Method for preparing binding-form ursodesoxycholic acid by two-step enzymatic method

A technology of ursodeoxycholic acid and chenodeoxycholic acid, which is applied in the field of bound ursodeoxycholic acid, can solve problems such as difficulty in simulation, and achieve the effects of simple preparation method, high conversion rate, and simple preparation process

Active Publication Date: 2013-03-27
SHANGHAI KAIBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Therefore, the synthesis of TUDCA in vivo is a rather complicated process involving 5 enzymes, so it is very difficult to simulate this process in vitro

Method used

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  • Method for preparing binding-form ursodesoxycholic acid by two-step enzymatic method
  • Method for preparing binding-form ursodesoxycholic acid by two-step enzymatic method
  • Method for preparing binding-form ursodesoxycholic acid by two-step enzymatic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0045] Implementation Case 1: Preparation of Chicken, Duck and Goose Gall Powder

[0046] Chicken gall, duck gall and goose gall are purchased from slaughterhouses. First determine the number and weight of gallbladder. Use 75% alcohol to sterilize parts of chicken gall, duck gall and goose gall respectively, and then dissect the bile separately. Put the bile taken out into different stainless steel round basins. Put the bile-filled disc into -40°C refrigerator for 24 hours, and finally put it into a freeze dryer and dry it for 48 hours to obtain chicken gall powder, duck gall powder, and goose gall powder respectively.

Embodiment example 2

[0047] Implementation Case 2: Content Analysis of TCDCA and TUDCA in Chicken Gall Powder, Duck Gall Powder and Goose Gall Powder

[0048] Take about 0.16g of chicken gall powder, duck gall powder, and goose gall powder, respectively, and place them in different measuring bottles (50mL). Dilute with methanol to the mark, shake well, filter, take the filtrate, that is. Take 10 μL of each of the above solutions and inject them into a high-performance liquid chromatograph, and the detector adopts an evaporative light scattering detector. Record the chromatogram ( figure 1 , figure 2 , image 3 ). The percentage contents of TCDCA and TUDCA were calculated respectively (Table 1). The reference substances TCDCA and TUDCA were from China Institute for the Control of Pharmaceutical and Biological Products.

[0049] Table 1 The contents of TCDCA and TUDCA in chicken gall powder, duck gall powder and goose gall powder detected by HPLC (%)

[0050]

Embodiment example 3

[0051] Example 3: Whole gene synthesis of 7α-HSDH and 7β-HSDH

[0052] The optimized 7α-HSDH gene sequence from Clostridium sardiniense (Clostridium sardiniense, ATCC27555) after gene sequence optimization is shown in SEQ ID No 1; The 7β-HSDH gene sequence is shown in SEQ ID No 2; the optimized 7α-HSDH gene sequence from Bacteroides fragilis ATCC25825) is shown in SEQ ID No 3; the optimized gene sequence is from the production The 7β-HSDH gene sequence of Collinsella aerofaciens ATCC25986 is shown in SEQ ID No 4. The above-mentioned codon-optimized gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the restriction sites were both BamH I and Not I.

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Abstract

The invention relates to a method for preparing binding-form ursodesoxycholic acid by a two-step enzymatic method, belonging to the field of biotechnology. Under the water solution state, a substrate, namely binding-form chenodeoxycholic acid, is converted into the binding-form ursodesoxycholic acid in the presence of 7alpha-HSDH and 7beta-HSDH, the separation is not needed in the intermediate step, thus the method disclosed by the invention is very simple, and furthermore, the conversion efficiency is very high. Especially chicken bile, duck bile or goose bile in a mixture form can be subjected to reaction without separating and purifying the binding-form chenodeoxycholic acid. The preparation process is simple and easy to implement, and the new, simple and convenient preparation method is provided for the binding-form ursodesoxycholic acid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a combination of 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) to catalyze bound chenodeoxycholic acid in a two-step method A method for conversion to conjugated ursodeoxycholic acid. Background technique [0002] Bear bile is a precious Chinese medicinal material, which is used to treat gallstone disease and various acute and chronic liver diseases, and has a good therapeutic effect. Tauroursodeoxycholic acid (TUDCA) is one of the main active ingredients of bear bile, a traditional Chinese medicine. However, the main method of obtaining traditional Chinese medicine bear bile powder is "draining live bears to obtain bile", which violates the wildlife protection law and is considered to be a very "humane" method of obtaining. However, there are many bile acids in the bile of poultry and livestock, especially the rich taurodeoxycholic acid (T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/00
Inventor 王伯初谭君娄德帅祝连彩刘绍勇岑小惜季庆治
Owner SHANGHAI KAIBAO PHARMA
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