2023 Volume 31 Pages 63-75
We investigated the feasibility of immobilized enzymes on silk fibers in which functional enzymes are fixed through tagging the silk-binding peptide (YN42), a fibroin-binding peptide discovered in 2011 by phage display. Fluorescent protein-fused enzymes (cellulase and β-glucosidase, derived from termites) containing YN42 peptide at the N-terminus were expressed in the silk glands of transgenic silkworm using the GAL4/UAS gene expression system. The resultant cocoons showed fluorescence, indicating successful expression of the introduced recombinant enzymes on the silk fibers. When the cocoons were immersed in assay solution containing substrate, their enzyme activities were confirmed by detecting the enzymatically degraded products. The enzyme activity in the cocoon was maintained after repeating 5 min reactions. Ten-round reaction assay was done by replacing the substrate after each cycle. Moreover, the activity was observed even after the cocoons had been incubated at 37°C for four days, demonstrating excellent immobilization performance of the enzymes onto silk. Considering their use in a bioreactor, we tested whether our material is tolerant of continuous flow reactions. Enzyme activity was maintained even after 10 repetitions of 24-h continuous flow reaction, demonstrating excellent immobilization ability on silk. These results demonstrated that silk fibers immobilized with enzymes through YN42 binding peptide can be useful as matrix for enzyme-based bioreactors.
