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High-purity steviol glycosides

A technology of steviol glycosides and steviol, which is applied in the direction of preparation of sugar derivatives, sugar derivatives, and sugar derivatives, and can solve problems such as unsuitable for commercial use

Pending Publication Date: 2020-05-01
PURECIRCLE USA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although methods for preparing steviol glycosides from Stevia rebaudiana are known, 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

[0163] Protein sequences of engineered enzymes used in biocatalytic methods

[0164] SEQ ID 1:

[0165] >SuSy_At, variant PM1-54-2-E05 (engineered sucrose synthase; source of WT gene: Arabidopsis)

[0166]MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELVNLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYL...

Embodiment 2

[0174] Expression and formulation of the SuSy_At variant of SEQ ID 1

[0175] 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.

[0176] 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.

[0177] Cells were harvested by centrifugation (3220xg, 20 minutes, 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 (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 minutes, 4°C). The supernatant was sterilized b...

Embodiment 3

[0180] Expression and formulation of UGTS1 variants of SEQ ID 2

[0181] The gene encoding the UGTS1 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.

[0182] 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.

[0183] Cells were harvested by centrifugation (3220xg, 20 minutes, 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 200 optical density (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 minutes, 4°C). The supernatant was sterilized by filtration...

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Abstract

Methods of preparing highly purified steviol glycosides, particularly rebaudiosides M, D, E and / are described. The methods include utilizing enzyme preparations and recombinant microorganisms for converting various staring compositions to target steviol glycosides. The highly purified rebaudiosides are useful as non-caloric sweetener in edible and chewable compositions, such as any beverages, confectioneries, bakery products, cookies, chewing gums, etc.

Description

technical field [0001] The present invention relates to methods of preparing steviol glycoside-containing compositions, including high purity steviol glycoside compositions. [0002] sequence listing [0003] A 15 kb data-sized text file titled "PC_71PROV_Seq_Listing_ST25.txt" created on May 15, 2017, filed concurrently with this document, is hereby incorporated by reference into this application in its entirety. [0004] Background of the invention [0005] High intensity sweeteners have sweetness levels many times that of sucrose. They are essentially non-caloric and commonly used in diets and reduced-calorie products, including food and beverages. High-intensity sweeteners do not elicit a glycemic response, making them suitable for use in products aimed at diabetics and others who want to control their carbohydrate intake. [0006] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub in the family Asteraceae (Co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23L27/30C07H15/256C12N1/19C12N9/10C12N15/52
CPCC07H15/256A23L27/30C12N9/1048C12N9/1062C07H1/00C12P19/56A23L27/36A23L2/60A23V2002/00
Inventor A·马科斯雅恩S·普尔卡亚萨C·拜尔A·沃格S·克普克S·巴特施B·布鲁彻C·菲勒S·肖纳特M·萨洛莫T·舒尔钦
Owner PURECIRCLE USA
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