Preparation method of D-chiro-inositol

A technology of chiral inositol and myositol, applied in the field of bioengineering

Active Publication Date: 2019-05-03
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Yoshida et al. (Yoshida K, et al, Genetic modification of Bacillus subtilis for production of D-chiro-inositol, an investigational drug candidate for treatment of type 2diabetes and polycystic ovary syndrome. Appl Environ Microbiol 72:1310-1315, 2006.) using genetically modified Fermentative conversion of myo-inositol to D-chiro-inositol by Bacillus subtilis, but with only 6% yield

Method used

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  • Preparation method of D-chiro-inositol
  • Preparation method of D-chiro-inositol
  • Preparation method of D-chiro-inositol

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Embodiment 1

[0057] The cloning of embodiment 1iolG and iolI gene

[0058]In this example, the thermostable myo-inositol dehydrogenase is from Geobacillus kaustophilus, and the NCBI accession number is WP_011231389; the thermostable inositol monoketone isomerase is also from Geobacillus kaustophilus, and the NCBI accession number is WP_042380806. Geobacillus kaustophilus was purchased from China General Microorganism Culture Collection Center (CGMCC). These two genes were obtained from genomic DNA by PCR using different primers: Geobacillus kaustophilus was inoculated in 5 mL liquid nutrient gravy agar medium (peptone 10 g / L, beef extract 3 g / L, NaCl 5 g / L, adjusted to pH 7.0), cultured at 55°C until the logarithmic growth phase, and the genome was extracted using DNA Kit (Tiangen Biochemical Technology Co., Ltd., China). Use primers iolG-F (containing BamHI restriction site): 5′-CGCGGATCCGATGACTCGGGTGAAAGTAGG-3′ and iolG-R (containing SalI restriction site): 5′-ACGCGTCGACTTATTTTGACGGAGCT...

Embodiment 2

[0060] The construction of embodiment 2 recombinant engineering bacteria

[0061] (1) Construction of pETDuet-iolG

[0062] Digest the pETDuet-1 plasmid and the iolG gene containing two restriction sites obtained by PCR in Example 1 with BamHI / SalI double enzymes at 37°C, and recover the double-digested target fragment and expression vector respectively , which was ligated at 22° C. for 5 h under the action of T4 ligase. The ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformants were selected for colony PCR and double enzyme digestion identification, and 2-3 positive transformants were selected for further verification by sequencing. The sequencing results showed that pETDuet-iolG recombination was successfully obtained carrier.

[0063] The plasmid map of pETDuet-iolG is as follows Figure 3A shown.

[0064] (2) Construction of pET29a-iolI

[0065] Use NdeI / KpnI double enzymes to respectively digest the pET-29a(...

Embodiment 3

[0072] The preparation of the whole cell of embodiment 3 recombinant engineering bacteria

[0073] Recombinant engineered bacteria BL21(DE3) / pETDuet-iolG, BL21(DE3) / pET29a-iolI and BL21(DE3) / pETDuet-iolG-iolI were picked and inoculated in LB medium containing ampicillin, cultured overnight at 37°C with shaking . The culture was transferred to fresh LB medium containing ampicillin at 1% inoculum size, and cultured with shaking at 37°C until OD 600 =0.6-0.8, use the final concentration of 0.1mM IPTG to induce overnight at 18°C, centrifuge at 5500rpm for 10min, discard the supernatant, and obtain whole cells expressing thermostable myo-inositol dehydrogenase, expressing thermostable myo-inositol monoketone isomer Enzymes as well as whole cells co-expressing thermostable myo-inositol dehydrogenase and thermostable myo-inositol monoketone isomerase.

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Abstract

The invention discloses a method for preparing D-chiro-inositol from microorganism whole cells and / or lysate thereof for catalyzing myo-inositol. The method comprises the steps of constructing engineering bacteria for expressing heat-resisting myo-inositol 2-dehydrogenase and / or heat-resisting inosose isomerase genes, performing cell membrane permeability treatment and / or breaking on the engineering bacteria, and then converting the myo-inositol into the D-chiro-inositol by using the permeability engineering bacteria or the lysate thereof. Compared with a conventional method for producing theD-chiro-inositol, the method disclosed by the invention has the advantages of being simpler in production technology, lower in production cost, higher in yield and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for preparing D-chiro-inositol, in particular to a method for converting myo-inositol into D-chiro-inositol by catalyzing whole cells of microorganisms and / or their lysates Methods. Background technique [0002] D-chiro-inositol (DCI) is an epimer of myo-inositol (MI) ( figure 1 ), which mainly exist in leguminous plants in nature. Studies have shown that D-chiro-inositol plays an important role in the process of insulin signal transmission and is a functional factor that regulates the body's blood sugar. It can improve the body's sensitivity to insulin and glucose metabolism, so it can be used to treat type II diabetes . In addition, because D-chiro-inositol can reduce the male hormone in the blood and promote ovulation, there are many brands of D-chiro-inositol health products in the US market for the treatment of polycystic ovary syndrome. In addition, D-ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/02C12N15/63C12N15/53C12N15/61C12N1/21C12N1/19
Inventor 张以恒李元刘珊
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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