Purification, crystallization, and characterization of neuraminidase from Micromonospora viridifaciens.

Aisaka, Kazuo; Igarashi, Ayuko; Uwajima, Takayuki

doi:10.1271/bbb1961.55.997

Cited by 16 publications

(12 citation statements)

References 2 publications

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“…All three products show similar values, values which were also similar to those determined for the neuraminidase produced in M. viridifaciens (2). These observations show that the three products have similar neuraminidase activities and stabilities.…”

Section: Resultssupporting

confidence: 82%

“…The deduced processing site agrees with the C-terminal end of the C. perfringens neuraminidase (24) when the sequences are aligned by their homology. The homology found between the M. viridifaciens and C. perfringens neuraminidases was also consistent with the observations that the properties of the two enzymes are similar in many respects (2,20).…”

Section: Discussionsupporting

confidence: 86%

“…As previously reported, M. viridifaciens produced neuraminidase only when either the substrate, colominic acid, or the product, N-acetylneuraminic acid, was added to the culture medium (2). When the concentration of colominic acid in the medium was increased, the level of neuraminidase excreted was increased.…”

Section: Resultssupporting

confidence: 67%

“…A similar observation was reported for the neuraminidase produced in M. viridifaciens, which produced a 41-kDa product when colominic acid was used as the inducer. However, when milk casein was used as the inducer, a 68-to 72-kDa product was mainly excreted (2). To understand the meaning of these processing reactions, each product (41, 52, and 68 kDa) was purified, and its properties were analyzed as described below.…”

Section: Resultsmentioning

confidence: 99%

“…viridifaciens was purified, and its properties were analyzed (2). Neuraminidase activity in M. viridifaciens was detected in the culture broth only when a substrate such as colominic acid or the product N-acetylneuraminic acid was present in the culture medium (2).…”

mentioning

confidence: 99%

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Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans

1992

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We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces fividans. This was accomplished by using the vector pU7O2 and BgM-BclI libraries of M. viridifaciens chromosomal inserts created in S. fividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BgI fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M, of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-XThr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. fividans. Neuraminidase (EC 3.2.1.18) is a glycosidase which cleaves an a-ketosidic linkage between sialic acid and the adjacent sugar residue on glycoproteins, glycolipids, and oligosaccharides. It is widely distributed in microorganisms such as viruses, bacteria, and actinomycetes and in various tissues from fowls and mammals. For myxovirus it is an important surface antigen and also plays a role in facilitating the movement of the virus both to and from the site of infection (1). Bacteria and actinomycetes which produce neuraminidase can release sialic acids from glycoconjugates for use as carbon and energy sources. In pathogenic microorganisms, neuraminidase activity has a strong correlation with pathogenesis (27).Recently, neuraminidase genes were cloned from several pathogenic microorganisms, including Clostridium sordellii G12 (25), Clostridium perfringens A99 (24), Salmonella typhimurium LT-2 (23), Vibrio cholerae 395 (29), and Bacteroides fragilis TAL2480 (26). From examination of their deduced amino acid sequences, a conserved sequence of 12 amino acids (Asp box) which was repeated at four or five positions was found (23). This conserved sequence shows similarity to the neuraminidase of influenza virus A H7N1 and H13N9. The structures of neuraminidases from nonpathogenic bacteria have not been characterized. In a recent report, neuraminidase was widely found in culture fluids of actinomycetes (3). For Micromonospora spp., 10 of the 39 strains examined exhibited neuraminidase activity. Micromonospora viridifaciens showed the highest neuraminidase activity among this group. Neuraminidase from M.viridifaciens was purified, and its properties were analyzed (2). Neuraminidase activity in M. viridifaciens was detected in the culture broth only when a substrate such as colominic acid or the product N-acetylneuraminic acid was present in the culture medium (2).We would like to understand the regulation of neuraminidase gene (ned4) expression and the properties of its * Corresponding author. product in M. viridifaciens and ...

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Section: Resultssupporting

confidence: 82%