title: Cloning, expression, and characterization of a novel sialidase from Brevibacterium casei.
abstract: 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 Kmand Vmaxfor the artificial substrate 2'-(4-methylumbelliferyl)-α-d-N-acetyl-neuraminic acid sodium salt hydrate were 1.69 × 10-3mM 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Xuedong
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Long
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Danhong
authors.initials: D
authors.lastname: Shen
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
mesh_terms: D001951:Brevibacterium / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D007700:Kinetics; D009439:Neuraminidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D009439:Neuraminidase
keywords: Brevibacterium casei; heterologous expression; monosialotetrahexosylganglioside; sialidase