Development of a reusable non-viable koji-type CHase biocatalyst from yerba mate residues

A koji biocatalyst with chlorogenate hydrolase (CHase, EC 3.1.1.42) activity was produced by solid-state fermentation of Aspergillus niger AKU 3302 using yerba mate processing residues as both support and inducer. The fungus was cultivated in traditional koji trays until complete mycelial colonization. Thermal treatment of the koji at 60 °C for 30 min effectively inactivated fungal viability while preserving CHase activity and yielded a non-viable but highly active biocatalyst (YMK_non-viable). Maximum CHase activity of the biocatalyst (approximately 31 EU/100 g_{dry material}) was obtained in a solid culture medium composed of discarded yerba mate powder supplemented with 7 g/L sucrose and 0.4% (w/v, dry basis) yerba mate extract after 4 days of fermentation. The YMK_non-viable exhibited good operational stability during two consecutive reaction cycles (up to approximately 330 min of continuous operation) and excellent storage stability, retaining 100% of its initial activity after 7 months at -20 °C. These results demonstrated that yerba mate processing residues could be efficiently valorized to produce a heat-inactivated, reusable CHase biocatalyst with good operational and storage stability, providing a simple and sustainable approach for the biotransformation of chlorogenic acid-rich extracts.