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Purification and characterization of a sialidase fromClostridium chauvoei NC08596

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Abstract

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg−1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn2+, Mg2+, and Ca2+ and bovine serum albumin have a positive effect on the sialidase activity, while Hg2+, Cu2+, and Zn2+, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.

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Abbreviations

FPLC:

fast protein liquid chromatography

NCTC:

National Collection of Type Cultures

ATCC:

American Type Culture Collection

MU-Neu5Ac:

4-methylumbelliferyl-α-d-N-acetylneuraminic acid

buffer A:

0.02m piperazine, 0.01m CaCl2, pH 5.5

buffer B:

0.02m piperazine, 0.01m CaCl2, 1.0m NaCl, pH 5.5

buffer C:

0.1m sodium acetate, 0.01m CaCl2, pH 5.5

SDS:

sodium dodecyl sulfate

PAGE:

polyacrylamide gel electrophoresis

Neu5Ac:

N-acetylneuraminic acid

BSM:

bovine submandibular gland mucin

GD1a:

IV3Neu5Ac, II3Neu5Ac-GgOse4Cer

GM1:

II3Neu5Ac-GgOse4Cer

MU-Neu4,5Ac2 :

4-methylumbelliferyl-α-d-N-acetyl-4-O-acetylneuraminic acid

TLC:

thin-layer chromatography

HPTLC:

high performance thin-layer chromatography

EDTA:

ethylenediamine tetraacetic acid

EGTA:

ethylene glycol bis(2-aminoethyl-ethen)-N,N,N′,N′-tetraacetic acid

BSA:

bovine serum albumin

Neu5Ac2en:

2-deoxy-2,3-didehydro-N-acetylneuraminic acid

IEF:

isoelectric focusing

IEP:

isoelectric point

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Authors and Affiliations

  1. Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, D-2300, Kiel, Germany

    Dorothee Heuermann, Peter Roggentin, Reinhard G. Kleineidam & Roland Schauer

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  1. Dorothee Heuermann
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  2. Peter Roggentin
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  3. Reinhard G. Kleineidam
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  4. Roland Schauer
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Heuermann, D., Roggentin, P., Kleineidam, R.G. et al. Purification and characterization of a sialidase fromClostridium chauvoei NC08596. Glycoconjugate J 8, 95–101 (1991). https://doi.org/10.1007/BF00731018

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  • DOI: https://doi.org/10.1007/BF00731018

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