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Protein, Cell Engineering for Biocatalytic Processes

01/08/2018 |

By Robert Kourist

Enzymes catalyse challenging chemical reactions under very mild reaction conditions. An optimization of synthetic applications requires a good knowledge of their structure and their mechanisms.

TU Graz-researcher Robert Kourist.
An essential feature of the cellular metabolism is its immense complexity. To cope with this complexity, nature developed biocatalysts with outstanding selectivity. This selectivity can be utilized for the development of efficient biotechnological processes. The possibility to discriminate between very similar molecules or to selectively produce one compound from several possible products allows the number of reaction steps or synthetic routes to be reduced and the separation and purification of the reaction products to be greatly facilitated. This results in tremendous savings in terms of cost, energy and waste accumulation. Biocatalysis thus makes a significant contribution to the development of sustainable processes for the chemical and pharmaceutical industries. The capacity of many enzymes to convert non-natural substrate allows their application in the synthesis of a wide spectrum of products. On top of this, many enzymes catalyse reactions that would not be possible or would be too challenging for chemical catalysts. This includes the selective introduction of oxygen atoms into unreactive molecules and the breaking of highly stable chemical bonds, such as those between carbon-carbon atoms. As in all catalytic processes, these advantages have to justify the cost and effort of the production of the catalyst. A large number of enzymes catalyze highly interesting reactions but do not meet all the requirements for a successful process. Nowadays, most synthetic applications of enzymes aim to manufacture high-value products, such as pharmaceutical ingredients, cosmetics and food and feed additives. The complexity of these chemicals and the high requirements regarding the purity of the products lead to considerable consumption of energy and accumulation of polluting waste. The potential of biocatalytic methods in the improvement of the environmental footprint is here very high. For the production of high-value products, biocatalysis is thus very competitive and often the method of choice. High-value products, however, are often manufactured in relatively small quantities. Due to higher cost pressure, processes for the production of specialty chemicals and commodities are usually more efficient and produce less waste. In turn, this allows less savings to be made by enzymes. Moreover, the lower price for mass products makes it more difficult to afford the cost for the catalyst production. This is an important factor in view of potential applications of biotechnological processes for the utilization of renewable resources in the future bioeconomy. In principle, enzymes are highly suitable for the conversion of bio-based molecules. The utilization of bio-based chemicals only has a beneficial effect when this can be done in large quantities. This requires a considerable increase in the efficiency of biocatalytic processes in terms of cost, waste accumulation and energy consumption.
© Robert Kourist
Knowledge-based and thus “rational” design of proteins and molecular optimization algorithms, such as “directed evolution”, are complementary approaches for the optimization of enzymes for biotechnological applications.

Optimization of enzymes

Enzymes have evolved to function optimally with their natural substrates and their natural reaction conditions. These often differ significantly from those that are required for industrial processes. In principle, the optimization of a biocatalyst can be done either by reaction engineering or by molecular design. In the latter, mutations in its gene alter the structure of an enzyme, which results in modified catalytic properties. Examples for successful molecular optimizations include an adjustment of the selectivity, an increase of the stability or an expansion of the substrate spectrum. Molecular design also allows new functions to be introduced to enzymes. In many cases, our understanding of enzyme catalysis on a molecular level is not sufficient to make accurate predictions on the outcome of mutations. Therefore, randomized optimization methods play an important role in biotechnology. Introduction of sets of amino acids whether randomly or site-specifically allows mutant libraries to be generated. An evaluation of the resulting combinatory diversity in high-throughput screening assays allows enzyme variants with the desired properties to be identified. A well-planned combination of randomized approaches with the available knowledge on structure and mechanism is in many cases straightforward for the generation of tailor-made enzyme variants for catalytic processes.
© Robert Kourist
Combining chemical and biocatalytic catalysts allows new eco-friendly biotechnological processes to be created. This example shows the combination of an enzymatic decarboxylation using olefin metathesis for the synthesis of bio-based antioxidants. The separation of the enzyme in polymeric capsules allows compatibility with the reaction conditions of the chemical reaction in the organic solvent to be achieved.

Enzymes as components

Since many enzymes show outstanding activities in water and under mild reaction conditions, their assembly regarding reaction cascades is often straightforward. This is different to chemical synthesis routes, where the reaction conditions differ considerably in many cases. This makes it very difficult to achieve a compatibility of the subsequent steps. Cascade reactions allow the work-up and purification of intermediate products to be saved. This results in a significant increase in the efficiency of the overall process. An extension of the concept is the combination of enzymes with chemical catalysts. This allows the strengths of both fields to be combined.
Enzymes from different organisms often show some differences regarding their optimal reaction conditions. Moreover, they often interact with the reagents of other reaction steps of the cascade. A successful establishment and scaling of a multi-step reaction cascade is therefore more difficult compared to single-step enzyme reactions. The key here is a successful integration of molecular methods, such as cell and protein engineering and process engineering. Despite these technical differences, the combination of enzymes with other catalysts is emerging as a particular strength of biocatalysis. The immense metabolic potential of reaction sequences in living cells and cell-free systems is still unexploited. Examples such as the combination of enzymes with chemical catalysts and the coupling of biocatalysts with photosynthesis as energy source underline the synthetic potential of biocatalysis. The higher complexity of these reaction systems, however, requires an interdisciplinary approach. Molecular engineering is a highly efficient tool to design biocatalysts with the desired activity, selectivity and stability for applications in cascade reactions.
© Robert Kourist
Research in the Biocatalysis and Enzyme Engineering working group at the Institute of Molecular Biotechnology.

This research project is attributed to the Field of Expertise „Human & Biotechnology“, one of TU Graz' five strategic areas of research.

Information

Robert Kourist is head of the Institute of Molecular Biotechnology and specializes in the molecular optimization of cells and enzymes and their use in eco-friendly biotechnological processes.