Chemistry is helpful in many areas of human life. It is used for the synthesis of products needed in medicine or pharmacology. One way to manufacture those is by means of biotransformation.
It is aimed at using enzymes as the catalysts of a chemical reaction.
This is done by means of transformation of a chosen substrate fragment into a desired product.
The idea of biotransformation as a chemical reaction was derived from biocatalytic transformations in microorganisms.
Products created as a result of a biotransformation usually do not interfere with the development of a cell.
Its natural enzymatic mechanism is often triggered artificially. Compounds, usually organic ones, are transformed by a cell as a result of providing it with such substances as the ones synthesised in a natural process. Biotransformation is a highly specific reaction in terms of direction, substrate specificity and stereospecificity. Thus, a biotechnologist needs to analyse the enzyme that allows them to conduct a chemical reaction depending on the main reagent.
The main purpose discussed in the work was to prove the thesis that the biotransformation of carboxylic acids is highly selective despite the constancy of the second reagent – 2-phenyl-3-oxobutanoate.
The work began with preparing three chemical reactions – esterifications (in the picture below). It is not possible to conduct the reactions in standard conditions due to the low reactivity of the substrates. The addition of 4 different enzymes: Mucor white, Rhizopus oryzae, Pseudomonas aeruginosa and Aspergillus oryzae (10 mg each), makes it possible to conduct the reaction. It doesn’t mean that the reaction will be immediate. The entire reaction system needs to be heated and constantly mixed by boiling chips for 24 hours. The provision of constant energy supply by heating the systems up to 50 °C is meant to additionally speed up the reaction process (by helping to break the chemical bonds between the reagents).
After 24 hours, three reaction systems were distilled to thicken the obtained product to the maximum. The process lasted also 24 hours at 50 °C and 0.9 mmHg (119.99 Pa). The vaporisation of water from the products makes them much easier to analyse.
Chromatography on TLC plates in the hexane:ethyl acetate 8:2 phase was used to analyse the products obtained in the biotransformation process. The conclusion was drawn on the basis of the analysis of the TLC plates using UV light (for the hexane:ethyl acetate 8:2 phase).
- Flask no. 1 – the reaction was partial; a substrate and the product are present in the flask.
- Flask no. 2 – the reaction was complete; the substrates underwent reaction to a considerable extent.
- Flask no. 3 – no product. No reaction.
The conclusion is that the adequate selection of biocatalysts facilitates the reaction process in flask no. 2 (3-phenylpent-4-enoic acid and 2-phenylo-3-oxobutanoate) and prevents the reaction in flask no. 3 (benzoic acid and 2-phenylo-3-oxobutanoate).
The positive result of the reaction in flask 2 is the result of selecting the reagents of proper specification. It is highly probable that the structure of 3-phenylpent-4-enoic acid is similar to the compound naturally transformed by organisms.
One can state that the absence of the reaction no. 3 results from the presence of another variable component (benzoic acid) that, in comparison with other acids (cinnamic acid and 3-phenylpent-4-enoic acid), produces the most volatile ester that probably exudes from the flask. It is the least non-polar ester – it is hardly soluble in toluene (non-polar solvent) and thus it is not visible on a TLC plate.
A well-visible product of the reaction no. 2 (on the right in the picture below), in the hexane:ethyl acetate 8:2 phase, is the result of an adequately low polarity of the system. The selected separation funnel allowed for the complete elimination of the product of the reaction no. 3.
THE FUTURE OF BIOTRANSFORMATION
As an organic synthesis, biotransformation often has an impact on people, especially on the metabolites of our bodies.
Regardless of the route of administration, chemical substances are subject to various processes in our bodies. All the processes determining the activity of the foreign substances in the body are referred to as xenobiotic metabolism.
In 1947, in his monograph entitled ‘Detoxifications Mechanisms’, Roger Williams presented for the first time the xenobiotic biotransformation as a process that has subsequent phases of so-called functionalisation and conjugation. Now, it is known that xenobiotic metabolism can be divided into four main processes: absorption by human tissues, distribution between autosomal barriers, biochemical transformation – biotransformation, and disposal from the body. The main place of compound biotransformation is the body – the skin, the lungs, the liver and the gastrointestinal tract.
An effective process of xenobiotic detoxification requires not only the coordinated but also sustainable activity of enzymes in each biotransformation phase. Thanks to that reaction, toxins and unwanted compounds can be removed from the body.