Purification and Characterization of N-Acetylneuraminate Lyase from Clostridium perfringens
S NEES, R SCHAUER, F MAYER, K EHRLICH - 1976 - degruyter.com
S NEES, R SCHAUER, F MAYER, K EHRLICH
1976•degruyter.com1) Clostridium perfringens cells were cultivated on a large scale using an automatic system.
2) TV-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria
by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by
precipitation with ammonium sulfate, filtration on Sephadex G-100, chromatography on
DEAE-Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel.
Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme …
2) TV-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria
by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by
precipitation with ammonium sulfate, filtration on Sephadex G-100, chromatography on
DEAE-Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel.
Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme …
Summary
1) Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) TV-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precipitation with ammonium sulfate, filtration on Sephadex G-100, chromatography on DEAE-Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein.
3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophoresis in 0.1% sodium dodecylsulfate or SM urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 IC^ M" 1 cm" 1 was determined. The enzyme exhibited aA" m value forTV-acetylneuraminic acid of 2.8 mM at its pH optimum of pH 7.2. The pH dependence of the Km value
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