The eobiotic compound indican lends itself to a compelling biocatalytic dyeing strategy for denim, in which the formation of corrosive by-products is avoided. However, the efficient and scalable production of indican remains a key bottleneck. This work focuses on the in vitro characterization of PtUGT1, a glycosyltransferase from Polygonum tinctorium that catalyzes the formation of indican via the glycosylation of indoxyl. Here, the buffer composition and enzyme concentration were identified as key parameters for enzyme activity and stability. The short lifetime of the enzyme under reaction conditions initiated an immobilization study. As a consequence, an amino-functionalized methacrylate resin was identified as a highly functional option for efficient immobilization of PtUGT1, allowing immobilization yields of > 98% for enzyme loadings up to 7.6 w-%. We further report a stabilization factor of 47 and significantly improved overall biocatalytic productivity. The straightforward handling and reuse of the described heterogeneous biocatalyst is demonstrated.

where I spent three months carrying out the immunomodulatory experiments described in the chapter 4. To Linette Willemsen, thanks for patiently explaining me, a thousand times, everything that I did not understood regarding immunology and immunomodulation. Thanks for showing me your particular philosophical approach to science, and for showing that we are fortunate to do what we love and to contribute from our position. To Gemma Dingjan, thank you for helping me with all the experiments, for working side by side with me even during weekends and specially for helping me make my stay in Utrecht and in the laboratory not only fruitful, but also funy, introducing me to the people and integrating me into the out work activities.

The eobiotic compound indican lends itself to a compelling biocatalytic dyeing strategy for denim, in which the formation of corrosive by-products is avoided. However, the efficient and scalable production of indican remains a key bottleneck. This work focuses on the in vitro characterization of PtUGT1, a glycosyltransferase from Polygonum tinctorium that catalyzes the formation of indican via the glycosylation of indoxyl. Here, the buffer composition and enzyme concentration were identified as key parameters for enzyme activity and stability. The short lifetime of the enzyme under reaction conditions initiated an immobilization study. As a consequence, an amino-functionalized methacrylate resin was identified as a highly functional option for efficient immobilization of PtUGT1, allowing immobilization yields of > 98% for enzyme loadings up to 7.6 w-%. We further report a stabilization factor of 47 and significantly improved overall biocatalytic productivity. The straightforward handling and reuse of the described heterogeneous biocatalyst is demonstrated.