Purification and Characterization of Streptomyces Sialidases

Abstract

Some strains of Streptomyces produce sialidases. Two sialidases were purified over 1,000-fold from a culture filtrate of two Streptomyces species. They had the same properties in molecular weight, behavior to ions and other reagents, and substrate specificity. They showed very small differences in kinetic properties, pH optima, and heat stability. These Streptomyces sialidases differed markedly from Clostridium perfringens sialidase in molecular weight, p-chloromercuribenzoate sensitivity, and substrate specificity. Approximate molecular weights of the sialidases from Streptomyces and C. perfringens were 32,000 and 57,000, respectively. p-Chloromercuribenzoate (10-I M) caused complete inhibition of C. perfringens sialidase but not of Streptomyces sialidases.Glycosidases called sialidase or neuraminidase (N-acetylneuraminate glycohydrolase [EC 3.2.1.18]) are widely distributed in a variety of microorganisms and animal tissues, and bacterial sialidases have been prepared in a highly purified state (1, 6). However, extensive purification has not been attempted for sialidase from other sources. Sialic acids are constituents of numerous glycoproteins, glycolipids, and oligosaccharides, and in many instances removal of sialic acid by sialidase is accompanied by a loss of biological activity (5,20). Thus, it is thought that a sialidase specifically reacts with these sialyl compounds might be useful in detecting its physiological role.In this communication we describe the purification and characterization of Streptomyces sialidase and compare it with Clostridium perfringens sialidase. MATERIALS AND METHODSOrganisms and growth conditions. Streptomyces strains, to which laboratory numbers (MB395-A5 and MB503-C1) had been given at the Institute of Microbial Chemistry, were studied in detail. Strains MB395-A5 and MB503-C1 were classified as Streptomyces griseus and Streptomyces purpeofuscus, respectively, by M. Hamada in our institute. They were shake-cultured at 130 rpm in a medium containing 1.0% glucose, 1.0% starch, 0.75% peptone, 0.75% meat extract, 0.3% NaCl, 0.1% MgSO4*7H2O, 0.1%

Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins

“…Prolonged incubation, for up to 8 h, was necessary to detect the low levels of sialidase activity in these fractions. As shown later, the molecular weight of the ATCC 10543 enzyme was similar to that reported previously (3,4,11). The molecular weight of the active hemagglutinin produced by strain ATCC 10543 was lower than that of either the active sialidase produced by this strain or the active hemagglutinin from strain CN3870.…”

“…All three enzymes were completely inhibited by 1 mM pchloromercuribenzoate. Other workers (11) have also observed inhibition of C. perfringens sialidase by this compound. No significant inhibition of sialidase activity was observed with 10 mM EDTA in our studies.…”

“…Other workers (3,4,10,11) reported the presence of only one active form of sialidase. It was therefore decided to examine supernatant fluid from strain ATCC 10543, the strain used by these workers, to see which sialidase enzymes were produced by this strain under our conditions.…”

“…In earlier studies these workers (3) had concluded that the molecular weight of this enzyme was 56,000 ± 6,000. Kunimoto et al (11) obtained a value of 56,000 to 58,000 for this enzyme. In a recent publication, Nees et al (15) purified sialidase from C. perfringens strain ATCC 10543.…”

Isolation of a neuraminidase gene from Actinomyces viscosus T14V

“…The resistance of 0-acetylated or N-substituted sialic acids to hydrolysis by other bacterial neuraminidases such as those from C. perfringens or Vibrio cholerae (14,26) and from A. viscosus DSM 43798 (36) has been described before. Thus, with respect to hydrolytic properties, the A. viscosus T14V neuraminidase shares certain similarities with neuraminidases from A. viscosus DSM 43798 (36), C. perfringens 7, and other bacteria (18,22,38) that have broader ranges of substrate specificity. It is of interest that the properties of neuraminidase from A. viscosus T14V are not identical to those of A. viscosus DSM 43798.…”