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Method for preparing D-chiro-inositol using microbes

A chiral inositol and inositol technology, applied in biochemical equipment and methods, chemical instruments and methods, and the use of vectors to introduce foreign genetic material, etc., can solve problems such as high price, poor economy, and difficult to separate by-products

Active Publication Date: 2016-01-06
(株)韩国迪外天然健康
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the price of D-pinitol or kasugamycin as a raw material is high, and there is also a well-known organic synthesis method (U.S.PatNo.5406005, W096 / 25381), because it is difficult to separate by-products, it is economically poor

Method used

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  • Method for preparing D-chiro-inositol using microbes
  • Method for preparing D-chiro-inositol using microbes
  • Method for preparing D-chiro-inositol using microbes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Cloning of the inositol transporter gene and preparation of a recombinant vector comprising the transporter

[0073] It has been reported that Bacillus subtilis, Salmonella typhimurium, and Agrobacterium tumefaciens, which use inositol as a carbon source, respectively cloned the major of myo-inositol transporter. Genes and minor genes.

[0074] In Bacillus subtilis (B.subtilis), the inositol transporter genes are iolT gene and iolF gene; in Salmonella typhimurium (S.typhimurium), the inositol transporter genes are iolT1 gene and iolT2 gene; In Bacillus (A. tumefaciens), the inositol transporter genes are Atu5935 gene and Atu2525 gene. The information of the above genes is shown in Table 1.

[0075] Table 1

[0076]

[0077] 1-1. Preparation of recombinant vectors pACYCD-BsiolT(F2) and pACYCD-BsiolT-BsiolF(F2)

[0078] In the case of the B. subtilis strain, the major transporter iolT and the minor transporter iolF were introduced into pACYCDuet-1 from t...

Embodiment 2

[0091] Example 2: Evaluation of the activity of the inositol transporter

[0092] As prepared in the above Example 1, each recombinant plasmid containing the inositol transporter derived from Bacillus subtilis (B. subtilis), Salmonella typhimurium (S. typhimurium), Agrobacterium tumefaciens (A. tumefaciens) , pACYCD-BsiolT(F2), pACYCD-BsiolT-BsiolF(F2), pACYCD-StiolT1(F2), pACYCD-StiolT1-StiolT2(F2), pACYCD-Atu5935(F2), pACYCD-Atu5935-Atu2525(F2) and containing The recombinant plasmid pCOLAD-sAtiep-sAtiepf, which converts inositol into D-chiro-inositol and the gene encoding inositol dehydrogenase and inositol isomerase, is transformed into Escherichia coli (E.coli) BL21 (DE3) together to prepare transformed strains.

[0093] 5 mL of the prepared recombinant transformed strain was cultured in a glass test tube with a diameter of 25 mm and a height of 150 mm. The culture conditions are as follows: utilize the M9 minimum culture medium that contains the inositol of 1% (w / v), th...

Embodiment 3

[0095] Example 3: Cloning of inositol dehydrogenase and inositol isomerase genes and preparation of recombinant vectors comprising them

[0096] Inositol is converted to D-chiro-inositol through the continuous reaction of the following reaction formula 1 to reaction formula 3. Inositol dehydrogenase (inositoldehydrogenase) catalyzes the reactions of reaction formula 1 and reaction formula 3, and inososeisomerase (inososeisomerase) catalyzes the reaction of reaction formula 2.

[0097] [Reaction 1]

[0098] Inositol + nicotinamide adenine dinucleotide (oxidized state) (NAD + ) 2-keto-inositol + nicotinamide adenine dinucleotide (reduced state) (NADH) + H +

[0099] [Reaction 2]

[0100] 2-Keto-Inositol 1-Keto-D Chiral Inositol

[0101] [reaction formula 3]

[0102] 1-Keto-D-chiro-inositol + nicotinamide adenine dinucleotide (reduced state) (NADH) + H + D-chiro-inositol + nicotinamide adenine dinucleotide (oxidized state) (NAD + )

[0103] Cloned from A. tumefaciens (...

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Abstract

The present invention relates to a method for preparing D-chiro-inositol from myo-inositol by using a transformed host cell which expresses enzymes such as a myo-inositol transporter, inositol dehydrogenase, and inosose isomerase. According to the method of the present invention, myo-inositol can be converted into D-chiro-inositol at a high yield.

Description

technical field [0001] The present invention relates to a method for producing D-chiro-inositol using microorganisms. In more detail, it relates to a method for producing D-chiro-inositol from myo-inositol based on transformed microbial host cells. Background technique [0002] D-chiro-inositol (cis-l,2,4-trans-3,5,6-cyclohexol) as myo-inositol (cis-l,2,3,5-trans -4,6-cyclohexanehexol) stereoisomer, which is the epimerized form of the 3rd hydroxyl group ( figure 1 ). According to reports, D-chiro-inositol is the main component of inositol phosphoglycan (IPG), and is an important mediator of insulin signal transmission. It is well known that the above-mentioned D-chiro-inositol has a therapeutic effect on type 2 diabetes. D-chiro-inositol is mainly found in eukaryotes, and its biosynthesis is achieved by epimerization of inositol. [0003] D-chiroinositol is mainly produced by hydrolysis of D-pinitol or kasugamycin (U.S. Pat No. 5827896, U.S. Pat No. 5091596, U.S. Pat No....

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/63C12N1/21C12P7/18
CPCC12N9/0006C12N9/90C12P7/18C12Y101/01018C12N1/20C12N15/52C12N15/63C07K14/195C07K14/255C07K14/32C12P7/02
Inventor 明贤君尹相活李炫抒
Owner (株)韩国迪外天然健康
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