A traditional chemical process involves organic solvents, high temperatures, sometimes high pressure and often precious metal catalysts. Wolf-Dieter Fessner, Professor of Organic Chemistry at TU Darmstadt, discovered that it could also be done differently at a conference in Freiburg in the mid-1980s. Amongst other topics, the conference programme included talks on biocatalysis. “What I learned there was a real revelation to me,” he remembers.
“I had never thought that complex molecules could be specifically produced under mild reaction conditions in water and at room temperature just by adding a few enzymes to the pot.” Fessner has followed one goal since then: He develops enzymes for industry to enable chemical processes designed in a more environmentally friendly way.
Enzymes are proteins that as biocatalysts drive almost all biochemical reactions, our metabolism as well as the process of photosynthesis in plants. Just like all catalysts, enzymes simplify and accelerate chemical reactions. Fessner emphasises that these biocatalysts have achieved an unparalleled level of perfection in the course of evolution: “Nature has chemistry firmly in its grip. It operates an optimal catalytic process.”
The chemical industry should learn from this example because, aside from the harsh reaction conditions and high consumption of energy in traditional processes, standard metal catalysts also have some other issues: They are not only expensive but also sensitive to handle and damage the environment. “In the mining of precious metals, millions of tonnes of heavy metals are released into the atmosphere,” points out Fessner. “By using biocatalysis we are developing more sustainable processes.”
EU project CarbaZymes
Fessner is coordinating the EU project CarbaZymes since 2015. It focuses on biocatalysts used for carbon-carbon bond forming reactions. These fundamental reactions that play a key role in almost all chemical processes combine small organic fragments with one another, such as for making precursors to mass-produced plastics. The components must come together in the correct spatial orientation, emphasises Fessner, thus selective reactions pose a challenge to chemists.
In contrast, enzymes easily control the reaction because they bind the starting molecules for a brief moment and position them perfectly to one another. It is exactly in this way, for example, that our bodies can build up complex hormones and other biomolecules from simple fragments. In the chemical and above all pharmaceutical industries, many enzymatic processes have already become established, yet the formation of carboncarbon bonds is an underdeveloped field according to Fessner: “The enzymes required for this purpose have long been thought to be too specific and thus not suitable for industrial application.”
The problem is that many of these types of enzymes only catalyse a certain reaction because they bind the starting molecules in a highly specific way according to the lock and key principle. However, tolerant enzymes also exist that can convert various compounds. “This is what we’re looking for,” explains Fessner, “plus we can now change enzymes so that they are suitable for a whole range of molecules, even for those that are unreactive under natural conditions.”
The blueprint for the enzyme is hidden in the genes of the cell
Fessner’s group use a fermentation process to obtain the enzymes out of cells of Escherichia coli, also known as coliform bacteria. The blueprint for the enzyme is hidden in the genes of the cell. Using molecular biology methods such as implanting artificial gene sequences, the researchers can change the blueprint and optimise the enzymes. Biocatalysts that do not occur naturally in coliform bacteria can also be produced in this way. In the search for and development of enzymes the method of directed evolution is helpful: It induces random gene changes and thus results in numerous related enzymes, from which the scientists can identify those variants best suited for the task.
This selection proceeds over multiple cycles. The researchers in Darmstadt are working closely together with Prozomix in England, one of their partners in the CarbaZymes project. This company provides them with hundreds of proteins that all catalyse the same reactions but originate from various different organisms.
These also include many microbes that live under extreme environmental conditions and cannot be cultivated in the laboratory – but which are particularly interesting for industry. Bacteria from hot springs, for example, have particularly heat-stable enzymes. From the pool of proteins provided by Prozomix, the scientists select suitable enzymes that can then be further refined as required.
A new biocatalytic process for the production of homoserine
Fessner already holds multiple patents for enzymatic processes, and the CarbaZymes project has also already resulted in two patent applications. In cooperation with their Spanish partners in the CarbaZymes project, the team at Darmstadt published a new biocatalytic process for the production of homoserine, a precursor for pharmaceutical ingredients and essential amino acids for animal feed, in early 2017. The chemical industry is showing great interest in biocatalysis says Fessner: “There are a lot of companies investing heavily in this area and a number of processes have already been converted.”
Instead of pure enzymes, living cells are often utilised at an industrial scale. Whole cell biocatalysis makes more sense economically because it is not necessary to isolate the enzyme. The use of whole cells is particularly advantageous for complicated syntheses that require multiple different enzymes. A classic example is the production of vitamin B2: The fermentation process using fungal cells produces the vitamin in one step from vegetable oil. In contrast, the old chemical synthesis required eight steps and caused more carbon dioxide emissions, more waste and higher costs.
The biocatalytic production of vitamins and other complex molecules is highly appealing to Fessner as a chemist but he prefers to dedicate himself to the enzymatic synthesis of basic chemicals: “Billions of tonnes of these chemicals are produced every year. If we could design these processes in a more environmentally friendly way, it would have a much bigger impact on the future of our planet.”