International Chemistry REU - Biocatalysis
Advisor
Dr. Florent Allais
Institution
AgroParisTech - CEBB
Department
Biochemistry
Time
11 weeks - Summer 2023
Background
What is a Biocatalysis?
Biocatalysis is the process of using purified enzymes to perform chemical conversions from a substrate to a product. Biocatalytic synthetic methods have grown considerably over the last few decades primarily due to their ability to create complex products with enzymatic stereoselectivity. These enzyme-driven reactions are (1) ecologically friendly, (2) customizable via tailor-made enzymes for greater efficiency and selectivity, and (3) are expanding the range of accessible chemical reactions through enzyme discovery tools (Bell et al., 2021). Some enzymes require complementary components called cofactors to undergo catalytic activity.
What is a Cofactor and Why is it Important?
Although some enzymes do not require any additional chemicals for their catalytic activity other than their amino acid residues, many require a complementary component called cofactor. To study or take benefit of all naturally occurring enzymes, it is necessary to have an affordable access to these cofactors. Although the use of inorganic ions seems straightforward, it is not so obvious when it comes to coenzymes. Indeed, their price can be prohibitive: 1600 €/g, 30 000 €/g or 1000 €/g for Coenzyme A (CoA), its disulfide (CoAS2) and S-adenosylmethionine (SAM) respectively.
​
What is S-adenosylmethionine (SAM)?
S-adenosylmethionine (SAM) is one of these key coenzymes and an essential metabolite in biological systems: SAM acts as a methyltransferase cofactor in the transmethylation of DNA, RNA, and proteins; is an important precursor in the transulfuration pathway; and is involved in polyamide synthesis (Malla et al., 2012). In pharmaceutical applications, SAM is an effective treatment for liver disease, Alzheimer’s, osteoarthritis, and depression (Papakostas et al., 2012; Linnebank et al., 2010). However, the prohibitive price of this cofactor limits its clinical and industrial applicability (1000€/g).
Issues with in vitro enzymatic SAM Synthesis?
Enzymatic synthesis of SAM results in severe product inhibition when the accumulation of SAM exceeds 1 mM (Niu et al., 2017). Previous studies demonstrate that product inhibition can be decreased by genetic engineering or the addition of p-toluene sulfonate, a "free salt" that can bind SAM (Niu et al., 2017). However, the latter method is not suitable for scaled-up industrial SAM production.
​
​
​
​
​
​Enzymatic Synthesis of S-adenosylmethionine (SAM) and its Optimization: The Implementation of a Resin-Assisted in-situ System
Previous work within this study focused on the optimization of enzymatic reaction conditions to access SAM. Following the production and purification of MAT, this enzyme was used for the biocatalytic production of SAM, and the optimal pH range, temperature range, and concentration of p-toluene sulfonate were determined using design of experiment (DoE) methods. The optimal biocatalytic reaction conditions were determined and a productivity of 5 g/L was achieved allowing the increase of SAM production by 26% compared to literature values.
To further improve the production of SAM I employed a resin-bound salt during the in-situ biocatalytic production of SAM. I presented proof of concept that these macroporous resins can selectively absorb SAM and desorb SAM using a sulfuric acid solvent, thereby decreasing product inhibition significantly. Overall, I completed my goal of allowing for a simpler purification of SAM by determining an optimal desorption solvent and processing conditions for SAM desorption without degradation. This work represents a significant improvement in the in vitro enzymatic synthesis of SAM, combining easier purification of the compound with the addition of an in-situ resin. This paves the way for larger-scale, sustainable, and cost-effective production of this coenzyme.
​
Techniques and Skills Developed:
​Enzyme Purification
AKTA Usage
HPLC Purification
FTIR Usage
​
The Benefit of International Research
My research site was the URD Agro-Biotechnologies Industrielles (ABI) at the CEBB (Center for Biotechnology and Bioeconomy) in Reims, France. This site was a blend of scientists of various disciplines of biology, chemistry, and engineering, working in close quarters to streamline the production of sustainable bio-based products. This environment was full of scientists of different nationalities and specialties collaborating on a day-to-day basis. I was not limited to working with only students in the biocatalysis laboratory from AgroParisTech; even within the office where I worked, various master's students and engineering preparatory school students from other prominent European Universities (CentraleSupélec and Urca) also worked and collaborated with one another. In the lab and the intern office, it was exciting to hear French, Arabic, and Spanish being spoken and to hone my listening skills over time to be attuned to certain scientific words in all these languages. Doing research abroad was an enriching experience for me because it took me out of my comfort zone in my research environment and my free time. I was able to travel on my weekends to places like Paris and Amsterdam and conduct exciting and innovative experiments throughout the week. My communication skills improved both inside and out of the lab by tailoring my vocabulary and phrasing to be as precise and understandable as possible. The most impactful things I will take away from this international REU are adaptability to new environments, conviction that I can work in a professional scientific setting in an autonomous manner, and the importance of communication and collaboration of different fields of science and different cultures.
​