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

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 Vmax 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°C 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 α2,6-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.

Autoren:		Jae Kweon Park, Doo Jin Choi, Sung Min Kim, Ha Na Choi, Joo Woong Park, Sung Jae Jang, Young Kug Choo, Choul Gyun Lee, Yong Il Park
Journal:		Biotechnology and Bioprocess Engineering
Jahrgang:		2012
DOI:		10.1007/s12257-011-0495-7
Erscheinungsdatum:		05.06.2012

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