Method for preparing L-ascorbyl-2-glucoside

A technology of sucrose and AA-2G, applied in the field of biocatalyst preparation of L-ascorbic acid-2-glucoside and preparation of L-ascorbic acid-2-glucoside, can solve the problems of poor water solubility, unfavorable conversion, high price problem

Active Publication Date: 2017-08-18
山东格得生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The third is that the glycosyl donor cyclodextrin has defects such as poor water solubility and high price, which is not conducive to conversion and high raw material cost
Although later US patents US20060216792 (2006) and US8759030 (2014) reported that in order to avoid the formation of AA-5G and AA-6G by-products in the catalytic reaction of CGTase, the use of α-isomaltosylglucose derived from Arthrobacter globiformis A19 The base sugar forming enzyme is used as a catalyst to prepare AA-2G, but the enzyme source is not easy to obtain

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Embodiment 1, the construction of sucrose phosphorylase clone strain pet28 a(+)-spase

[0097] The cryopreserved Bifidobacterium longum was revived and subcultured twice, and the bacterial liquid was used as a DNA template. A pair of primers were designed using the sucrose phosphorylase gene sequence of Bifidobacterium longum on NCBI as a template:

[0098] Forward primer (spase-bam-F): 5’ CGCGGATCCATGAAAAACAAAGTGCAGCTCATC 3’

[0099] Reverse primer (spase-hind-R): 5’ CCCAAGCTTGTCGATATCGGCAATCGG 3’

[0100] Then carry out PCR reaction, reaction conditions: 95°C pre-denaturation for 1min, then 30 cycles (95°C 10S, 49°C-64°C 12S, 72°C 22S), and 72°C extension for 5min.

[0101] In the site-directed mutagenesis experiment of sucrose phosphorylase, different mutation primers were designed and the above-mentioned PCR reaction was carried out.

[0102] After the PCR reaction is completed, after gel recovery, verification and purification, the product of the PCR reaction an...

Embodiment 2

[0104] Embodiment 2, the construction of sucrose phosphorylase clone strain pet28 a(+)-spase

[0105] The cryopreserved Bifidobacterium longum was revived and subcultured twice, and the bacterial liquid was used as a DNA template. A pair of primers were designed using the sucrose phosphorylase gene sequence of Bifidobacterium longum on NCBI as a template:

[0106] Forward primer (spase-bam-F): 5’ CGCGGATCCATGAAAAACAAAGTGCAGCTCATC 3’

[0107] Reverse primer (spase-hind-R): 5’ CCCAAGCTTGTCGATATCGGCAATCGG 3’

[0108] Then carry out PCR reaction, reaction conditions: pre-denaturation at 92°C for 2 minutes, then 40 cycles (92°C for 10S, 49°C to 64°C for 10S, 68°C for 25S), and extension at 68°C for 7 minutes.

[0109] In the site-directed mutagenesis experiment of sucrose phosphorylase, different mutation primers were designed and the above-mentioned PCR reaction was carried out.

[0110] After the PCR reaction is completed, after gel recovery, verification and purification, the...

Embodiment 3

[0112] Embodiment 3, the construction of sucrose phosphorylase clone strain pet28 a(+)-spase

[0113] The cryopreserved Bifidobacterium longum was revived and subcultured twice, and the bacterial liquid was used as a DNA template. A pair of primers were designed using the sucrose phosphorylase gene sequence of Bifidobacterium longum on NCBI as a template:

[0114] Forward primer (spase-bam-F): 5’ CGCGGATCCATGAAAAACAAAGTGCAGCTCATC 3’

[0115] Reverse primer (spase-hind-R): 5’ CCCAAGCTTGTCGATATCGGCAATCGG 3’

[0116] Then carry out PCR reaction, reaction conditions: 98°C pre-denaturation for 0.5min, then 20 cycles (98°C 15S, 49°C-64°C 15S, 75°C 18S), 75°C extension for 3min.

[0117] In the site-directed mutagenesis experiment of sucrose phosphorylase, different mutation primers were designed and the above-mentioned PCR reaction was carried out.

[0118] After the PCR reaction is completed, after gel recovery, verification and purification, the product of the PCR reaction and th...

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Abstract

The invention relates to a method for preparing L-ascorbyl-2-glucoside by virtue of an engineering strain of recombinant sucrose phosphorylase. The engineering strain of the sucrose phosphorylase is preserved in Wuhan University in Wuhan of China, postcode 430072, with preservation number of CCTCC M2016496. The engineering strain of the sucrose phosphorylase is constructed by virtue of gene recombination and site-specific mutagenesis technologies. With strain cells, obtained from fermentation, serving as a biological catalyst, AA-2G is prepared from sucrose and L-ascorbic acid in a trisodium citrate buffer solution system. According to the method provided by the invention, the sucrose, which is directly used as a glycosyl donor, is easily soluble in water and is cheap and easily available. Meanwhile, the reaction product, which mainly includes the AA-2G, is single; a yield reaches 65% or above; and additional treatment of adding saccharifying enzyme is avoided, so that production cost is reduced.

Description

[0001] (1) Technical field [0002] The invention relates to the technical field of biocatalyst preparation of L-ascorbic acid-2-glucoside, in particular to a method for preparing L-ascorbic acid-2-glucoside. [0003] (2) Background technology [0004] L-Ascorbic acid-2-glucoside, namely 2-O-α-D-glucopyranosyl-L-ascorbic acid (2-O-α-D-glucopyranosyl-L-ascorbid acid, AA-2G), is produced by A glucoside derivative of L-ascorbic acid (i.e. vitamin C) jointly discovered by Hayashibara Institute of Biochemistry in Japan and the Department of Pharmacy, Okayama University (J. Biochem. 107,222-227 (1990); Agric. Biol. Chem. , 54 (7), 1697-1703 (1990); US5084563 (1992)). It has the physiological activity of ascorbic acid. At the same time, because the hydroxyl group on the 2-position C of L-ascorbic acid is connected with glucose by α-1, 4 glycosidic bonds to form a stable and stable glycosidic bond, it significantly reduces the reducibility and stability of L-ascorbic acid. Greatly im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/10C12P19/60C12R1/19
CPCC12N9/1051C12N15/70C12N2800/101C12P19/60C12Y204/01007
Inventor 黄爱清王永桥宋长红王金才朱嘉震王洪果史峻嵩
Owner 山东格得生物科技有限公司
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