Latest revision as of 03:25, 2 November 2017

Project

Project Description

Demonstration

Project Description

Background

Bear bile, one of the most famous animal drugs in Traditional Chinese Medicine (TCM), has been recorded in ancient Chinese medicine book as a significant method to treat hepatic and biliary disorders. UDCA, the effective ingredient of bear bile.Aside from the traditional use of bear bile in Chinese medicine, UDCA(ursodeoxycholic acid), the effective ingredient of bear bile acid, has a much larger pharmaceutical application. As well as the usage of UDCA in dissolving gallstone, its efficacy in primary biliary cirrhosis and primary sclerosing cholangitis (PSC) as an adjunct to medical therapy has been well established. Newer indications include its use in the management of chronic hepatitis, cirrhosis, post liver transplant rejection, graft-versus-host disease and acute viral hepatitis, where it not only relieves symptoms of cholestasis but also arrests ongoing hepatocyte necrosis. However, the increasing demand for bear bile has caused bears to be in an endangered state: bear poaching and illegal animal trade have greatly dwindled the number of the wild Asiatic black bear. Apart from that, bear bile farming industry in Asia extracts bile through “milking” from the bears, which is operated through surgically implanting a permanent catheter in the animal's gallbladder to obtain the drips. It is unquestionable that the bear bile farming process will lead to both physical and psychological damage in bears.

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Purpose

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To find substitute or alternative for bear bile farming, our team will be working on the biological synthesis of the main effective component of this important medicine, UDCA(Ursodeoxycholic Acid). This biological approach will not only be more efficient but also be cheaper than the original chemical approach, which is widely used in the current UDCA synthesis industry.

Overview of the project

We found that it is possible to convert the main component of poultry bile, CDCA(Chenodeoxycholic Acid), into UDCA, by employing two enzyme-catalyzed the reactions. First, two enzymes was employed to manage the transformation of CDCA to 7oxo-LCA. In the present of 7a-HSD(7alpha-hydroxysteroid dehydrogenase), CDCA is transformed in to 7oxo-LCA by loosing a pair of hydrogen(2H+ and 2e-), the pair of hydrogen is added to NAD+, the cofactor and the acceptor. The NAD+ is transformed into NADH during the reaction. To regenerate the NAD+ and recycle the reaction, to , the LDH(Lactate dehydrogenase) works on pyruvate and take the pair of hydrogen from NADH and transform the pyruvate to lactate and NADH to NAD+.

In the second step, the 7oxo-LCA is transformed to UDCA by 7β-HSDH(7beta-hydroxysteroid dehydrogenase)and GDH(glutamate dehydrogenase).The 7β-HSDH works on 7oxo-LCA and take a pair of hydrogen from NADPH(the cofactor for the second step)and add it to 7oxo-LCA and form a beta position 7-hydroxyl group, which is our target product UDCA. The GDH works on glucose and take a pair of hydrogen from it and add it to the NADP+, to form NADPH to manage the regeneration of cofactor NADPH for the second step.

Our goal

Our mission is to expression of the four enzymes 7α-HSDH (from ecoli DH5a), 7β-HSDH (from Ruminococcus Torques), GDH (from Bacillus subtilis), and LDH from (Lactobacillus delbruechii subsp. Bulgaricus)and test their activities. By adding the CBD( cellulose binding domain)sequnence to the plasmid we construct, we manage to bind our enzyme on gauze. This specific design excels in 3 specific ways: first, by controlling the presence of the gauze in the solution, we can control the process of the reaction. Second, when the target enzyme is bound to cellulose we manage to purify the protein we express. Third, the enzyme binding gauze is employed to a machine including the reaction efficiency measuring system and the enzyme addition controlling system .

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Demonstration

Our purpose

The purpose of our experiment is to employ 4 enzymes to achieve the transformation of CDCA to UDCA. Our experiment is divided into two steps. The first step is to oxidize CDCA into 7oxo-LCA, and the second step is to reduce 7oxo-LCA to UDCA.

Adding CBD sequence

In order to achieve the purification of the enzymes and automatic control of the start and cease of the reaction and the recycling usage of enzyme, we added the sequence of CBD（cellulose binding domain) when we construct plasmid and manage to express the enzyme that able to bind with gauze(made of cellulose) , before the reaction, we employed ddH2O to wash over the gauze for 3 times in order to purify the enzymes .the gauze is then employed on an enzyme slot.

The two step reaction

The first step

Overview

In the first reaction 7a-HSDH(7alpha-hydrosteroid dehydrogenase) was employed to transform CDCA into 7oxo-LCA, with the presents of cofactor NAD+ that works as the acceptor of a pair of hydrogen(2H+and 2e-) from CDCA, the LDH(lactate dehydrogenase) works on pyruvate that take a pair of hydrogen from NADH and add it to the pyruvate to form Lactate and manage to transform the NADH back to NAD+. By doing so, we manage the regeneration of cofactor of the first step reaction.

Our experiment to testify the function of 2 enzyme of the first step

Oxidation of CDCA to 7-oxo-LCA using E. coli 7˛-HSDH and NAD+ regeneration

CDCA was converted in a 3mL solution containing 150 mM phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium pyruvate, 0.2mM NAD+, combined with 3U/ml LDH and1 1 U/ml E. coli DH5a 7a-HSDH-CBD at room temperature.

The biotransformation experiments were monitored via ultraviolet spectrophotometer at 340nm and HPLC measurements by UV detection at 210 nm, using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using C18

Result

As shown in figure, the transformation was complete after 2.5h.

The Peak area of 7oxo-LCA sample in different concentration

The concentration of 7oxo-LCA	Peak Area
5mM	7565117
10mM	7569983
15mM	7574421
20mM	7580012

The HPLC result of the solution Before the first step reaction.

The HPLC result of the solution after 150min of reaction.

Interpretation

According to the HPLC after 150minute, there is no significant increase of 7oxo-LCA, as a result, most of the CDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 94%.

According to the Absorbance of NADH that shown in figure, the absorbance is decreased significantly after 150 minute dual to the depleted CDCA that stop the conversion of CDCA to 7oxo-LCA and NADH synthesize. Because of the abundant amount of pyruvate in the solution, the LDH that works on pyruvate still regenerate the NAD+ by taking a pair of hydrogen from NADH until most of the NADH synthesized transformed into NAD+.