Team:SDSZ-China/Project
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.
Purpose
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 .
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+.
The second step
Overview
in the second step the 7β-HSDH（7 beta-hydrosteroid dehydrogenase) was employed to transform 7oxo-LCA to UDCA. The 7oxo-LCA is transformed to UDCA by adding a pair of hydrogen provide from the cofactor for the second step, NADPH. The NADPH loose a pair of hydrogen and form NADP+ and the hydrogen is added to 7oxo-LCA by 7β-HSDH and form a beta position 7-hydroxyl group which is our target produce UDCA.
Our experiment to testify the function of 2 enzymes of the second reaction
Reduction of 7-oxo-LCA to UDCA using 7β-HSDH and GDH(NADPH regeneration)
The 3mL reaction solution containing 150 mM phosphate buffer(pH 8.0), 10 mM UDCA, 30 mM glucose, 0.2mM NADP+, combined with 2U/ml 7β-HSDH and 5U/ml GDH at room temperature.
The bioconversion experiment was monitored via HPLC measurements. The sample was analyzed by UV detection at 210nm. We testify the synthesize of 7-oxo-LCA and the decrease of UDCA. Using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using C18 .
Result
As shown in the figure below the reaction was complete at 90min
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 start of the second reaction
The HPLC result of the solution after 90 min of second reaction
Interpretation
According to the HPLC result after 90minute, there is no significant increase of 7oxo-LCA, as a result, most of the UDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 93%.
According to the Absorbance of NADPH that shown in figure, the absorbance is decreased significantly after 90 minute dual to the depleted UDCA that stop the conversion of UDCA to 7oxo-LCA and NADP+ synthesize. Because of the abundant amount of glucose in the solution, the GDH that works on glucose still regenerate the NADPH by taking a pair of hydrogen from glucose until most of the NADP+ transformed into NADPH.
The implication of the enzyme binding gauze
Description of the purpose of our machine
Because of the fact that if the two reactions proceed at the same time, the equilibrium of the reactions will shifted towards the reactant, CDCA. Thus because of this, we cannot allow the two reactions to proceed at the same time and in the same space. To separate the 2 reaction, we design our reaction vessel in the following way: After the first reaction is fully completed, the enzyme slot with gauze bond by 7a-HSDH and LDH will be raised up, and in this manner, the first reaction is ceased. After this process, we will start the second reaction by dropping the enzyme slot with gauze containing 7β-HSDH and GDH.
Testification of our own NADH optical detector
To testify the function of our NADH optical detector, the change in the concentration of NADH is an essential indicator of the course of the reaction, our devices is design to examine the absorbance of NADH at 340nm ultraviolet light. By adding NADH in different concentration. We testify the the absorbance of ultraviolet ray of NADH under a 3 mL solution.
Interpretation of the testification
A liner function can be demonstrated base on the date we collect of absorbance of NADH in different concentration. Their test values are close to the theoretical value of absorbance. Which means our devices manage to detect the absorbance of NADH and its concentration.
The relationship between our experiment result and the controlling of our devices
Since the change in the concentration of the cofactor NADH is an essential indicator of the course of the first step of the reaction, and solutions with different concentration of NADH absorb different proportion of the 340nm ultraviolet ray. So in this way we are able to determine the course of the reaction just by tracking the change in the solution’s absorbance of 340nm ultraviolet ray.
Interpretation
We manage to find out that when there is a significant decrease shown on the absorbance of NADH , the transformation of CDCA reach its end consider no significant increase of 7oxo-LCA shown in the HPLC result. So that we can determine the ceasing point of the reaction.As soon as our devices testify the significant decrease of absorbance of NADH, we will raise the first enzyme slot from the reaction vessel, and thus in this way end the first reaction. The next step is to drop the second enzyme slot and start the second reaction
