Process considerations for protein engineering of ω-Transaminase

Over the past decades, the use of biocatalysis in the chemical and pharmaceutical industry has significantly increased. In parallel and contributing to this trend, many enzymes have been discovered and isolated from different biological sources. This has broadened the scope of biocatalysis and nowadays allows the green regio- and enantio-selective synthesis of many compounds, potentially with less time and energy demand and avoiding the use of toxic reagents.

The technology therefore has many advantages over classical chemical synthesis to prepare fine chemical and pharmaceutical intermediates. However, often wild type enzyme does not fit the requirements of the process conditions, where high substrate and product concentrations as well as high productivity demands of the catalyst (g product per g biocatalyst), are key to economic feasibility.

The question thus arises whether to fit the process to the catalyst or the other way around. Modern biotechnology has indeed seen a tremendous development in the last decades which in fact makes it possible to improve many of the enzyme properties needed, such as, tolerance to pH and temperature, substrate and product inhibition and finally the enantio specificity (e.e).

However, it is critical that this is done in parallel process development to make sure that the properties developed also fit the process requirements. As an example, ω -transaminases (EC 2.6.1.18) can be used to produce optyically pure chiral amines (with 100% theoretical yield) which are important building blocks for the chemical and pharmaceutical industries.

On the other hand, there are a number of challenges associated with the use of this enzyme for instance substrate and product inhibition, and a potentially unfavorable equilibrium. In the present work it was investigated how changes to a wild type transaminase through protein engineering changed the characteristics of the biocatalyst and the implications this would have on a process.

A methodology for characterizing the biocatalyst was developed which was subsequently applied to the wild type and 5 mutants selected. It was seen that the mutants had a better tolerance to the substrate and to higher temperature as well as displaying a broader pH tolerance. Based on the improved properties it could be shown that the feasibility of the process was significantly improved and that these properties opened up the potential for improvements in the process, such as operating at a higher pH for facilitated in-situ product removal.