Method of preparing tauroursodeoxycholic acid by biotransformation and application of method

A technology of tauroursodeoxycholic acid and biotransformation, applied in biochemical equipment and methods, botanical equipment and methods, chemical instruments and methods, etc., can solve the problem of low substrate concentration, difficult industrial production, taurine 7 -The problem of high ketolithocholic acid content

Active Publication Date: 2019-03-01
JIANGSU BANGZE BIOLOGICAL MEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this kind method substrate concentration is low, conversion rate is low, and reaction intermediate taurine 7-

Method used

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  • Method of preparing tauroursodeoxycholic acid by biotransformation and application of method
  • Method of preparing tauroursodeoxycholic acid by biotransformation and application of method
  • Method of preparing tauroursodeoxycholic acid by biotransformation and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Escherichia coli containing the recombinant plasmid fermented and expressed in the Erlenmeyer flask

[0083] Take 20 μL of Escherichia coli BL21(DE3) strain containing the recombinant plasmid, inoculate it into 200 mL of ampicillin-resistant LB medium, and culture it overnight at 37°C, 220 rpm, with an OD600 value of 2.5-4.0. Inoculate 20 mL of culture solution into 1 L of ampicillin-resistant medium, culture at 37°C and 140 rpm for 3 hours, and when the OD600 value is 1, add 0.5 mM IPTG to induce overnight expression. Bacteria were collected by centrifugation. A small amount of bacteria was resuspended in 100mM phosphate buffer, and ultrasonically disrupted to obtain crude enzyme solution. Enzyme activity was determined according to the method in the technical scheme.

Embodiment 2

[0084] Example 2 Escherichia coli containing recombinant plasmid fermented and expressed in a fermenter

[0085] Take 20 μL of Escherichia coli BL21(DE3) strain containing the recombinant plasmid, inoculate it into 200 mL of ampicillin-resistant LB medium, and culture it overnight at 37°C, 220 rpm, with an OD600 value of 2.5-4.0. Inoculate 20 mL of culture solution into 1 L of ampicillin-resistant medium, and culture overnight at 37°C and 140 rpm. Aseptically inoculate 10L of the seed solution into a fermenter equipped with 200L of Escherichia coli high-density fermentation medium, culture at 37°C with aeration and stirring for 8 hours. Escherichia coli high-density fermentation medium contains: 18g / L dipotassium hydrogen phosphate dodecahydrate, 6.8g / L potassium dihydrogen phosphate, 0.7g / L anhydrous sodium sulfate, 0.48g / L magnesium sulfate, 2.25 g / L glycerin, 2.5g / L yeast powder, 5g / L peptone. After cultivating with aeration and stirring for 8 hours, add an IPTG solution ...

Embodiment 3

[0086] Example 3 Transformation of tauroursodeoxycholic acid with single gene expression protein in 1L reaction system

[0087]Dissolve 250g taurochenodeoxycholic acid in 700mL of 100mM glycine buffer, add 0.25mM NAD + , add the sodium pyruvate of 60g / L, add the 7α-steroid dehydrogenase (containing pure enzyme about 5g) and lactate dehydrogenase (containing pure enzyme about 5g) of purified or partially purified enzyme solution or cell lysate or bacterial suspension Pure enzyme is about 2g), add 100mM glycine buffer to 1L, adjust the pH to 7.5 with 5M NaOH, and react for 6-18h at 25°C. Add 100g / L of glucose, 7β-steroid dehydrogenase (containing about 5g of pure enzyme) and glucose dehydrogenase (containing about 5g of pure enzyme) of purified or partially purified enzyme solution or cell lysate or bacterial suspension 2g) of Escherichia coli, adjust the pH to 7.5 with 5M NaOH. React at 25°C for 6-18h. The substrate conversion rate is over 98%, the finished product content is...

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Abstract

The invention discloses a method of preparing tauroursodeoxycholic acid by biotransformation and application of the method. Biotransformation includes genetic codon optimization, engineered bacteria construction, engineered bacteria cultivation, substrate transformation and product preparation. Tauroursodeoxycholic acid is prepared by transforming a substrate through direct fermentation of engineered bacteria; the substrate is taurochenodeoxycholic acid. The substrate may reach 250 g/L in concentration; the reaction time is short; substrate transformation rate reaches 98% and above; the obtained product reaches 99% and above in purity; cyclic regeneration of NAD+ (nicotinamide adenine dinucleotide +) in the reaction system helps greatly reduce the usage of the coenzyme NAD+; the cost of enzymic catalytic reaction is reduced; industrial amplification is benefited. hydroxysteroid dehydrogenase and the regenerated coenzyme are connected via a flexible polypeptide sequence to form a protein fusion polymer; binding distances to the substrate and coenzyme are shorter; transformation progress is more facilitated; the number of fermenting times in industrial production is decreased; the process is simplified; time cost and material cost are saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for efficiently catalyzing the biotransformation of tauroursodeoxycholic acid after transforming biological enzymes by means of genetic engineering, specifically a method for preparing tauroursodeoxycholic acid through biotransformation and its application. Background technique [0002] Tauroursodeoxycholic acid, whose chemical name is 3α,7β-dihydroxycholanoyl-N-taurine, has antispasmodic, anticonvulsant, anti-inflammatory and gallstone-dissolving effects. Tauroursodeoxycholic acid is mainly present in the bile of black bears and is the symbolic active ingredient in bear bile. In 2007, Tauroursodeoxycholic Acid Capsules with the trade name of Taurote were approved for sale in China. It is mainly used to dissolve cholesterol gallstones. Ursodeoxycholic acid is a hydrophilic bile acid with limited stone dissolution rate, good safety and few side effects, and has been widely u...

Claims

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Application Information

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IPC IPC(8): C12P33/00C12N15/70C12N15/53C12N15/62
CPCC12N9/0006C12N15/62C12N15/70C12P33/00C12Y101/01159C12Y101/01201C07K2319/00C12N2800/22
Inventor 赵志斌王丹丹郑祥艳李清秦松柏丁峰陶京兰陈潘海曹海兵
Owner JIANGSU BANGZE BIOLOGICAL MEDICINE TECH CO LTD
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