Christian Hugo Grun

Mechanism of action of the endo‐(1 → 3)‐α‐glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum

(1 → 3)‐α‐Glucanases catalyze the hydrolysis of fungal cell wall (1 → 3)‐α‐glucan, and function during cell division of yeasts containing this cell wall component or act in mycoparasitic processes. Here, we characterize the mechanism of action of the (1 → 3)‐α‐glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum. We observed that MutAp releases predominantly β‐glucose upon hydrolysis of crystalline (1 → 3)‐α‐glucan, indicating inversion of the anomeric configuration. After having identified (1 → 3)‐α‐glucan tetrasaccharide as the minimal substrate for MutAp, we showed that reduced (1 → 3)‐α‐glucan pentasaccharide is cleaved into a trisaccharide and a reduced disaccharide, demonstrating that MutAp displays endo‐hydrolytic activity. We propose a model for the catalytic mechanism of MutAp, whereby the enzyme breaks an intrachain glycosidic linkage of (1 → 3)‐α‐glucan, and then continues its hydrolysis towards the non‐reducing end by releasing β‐glucose residues in a processive manner.

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.