title: [Molecular cloning and characterization of a N-acetylneuraminate lyase gene from Staphylococcus hominis].
abstract: A N-acetylneuraminate lyase gene (shnal) from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45 degrees C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 degrees C for 2 h but decreased rapidly over 50 degrees C. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being > 70% when the enzyme was incubated in different buffers at 4 degrees C for 24 h. Its K(m) towards N-acetylneuraminic acid, pyruvate and ManNAc were (4.0 +/- 0.2) mmol/L, (35.1 +/- 3.2) mmol/L and (131.7 +/- 12.1) mmol/L, respectively. The k(cat)/K(m) value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol x s), 0.08 L/(mmol x s) and 0.08 L/(mmol x s), respectively.
authors.affiliation: College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China.
authors.forename: Chuanhua
authors.initials: C
authors.lastname: Zhou
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Jinhui
authors.initials: J
authors.lastname: Feng
authors.forename: Dongguang
authors.initials: D
authors.lastname: Xiao
authors.forename: Qiaqing
authors.initials: Q
authors.lastname: Wuz
authors.forename: Dunming
authors.initials: D
authors.lastname: Zhu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D041181:Staphylococcus hominis / Q000201:enzymology*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase