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Molecular cloning and characterization of a N-acetylneuraminate lyase gene from Staphylococcus hominis / 生物工程学报
Chinese Journal of Biotechnology ; (12): 480-489, 2013.
Article in Chinese | WPRIM | ID: wpr-233228
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.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Temperature / Bacterial Proteins / Enzyme Stability / Recombinant Proteins / Cloning, Molecular / Staphylococcus hominis / Escherichia coli / Genetics / Hydrogen-Ion Concentration / Oxo-Acid-Lyases Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2013 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Temperature / Bacterial Proteins / Enzyme Stability / Recombinant Proteins / Cloning, Molecular / Staphylococcus hominis / Escherichia coli / Genetics / Hydrogen-Ion Concentration / Oxo-Acid-Lyases Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2013 Type: Article