Team:iBowu-China/Implementation
Proposed Implementation
Glycerβ for Implementation
In the past few months, we gradually came up with some aspects of our project as of how we could implement it in the real world for real users and in wider realms. In our project, one product is centered around glycyrrhetinic acid which can serve as drugs or can be further functionalized and used in cosmetic, food additive, and pharmaceutical industry; and the other product is β-glucuronidase as a versatile enzyme that not only can convert the extract of licorice but has the potential for more herbal ingredients.
Figure 1. Proposed implementation. The road map for our product before it reaches the end users.
Glycyrrhetinic acid (GA) can be decorated with functional groups for cosmetics, food additives and medine.
Glycyrrhetinic acid is a versatile compound that can be incorporated into a vast number of products to enhance potency. It finds wide applications in cosmetic industry, food industry and pharmaceutical industry. According to our research and data gathered from integrated human practice, GA has an increasing demand in the above industries.
With the success of synthesizing GA using the enzyme, the natural resource limit can be lifted and the production quantity can quickly rise to meet the increasing demand, with a lowering production cost. Highly efficient production wouldn't be achieved overnight due to a few obstacles along the road for the synthesis method to be expanded to an industrial scale, but the future looks promising.
GA synthesized in such a method is easier to be purified due to the lack of harsh acid or base mixed into the final product. The purified GA can be delivered to factories that would make GA-based derivatives by reacting GA with other small molecules. The safety for the production, transportation, and downstream reactions are similar to the situations of factory chemicals, and in fact GA is much lower in toxicity compared to most chemicals transported and used by labs and factories nowadays. As long as similar and necessary regulations are observed, the safety risk is very low.
Glycyrβ : a versatile Enzyme
In our project, we focused on the experiments to use the β-glucuronidase to produce the essence in licorice. In fact, the power of β-glucuronidase to convert herbal essence goes well beyond this one case. Its power to remove glucuronic acid groups can find applications in many of the active ingredients for herbal medicine.
Expression of the enzyme has been successful in our lab, and if the project had more time for experimental exploration, we would have found better conditions to increase the protein yield and we then would be able to extract and purify the protein. To immobilize the protein on a fixed substrate is a technique of common practice. We have researched many immobilization methods, from the textbook methods where the ceramic beads are used to remove the lactose in milk, to the recently published methods of using magnetic materials. In one of our collaboration/partnership efforts, SHSBNU-China team successfully constructed a new part that can attach to "legs" to our enzyme, expressed with a T7 promoter in BL21(DE3) strain. These legs can then attach onto cellulose fibers to realize enzyme immobilization.
The system then can be cleaned in a factory or lab and then transported to our users: the factories that need the enzyme for the production of various active ingredients from herbal medicines. The fixed substrate can effectively lower the production cost because it can be reused multiple times before the enzyme loses its activity.
Immobilized enzyme system: make synthesizing neat and cheap
It is widely known that the synthesizing methods of compounds used for medical and research purposes are mostly traditional, chemical ones. Such methods include not only expensive and possibly toxic solutions like sulfuric acid, hydrochloric acid, and ethanol but also wastes that are harmful to environments and hard to deal with. What’s more, even if so much effort above are given out, the yields of products are still significantly low. The high cost and pessimistic yields of traditional chemical synthesizing methods also elevate the price of the products, increasing the difficulty for researchers to obtain the reagents they need. In nearly all the experiments we did through the iGEM session, the prices of the reagents are always a major factor that affects our progress of researching. Identifying such issues, we aim to establish immobilized enzyme systems that can elevate the efficiency and yield of synthesizing processes as well as lower the negative effects brought to the environment.
Design
First, let’s get to know some basic concepts of enzyme immobilization. Our immobilized enzyme system is dedicated to fulfilling several goals: 1. Easy production, repairing and replacement. 2. Efficient and inexpensive synthesizing. 3. Minimized effects brought to the environment. To reach these goals, we adopt some specific designs. By browsing relevant journal articles and consulting experts in enzyme immobilization industries, we realize that adsorption is not an optimal method since the surface area is too small to let the enzyme and substrate react effectively. Also, Encapsulation is not practical because it is relatively difficult to perform the attachment of our enzyme to the semi-permeable membrane in our laboratory. Therefore, we chose entrapment-trapping enzyme inside a porous matrix. We plan to produce immobilized enzyme products based on chitosan gel and in a hard package that can be transported without damaging the gel. It is easy to produce since both gel production and enzyme attachment are fast and uncomplicated. Moreover, even if the apparatus encounters malfunctions, it can be replaced by a new one within a couple of minutes.
Safety
Pursuing minimum influence on the environment, we imposed certain designs to make the apparatus fully natural degradable. We plan to use biodegradable PET or PLA to make a shell and semipermeable membrane in both ends of the shell that allows glycyrrhetinic acid and water to pass but stops β-glucuronidase. Therefore, After denaturing the enzyme at a high temperature, which can be done by simply boiling or microwaving the gel, the whole apparatus can be thrown or buried in the soil to provide fertilizer for plants.
Problems
However, we still face some problems such as the products may aggregate in the gel and lower the yield and efficiency of the reaction. We plan to solve the issue by altering the pore diameter of the gel.
References
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