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 K m and V max 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.