High Production of Neuraminidase by a Vibrio cholerae Non-O1 Strain—the First Possible Alternative to Toxigenic Producers

Abstract

Vibrio cholerae neuraminidase (VCNA) is widely used in biochemical and medical research, in processes for preparing homogenous sialoconjugates, and in the pharmaceutical industry. Its production by non-toxigenic strains is quite desirable, in order to avoid the expensive safety measures. Here, we report the first method for highly effective production of a novel, purified V. cholerae extracellular neuraminidase from a non-toxigenic strain. The enzyme is highly active, and its properties, as well as the responsible gene nanH, are practically identical with those of the toxigenic strains. It cleaves α,2 → 3 and α,2 → 6 glycosidic bonds with highest affinity (K M 1.7 × 10(-5) μM) for human transferrin. The deduced amino acid sequence of the enzyme reveals three binding sites for Ca(2+) and one for sialic acid. The sequence analysis of the nanH gene, being the first for a V. cholerae non-O1 strain, shows 99% identity with a new nanH allele of an O1 Vibrio strain. The simple laboratory technology for efficient production of the new VCNA is based on the use of common and cheap nutrient media and easily available inducer--glycomacropeptide. The rapid purification consists of salting-out and diethylaminoethanol (DEAE) and Q-Sepharose chromatography steps. Purified preparation contains no aldolase and protease, which gives the production scheme a great potential for industrial application.

Safe Sialidase Production by the Saprophyte Oerskovia paurometabola: Gene Sequence and Enzyme Purification

“…In the present study, the isolation of a sialidase from O. paurometabola in an electrophoretically pure preparation is presented for the first time. After culturing the producer strain O129 for 24 h in a common nutrient medium, the sialidase activity reached levels comparable to those of our previously described strain V13 of the typical sialidase producer V. cholerae [28]. We used a simple laboratory purification scheme to obtain a homogenous enzyme preparation with a high specific activity.…”

“…It should be noted that the O129 enzyme degraded colominic acid (homopolymer of α(2→8) bounded sialic acids) with relatively high efficiency. Most of the bacterial sialidases are weakly active or completely inactive towards this compound [28,34]. The lower activity towards horse serum could be explained by the presence of sialic acids that are O-acetylated, a modification known to reduce the degree of hydrolysis by sialidases [26,35].…”

“…pneumoniae, V. cholerae non O1/13, Aeromonas sp. A40/02, C. perfringens [28,[37][38][39][40]. The addition of GMP to the semisynthetic medium leads to more than a 2-fold increase in enzyme activity.…”

“…The inductive effect of this compound was documented in the synthesis of sialidases from Erysipelothrix rhusiopathiae, V. cholerae non O1/13, and Aeromonas sp. A40/02 strains [28,39,41].…”