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Method for synthesizing ursodeoxycholic acid and high-chiral-purity D-amino acid based on enzyme-method coupling technology

A technology of ursodeoxycholic acid and chenodeoxycholic acid, which is applied in the field of biocatalysis, can solve the problems of complicated treatment process, large environmental pollution, and high cost, achieve mild reaction conditions, simplify the reaction process, and reduce the cost of enzymes Effect

Pending Publication Date: 2018-12-21
HUNAN BAOLISHI BIOTECH
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Problems solved by technology

The chemical method prepares UDCA through the oxidation and reduction reactions of CDCA. A large amount of organic solvents and redox reagents are used in the middle, and the treatment process is complicated, and the waste water produced is difficult to treat.
Chinese patent CN105861613A reports the method of preparing UDCA by enzymatic method, but a large amount of organic reagents are added to the reaction system, and the solvent needs to be recovered during the production process
At present, there are mainly two ways to prepare D-amino acid by chemical method and enzymatic method. Among them, the chemical method has severe reaction conditions, great environmental pollution, high cost, and is not suitable for large-scale industrial production.
The enzymatic preparation technology is mainly divided into the following two types: first, using D and L amino acids as raw materials, adopting enzymatic resolution technology, this method requires splitting reagents, and the yield is not high; the second is using hydra as raw material , prepared by the joint action of hydantoinase and carbamoyl hydrolase. The disadvantage of this method is that the product has limitations, and it is more suitable for D-amino acids with benzene rings, and the enzymes used are easily oxidized and inactivated

Method used

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  • Method for synthesizing ursodeoxycholic acid and high-chiral-purity D-amino acid based on enzyme-method coupling technology
  • Method for synthesizing ursodeoxycholic acid and high-chiral-purity D-amino acid based on enzyme-method coupling technology
  • Method for synthesizing ursodeoxycholic acid and high-chiral-purity D-amino acid based on enzyme-method coupling technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] 1. Add 40.0g CDCA to the reactor, add part of deionized water to stir, adjust the pH to 8.0 with liquid caustic soda to dissolve, then add 1.2 times the molar amount of CDCA butanic acid, add deionized water to the concentration of CDCA 4%, then add 7α-HSDH liquid enzyme, DAADH liquid enzyme and NADP to react, wherein the amount of 7α-HSDH liquid enzyme added is 0.5 times the mass of CDCA*1Mu / kg, and the amount of DAADH liquid enzyme added is 0.5 times the mass of CDCA *1Mu / kg, the dosage of NADP is 2‰ of the mass of CDCA, the reaction temperature is controlled at 30°C, pH8.0, the process is monitored by HPLC until the CDCA reaction concentration is ≤0.1mg / ml, and the total reaction is 6h.

[0067] 2. The above-mentioned conversion solution is separated by a 5K ultrafiltration membrane, and the concentrated liquid enzyme is used to catalyze the next batch of CDCA. Adjust the pH of the dialysate to 3 to crystallize, and obtain a wet powder containing 7-KLCA after filtrat...

Embodiment 2

[0072] 1. Add 50.0g CDCA to the reactor, add part of deionized water to stir, adjust the pH to 8.0 with liquid caustic soda to dissolve, then add pyruvic acid with 2.5 times the molar amount of CDCA, and add deionized water to make the concentration of CDCA to be 5%, then add 7α-HSDH liquid enzyme, DAADH liquid enzyme and NADP for reaction, wherein the amount of 7α-HSDH liquid enzyme added is 0.6 times the mass of CDCA*1Mu / kg, and the amount of DAADH liquid enzyme added is 0.6 times the mass of CDCA* 1Mu / kg, the dosage of NADP is 2‰ of the mass of CDCA, the reaction temperature is controlled at 30°C, pH8.0, the process is monitored by HPLC until the CDCA reaction concentration is ≤0.1mg / ml, and the total reaction is 3h.

[0073] 2. The above-mentioned conversion solution is separated by a 5K ultrafiltration membrane, and the concentrated liquid enzyme is used to catalyze the next batch of CDCA. Adjust the pH of the dialysate to 3 to crystallize, and obtain a wet powder contain...

Embodiment 3

[0078] 1. Add 40.0g CDCA into the reactor, add part of deionized water to stir, adjust the pH to 8.0 with liquid caustic soda to dissolve, then add phenylpyruvate with 1.2 times the molar amount of CDCA, and add deionized water to set the volume to the concentration of CDCA 4%, then add 7α-HSDH liquid enzyme, DAADH liquid enzyme and NADP to react, wherein the amount of 7α-HSDH liquid enzyme added is 0.6 times the mass of CDCA*1Mu / kg, and the amount of DAADH liquid enzyme added is 0.6 times the mass of CDCA *1Mu / kg, the dosage of NADP is 2‰ of the mass of CDCA, the reaction temperature is controlled at 25°C, pH8.0, the process is monitored by HPLC until the CDCA reaction concentration is ≤0.1mg / ml, and the total reaction is 4h.

[0079] 2. The above-mentioned conversion solution is separated by a 5K ultrafiltration membrane, and the concentrated liquid enzyme is used to catalyze the next batch of CDCA. Adjust the pH of the dialysate to 6 to crystallize, and obtain a wet powder ...

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Abstract

The invention discloses a method for synthesizing ursodeoxycholic acid (UDCA) and high-chiral-purity D-amino acid based on an enzyme-method coupling technology. The method comprises the following steps: putting chenodeoxycholic acid and alpha-ketonic acid into a solution system containing 7alpha-HSDH (Homoserine Dehydrogenase), DAADH and NADP (Nicotinamide Adenine Dinucleotide Phosphate) and carrying out enzyme catalysis reaction; separating a reaction solution by adopting an ultra-filtration membrane to obtain a concentrated mixed enzyme solution; regulating the pH (Potential of Hydrogen) ofa dialysis solution and crystallizing; filtering and separating to obtain 7-KLCA wet powder and filtrate; carrying out chromatographic treatment on the filtrate to obtain the D-amino acid; putting the7-KLCA wet powder into a solution system containing glucose, the NADP, the 7alpha-HSDH and GDH (Glutamate Dehydrogenase) and carrying out enzyme catalysis reaction; separating the reaction solution by adopting the ultra-filtration membrane to obtain the concentrated mixed enzyme solution; crystallizing, filtering and separating the dialysis solution, so as to obtain ursodeoxycholic acid. By adopting the method provided by the invention, UDCA and the high-chiral-purity D-amino acid can be obtained at the same time, the enzyme utilization rate is high, synthesis steps are simple and the cost isreduced; meanwhile, a metal reduction reagent and an organic solvent do not need to be added in a reaction process and conditions are mild; the method is environmentally friendly and is suitable forindustrial production.

Description

technical field [0001] The invention relates to a method for synthesizing ursodeoxycholic acid and D-amino acid, in particular to a method for synthesizing ursodeoxycholic acid (UDCA) and D-amino acid with high chiral purity by enzymatic coupling technology, which belongs to biocatalysis technology field. Background technique [0002] Ursodeoxycholic acid (Ursodeoxycholic Acid, UDCA), chemically named 3α, 7β-dihydroxy-5β-cholanic acid, was discovered from bear bile. At present, UDCA is widely used clinically to treat various gallstone diseases, various acute and chronic liver diseases, and can also improve the survival rate after liver transplantation. [0003] Natural ursodeoxycholic acid is derived from bear bile, which is drained from live bear bile. However, as the market demand continues to increase and live bear bile harvesting violates wildlife protection laws, natural ursodeoxycholic acid has long been unable to meet market demand. Therefore, it is becoming more a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/02C12P13/04
CPCC12P13/04C12P33/02
Inventor 王胜锋黄毅曾红宇许岗
Owner HUNAN BAOLISHI BIOTECH
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