Exploiting yeast surface display for whole cell biocatalysis in Komagataella phaffii

Recombinant enzymes are usually secreted into the cultivation medium, from which they are purified at the end of fermentation; however, this process can be labor-intensive and costly. Employing whole cell biocatalysis can reduce the number of purification steps required to cell isolation, buffer exchange, and, when necessary, a method to improve long-term stability (e.g., freeze-drying). Whole-cell biocatalysts can also be easily removed from the reaction mixture, facilitating its recycling in industrial processes. Whole cell biocatalysis suffers from the presence of the cell membrane, that can act as a barrier, slowing down or prohibiting substrate and product exchange. By employing yeast surface display, the enzyme is attached to the outer cell wall, thereby overcoming membrane permeability limitations. This approach has also been shown, in some cases, to increase enzymatic stability. In this work, we attempted to immobilize an inulinase from the yeast Kluyveromyces marxianus on the cell wall of Komagataella phaffii using a native K. phaffii GPI anchor. Its activity and stability were then compared with those of the secreted enzyme. The ratio of cell-associated enzymatic activity to that detected in the supernatant was greatly increased in the presence of the anchor, suggesting that immobilization was successful. A bacterial cellulosome-derived cohesin–dockerin system was also tested to increase the number of enzymatic monomers attached to each anchor, theoretically enhancing the catalytic activity associated with each cell.