventor A·马科斯雅恩S·Y·周K·尼扎姆宾纳维K·克汗M·阿弗扎尔宾哈希姆S·A/L·拉曼达赫
Owner PURECIRCLE USA
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