Chromogenic Plate Assay and Cell Wall Detachment Protocol for Extracellular β-Xylosidase from Pichia pastoris

Abstract

This study improved the selection and recovery of recombinant Komagataella phaffii transformants producing β-xylosidase using an X-Xyl activity-based plate assay, leading to identification of a high-producing clone. Methanol induction under the AOX1 promoter enabled increasing enzyme activity over 120 hours, confirming successful secretion. However, total extracellular protein exceeded enzymatic activity, indicating the presence of inactive or non-target proteins. A significant fraction of β-xylosidase remained associated with the yeast cell surface, reducing apparent extracellular yield. Detachment experiments showed that mannose, glucose, and DTT most effectively released the enzyme, suggesting carbohydrate- and redox-related interactions.