Purification and characterization of a polysialic acid-specific sialidase from Pseudomonas fluorescens JK-0412

• Published in: Biotechnology and bioprocess engineering 2012, 17(3), , pp.526-537
• Main Authors: Park, J.K., Gachon University of Medicine and Science, Incheon, Republic of Korea, Choi, D.J., The Catholic University of Korea, Bucheon, Republic of Korea, Kim, S.M., The Catholic University of Korea, Bucheon, Republic of Korea, Choi, H.N., The Catholic University of Korea, Bucheon, Republic of Korea, Park, J.W., The Catholic University of Korea, Bucheon, Republic of Korea, Jang, S.J., The Catholic University of Korea, Bucheon, Republic of Korea, Choo, Y.K., Wonkwang University, Iksan, Republic of Korea, Lee, C.G., Inha University, Incheon, Republic of Korea, Park, Y.I., The Catholic University of Korea, Bucheon, Republic of Korea
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Bacteria; Biodegradation; Bioengineering; Biomedical materials; Biotechnology; Carbon; Chemistry; Chemistry and Materials Science; Chitin; Engineering; Enzymes; Filtrate; Gels; hexamers; Homology; Industrial and Production Engineering; Industrial production; Influenza; Molecular weight; Nitrogen; oligosialic acids; pH effects; polysialic acid; Proteins; PSEUDOMONAS FLUORESCENS; Research Paper; Serratia marcescens; sialidase; Studies; 생물공학
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-011-0495-7

An enzyme with polySia degrading activity was purified from a culture filtrate of Pseudomonas fluorescens JK-0412 to apparent homogeneity using DEAE-Sepharose CL-6B column chomatography and fast performance liquid chomatography separation on a Mono-Q column. The molecular mass of the purified enzyme (tentatively named Endo-PS) was approximately 20 kDa on SDS-PAGE and 120 kDa on native-PAGE gels, suggesting that the active form is a hexamer. Although 12 residues of the Endo-PS N-terminal amino acid sequence showed 75% homology to the 21 kDa chitin binding protein (CBP21) of Serratia marcescens 2170, no significant similarity to other known proteins was observed. Apparent K∧m and V∧max values of Endo-PS toward the artificial substrate 4-methylumbelliferyl-sialic acid (4-MU-Neu5Ac) were 0.08 mM and 16 nmol/mg/min, respectively. The enzyme was maximally active at 37℃ and pH 8.0. Interestingly, the enzyme was shown to hydrolyze the natural substrate, α2,8-linked polySia (colominic acid), in an endo-acting manner. However, no activity toward α2,3- or α 2,6-sialyllactose was observed. Under optimal conditions, oligoSia ranging from 2 to 30 residues long were liberated by the cleavage of polySia, as identified by HPAEC-PED. Therefore, the purified enzyme Endo-PS was found to be a polySia-specific sialidase. This is the first report to describe the properties of a bacterial polySia-specific sialidase. Therefore, this enzyme may be a useful tool for both industrial oligoSia production and research on the structure and biological functions of polySia in nature.