Utilization of Ganglioside-Degrading Paenibacillus sp. Strain TS12 for Production of Glucosylceramide

Abstract

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a 3 asialo GM1 3 asialo GM2 3 lactosylceramide 3 glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, ␤-galactosidases, and ␤-hexosaminidases. TS12 also produced ␤-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.

Molecular Cloning and Catalytic Mechanism of a Novel Glycosphingolipid-degrading β-N-Acetylgalactosaminidase from Paenibacillus sp. TS12

“…TS12 was isolated as a GSL-degrading bacterium (10), and the glycosidases produced by TS12 were found to hydrolyze GSLs (11,12). Thus, to elucidate the specificity of the enzyme, various GSLs were tested as substrates.…”

“…TS12 was found to decompose various GSLs added to culture (10). For example, polysialogangliosides, GSLs with several NeuAc residues, were sequentially degraded in TS12 culture as follows: polysialogangliosides 3 GM1a 3 …”

“…TS12, that efficiently degrades various GSLs when added to cultures by producing a series of exoglycosidases (10). Subsequently, the molecular cloning of the TS12 glucocerebrosidase (11) and two ␤-HEXs (12), all of which are capable of hydrolyzing GSLs, was described.…”

Molecular Cloning and Crystal Structural Analysis of a Novel β-N-Acetylhexosaminidase from Paenibacillus sp. TS12 Capable of Degrading Glycosphingolipids

“…TS12 that degraded various GSLs efficiently by producing a series of exoglycosidases. 3,4 Furthermore, TS12 was found to be useful to produce or GlcCer from gangliosides as Fig. 1 (legend on next page) a biocatalyst, implying the potential industrial significance of TS12.…”

“…1 (legend on next page) a biocatalyst, implying the potential industrial significance of TS12. 3 Recently, O-glycoside hydrolases, which hydrolyze the O-glycosidic linkage between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety, have been classified into 115 glycoside hydrolase (GH) families based on amino acid similarity but not substrate specificity. 5,6 β-N-Acetylhexosaminidase (β-HEX; EC 3.2.1.52) is an exo-type enzyme that hydrolyzes both β-Nacetylgalactosamine (β-GalNAc) and β-N-acetylglucosamine (β-GlcNAc) linkages in oligosaccharides, glycoproteins, glycolipids, and glycosaminoglycans.…”