Molecular cloning and characterization of a N-acetylneuraminate lyase gene from Staphylococcus hominis.
- Author:
Chuanhua ZHOU
1
;
Xi CHEN
;
Jinhui FENG
;
Dongguang XIAO
;
Qiaqing WUZ
;
Dunming ZHU
Author Information
1. College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China.
- Publication Type:Journal Article
- MeSH:
Bacterial Proteins;
genetics;
metabolism;
Cloning, Molecular;
Enzyme Stability;
Escherichia coli;
genetics;
metabolism;
Hydrogen-Ion Concentration;
Oxo-Acid-Lyases;
genetics;
metabolism;
Recombinant Proteins;
genetics;
metabolism;
Staphylococcus hominis;
enzymology;
Temperature
- From:
Chinese Journal of Biotechnology
2013;29(4):480-489
- CountryChina
- Language:Chinese
-
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.
