Kinetic and biophysical characterization of a lysosomal α-L-fucosidase from the fresh water mussel, Lamellidens corrianus

Kinetic and biophysical studies have been carried out on a lysosomal a-L-fucosidase purified from the fresh water mussel, Lamellidens con-Janus. The enzyme migrates as a single band in SDS-PAGE as well as native PAGE corresponding to a M-r of 56 kDa. Mass spectrometric analysis yielded a molecular mass of 56175.1 Da for the enzyme, and peptide mass fingerprinting studies showed that it shares sequence homology with other fucosidases. Zymogram analysis showed that the a-L-fucosidase hydrolyzed 4 methyl umbelliferyl a-L-fucopyranoside. The pH and temperature optima of the enzyme were found to be 5.0-6.0 and 60 degrees C, respectively. The K-M, V-max and k(cat) values of the enzyme estimated with p-nitrophenyl fucopyranoside are 0.85 mM, 27.20 mU/mL and 1.01 s(-1), respectively. The inhibition constant (KO of the enzyme towards L-Fucose is 1.09 mM. CD spectral analysis has shown that the protein contains predominantly beta-sheets in its secondary structure. Chemical unfolding studies indicate that a-L-fucosidase unfolds in a broad sigmoidal, cooperative unfolding transition, centered at similar to 2.2 M for both guanidinium chloride and guanidinium thiocyanate. The present results obtained with the L. conianus alpha-L-fucosidase are expected to provide further insights into the various biological processes associated with fucosidases and help in exploiting this enzyme in therapeutic applications. (C) 2017 Elsevier B.V. All rights reserved.