Cloning, expression, and characterization of a novel sialidase from Brevibacterium casei.

The sialidase gene from Brevibacterium casei was cloned in pET28a and overexpressed as a histidine-tagged protein in Escherichia coli BL21(DE3). The histidine-tagged sialidase protein was purified and characterized from the crude cell extracts of isopropyl-β-d-thiogalactopyranoside-induced cells using Ni-NTA agarose chromatography. SDS-PAGE using the purified sialidase indicated a single band at 116 kDa. This sialidase showed maximum activity at a pH of 5.5 and temperature of 37 °C. The kinetic parameters Km and Vmax for the artificial substrate 2'-(4-methylumbelliferyl)-α-d-N-acetyl-neuraminic acid sodium salt hydrate were 1.69 × 10(-3) mM and 244 mmol·Min(-1) ·mg(-1) , respectively. The sialidase may catalyze the hydrolysis of terminal sialic acids linked by the α-(2,3) and α-(2,8) linkage of polysialogangliosides, but it does not act on monosialotetrahexosylganglioside (GM1), which offers it a great potential for commercially producing GM1 from polysialogangliosides.