Partial purification and properties of neuraminidase from Bifidobacterium lactentis.

Abstract

Bifidobacterium lactentis 659 a gram-positive, nonsporeforming anaerobic bacterium originally isolated from the feces of breast-fed infants produces neuraminidase after enzyme induction with 2mM N-acetylmannosamine added to the culture medium. Bacteria were transferred and grown in a medium containing casein hydrolysate, lactose, sodium acetate, amino acids, vitamins, salts and 2% human milk for 48 h at 37 degrees C under N2/CO2 atmosphere. Two subcultures derived from the original strain B. lactentis 659 were investigated separately because of different growth characteristics. However, their taxonomic identity was not doubtful. Neuraminidase was liberated from the bacterial sediments by ultrasonic treatment in 0.15M NaCl and was isolated by 60% ammonium sulfate precipitation, dialysis, concentration, and column chromatography on Sepharose CL-6B and Sephadex G-100. The enzyme exhibits a molecular weight of 38000 and a pH optimum in the range of pH 5--6 in barbital/acetate buffers. Starch gel electrophoresis and neuraminidase-specific staining with NeuAc alpha 2 leads to (3-methoxyphenyl) glycoside revealed two major and three minor enzyme bands. Michaelis constants (Km) were determined to be 7.1 mM for the (alpha 2 leads to 3) linkage of II3NeuAc-Lac, 7.5mM for the (alpha 2 leads to 6) linkage of II6Neu-Ac-Lac and 15mM for the (alpha 2 leads to 8) linkage in II3(leads from 2NeuAc8)2-Lac. Among the different groups of naturally occurring NeuAc-containing substrates, i.e. glycoproteins, gangliosides and oligosaccharides, B. lactentis neuraminidase cleaves oligosaccharides preferentially without remarkable differences between (alpha 2 leads to 3) and (alpha 2 leads to 6) linkages. Globular glycoproteins and mucins are poor substrates and gangliosides are practically not affected. In contrast, enzyme activity towards synthetic NeuAc glycosides is very high. The enzyme is activated by 50% with 50mM Ca2 and inhibited by 20mM EDTA accordingly. In general, B. lactentis neuraminidase shows a substrate specificity pattern similar to those found in other non-pathogenic and non-invasive representatives of the human bacterial flora. The potential biological role of intestinal Bifidobacteria will be discussed.