Natural Pigments in Light-Driven Biocatalytic Redox Cascades

Light-driven reactions have been recognized as a powerful approach to chemical synthesis based on the fascinating ability of natural photosynthetic systems to convert solar energy into chemical energy. Photo-redox catalysis has been used to mediate the transfer of electrons between chemical compounds employing the excitation by light of small amounts of a light-sensitive compound. Enzymatic catalysis is advantageous compared to non-enzymatic catalysis concerning effectiveness, enantioselectivity and environmental friendliness. The flavin cofactor plays an essential role in catalysis when in its fully reduced form (both in vitro and in vivo). The photoreduction of flavin derivatives by electron donors is a well-known process. Betanin (Bn) is a water-soluble and non-toxic natural pigment easily extracted from beetroot, which changes the color from magenta to yellow when oxidized. The high antioxidant activity and the low E ox for the first oxidation process of Bn indicates that this natural pigment might be a good sacrificial electron donor for the photochemical reduction of flavins. Most of the detection methods for controlling biotransformations described in the literature are usually substrate-dependent, which limits their application. Either in novel enzyme discovery campaigns or in enzyme engineering/enzymology studies, it is necessary to apply functional tests for high-throughput screening able to detect enzyme activity with high selectivity and sensitivity. Therefore, the present project proposes to combine the use of Bn as sacrificial electron donor in a photo-redox regeneration method for flavin-dependent monooxygenases (e.g. BVMOs) and reductases (e.g. EREDs) to the development of a versatile substrate-independent catalytic activity test (SICAT) for the same class of enzymes. By linking this catalytic activity test to a hydrogen peroxide detection assay, the uncoupling intrinsic to flavin-dependent enzymes can be determined and, in addition, avoid false positive results. As a result, the combination of the two assays leads to the design of a substrate-independent catalytic integrity test (SICIT).

The use of enzymes for synthetic applications is a powerful and environmentally-benign approach in the synthesis of chemical and pharmaceutical building blocks for chemical production. However, broader application of enzymes is limited by the requirement of expensive chemicals and low operational enzyme stability. The aim of this project was to reduce the operational costs of the use of isolated enzymes in the preparation of building blocks for chemical production using a light-driven process and natural pigments. In addition, the reaction progress should be monitored by the change in the color of the natural pigment, establishing a new detection method for the employment of enzymes. Light-driven activation of enzymes emerged as an expeditious and low-cost method in organic synthesis. The activation of enzymes by light results in chemical transformations applicable in the synthesis of chemical and pharmaceutical building blocks. With this project, it was possible to successfully improve enzyme stability, one of the major limitations for the broad use of enzymes in the industry. It was also possible to understand the effect of the reaction components on the light-driven process and on the enzyme stability to develop more efficient processes. Enzymes are also key components of biological processes implicated in diseases. Thus, our results will contribute not only to expand the application of the field but also to understand the dynamics of enzymatic transformations involving electronic excited states.