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A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzy-matically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/ MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from 17. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal gly-cosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catab-olism of β-1,3-glucan after its release in the extracellular medium. [ABSTRACT FROM AUTHOR]

A β-1,3-galactosyl-N-acetyIhexosamine phosphorylase (GaIGIyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GIcNAc and GalNAc as acceptors in the presence of α-o-galactose-l-phosphate as a donor to form lacto-N-biose I (LNB) (Galβ1 → 3GIcNAc) and galacto-N-biose (GNB) (GaIβ1 → 3GaINAc), respectively. GIcNAc was a much better acceptor than GaINAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the for LNB was approximately 60 times higher than the for GNB. This result indicated that the enzyme was remarkably different from GaIGIyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GaIGIyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated Iacto-N-biose I phosphorylase. The discovery of an LNB-specific GaIGIyNAcP resulted in recategorization of bifidobacterial GaIGIyNAcPs as galacto-N-biose/ lacto-N-biose I phosphorylases. [ABSTRACT FROM AUTHOR]