Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus.

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain β-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC ₅₀ values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC ₅₀ values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters K _M and V _max for HexA and B with p-nitrophenyl N-acetyl-β-d-glucosaminide are 4 mM, 0.23 μmol·min ^-1 ·mL ^-1 and 2.86 mM, 0.29 μmol·min ^-1 ·mL ^-1 , respectively, and with p-nitrophenyl N-acetyl-β-d-galactosaminide are 4.5 mM, 0.054 μmol·min ^-1 ·mL ^-1 and 1.4 mM, 0.14 μmol·min ^-1 ·mL ^-1 , respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.
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