May 25, 2006
Designer biocatalysts - Biochemists, chemists and engineers join forces

Enzymes are nature’s catalysts, playing a vital role in a myriad of biochemical processes in microorganisms, plants, animals and in the human body. In addition, enzymes function as remarkable biocatalysts for chemical reactions in the lab. These biocatalysts are often superior to chemical catalysts in terms of selectivity and they are also more energy efficient and environmentally acceptable, involving no toxic reagents or byproduct waste issues.

UCD Conway Institute and CSCB investigators, Professor Paul Engel and Dr Francesca Paradisi, have developed genetically engineered enzymes which catalyse the asymmetric synthesis of non-natural amino acids. Amino acids have both L and D enantiomers, but only the L-amino acids are found in nature. D-amino acids and also non-natural  L-amino acids are increasingly in demand as precursors by the pharmaceutical industry for peptidomimetic and other single-enantiomer drugs.

"Suitable enzymes for this process may be obtained from various bacterial sources and an example is phenylalanine dehydrogenase (PheDH). We have successfully engineered PheDH from Bacillus sphaericus to obtain versatile biocatalysts with broadened substrate specificity,” explains Professor Engel, of UCD School of Biomolecular and Biomedical Science.

In preliminary experiments in the lab the engineered enzymes demonstrated markedly improved activity with novel substrates which were synthesised in the Department of Chemistry at UCC by Professor Anita Maguire's group. “The potential for use of the engineered enzymes as biocatalysts for the production of non-natural amino acids is clear, with the catalysts showing enhanced activity, and significantly, without compromising the enantioselectivity,” says Dr Paradisi, a lecturer at the UCD School of Chemistry and Chemical Biology.

One anticipated limitation was the poor solubility of the substrates in water, as opposed to the enzymes’ preference for aqueous media. Performing the enzymatic reaction in the presence of organic solvents was the ideal solution. This line of investigation extended the collaboration further to Professor Daria Giacomini’s chemistry group at the University of Bologna. They found that the new enzymes were remarkably tolerant to organic solvents; this meant that an efficient coenzyme recycling system could be utilized using alcohol dehydrogenase (ADH) in ethanol.

"At this stage of the project, we had optimised our system in the lab but to make it attractive and viable for industry, we needed to call in the expertise of chemical engineers to develop the tools for pilot-scale production,” continues Dr Paradisi.

A team in the UCD School of Chemical and Bioprocess Engineering led by Dr Brian Glennon developed a fed-batch process for the high cell density cultivation of the E. coli strain and for the production of the recombinant protein PheDH. Scale-up of the process from a shake flask to a 20 litre bioreactor ensured that the important operating parameters, such as oxygen and glucose supply, were controlled to optimise the biocatalyst production.

According to Dr Glennon, optimisation has been successful: "High protein expression levels in bacterial cultures and high biomass concentrations have been obtained with a high intracellular product concentration of recombinant protein."

"Our work represents an outstanding example of how a system can be optimised to produce enzymes, not only in high density but with high activity and stability,” concludes Dr Paradisi. “Our present range of biocatalysts and their successors promise to offer a truly versatile range for amino acid production in the manufacturing industry.”

The three researchers will present their work in the Conway lecture theatre at 12:30pm on Friday May 26.

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