Molecular cloning and characterization of a N-acetylneuraminate lyase gene from Staphylococcus hominis
来源于葡萄球菌的N-乙酰神经氨酸裂合酶的基因克隆及性质

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 °C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 °C for 2 h but decreased rapidly over 50 °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 °C for 24 h. Its Km 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 kcat/Km value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol·s), 0.08 L/(mmol·s) and 0.08 L/(mmol·s), respectively.