Purification and characterization of a polysialic acid-specific sialidase from Pseudomonas fluorescens JK-0412
- Authors
- Park, Jae Kweon; Choi, Doo Jin; Kim, Sung Min; Choi, Ha Na; Park, Joo Woong; Jang, Sung Jae; Choo, Young Kug; Lee, Choul Gyun; Park, Yong Il
- Issue Date
- Jun-2012
- Publisher
- KOREAN SOC BIOTECHNOLOGY & BIOENGINEERING
- Keywords
- polysialic acid; sialidase; oligosialic acids; Pseudomonas fluorescens
- Citation
- BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, v.17, no.3, pp.526 - 537
- Journal Title
- BIOTECHNOLOGY AND BIOPROCESS ENGINEERING
- Volume
- 17
- Number
- 3
- Start Page
- 526
- End Page
- 537
- URI
- https://scholarworks.bwise.kr/gachon/handle/2020.sw.gachon/16361
- DOI
- 10.1007/s12257-011-0495-7
- ISSN
- 1226-8372
- Abstract
- 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 37A degrees C and pH 8.0. Interestingly, the enzyme was shown to hydrolyze the natural substrate, alpha 2,8-linked polySia (colominic acid), in an endo-acting manner. However, no activity toward alpha 2,3- or alpha 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.
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