title: The yeast Wickerhamomyces anomalus AS1 secretes a multifunctional exo-β-1,3-glucanase with implications for winemaking.
abstract: A multifunctional exo-β-1,3-glucanase (WaExg2) was purified from the culture supernatant of the yeast Wickerhamomyces anomalus AS1. The enzyme was identified by mass spectroscopic analysis of tryptic peptide fragments and the encoding gene WaEXG2 was sequenced. The latter codes for a protein of 427 amino acids, beginning with a probable signal peptide (17 aa) for secretion. The mature protein has a molecular mass of 47 456 Da with a calculated pI of 4.84. The somewhat higher mass of the protein in SDS-PAGE might be due to bound carbohydrates. Presumptive disulphide bridges confer a high compactness to the molecule. This explains the apparent smaller molecular mass (35 kDa) of the native enzyme determined by electrophoresis, whereas the unfolded form is consistent with the theoretical mass. Enzymatic hydrolysis of selected glycosides and glycans by WaExg2 was proved by TLC analysis of cleavage products. Glucose was detected as the sole hydrolysis product from laminarin, underlining that the enzyme acts as an exoglucanase. In addition, the enzyme efficiently hydrolysed small β-linked glycosides (arbutin, esculin, polydatin, salicin) and disaccharides (cellobiose, gentiobiose). WaExg2 was active under typical wine-related conditions, such as low pH (3.5-4.0), high sugar concentrations (up to 20% w/v), high ethanol concentrations (10-15% v/v), presence of sulphites (up to 2 mm) and various cations. Therefore, the characterized enzyme might have multiple uses in winemaking, to increase concentrations of sensory and bioactive compounds by splitting glycosylated precursors or to reduce viscosity by hydrolysis of glycan slimes.
authors.affiliation: Institute for Microbiology and Wine Research, Johannes Gutenberg University, Mainz, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Schwentke
authors.forename: Andrea
authors.initials: A
authors.lastname: Sabel
authors.forename: Anna
authors.initials: A
authors.lastname: Petri
authors.forename: Helmut
authors.initials: H
authors.lastname: König
authors.forename: Harald
authors.initials: H
authors.lastname: Claus
mesh_terms: D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014920:Wine / Q000382:microbiology*
chemical_list: D004271:DNA, Fungal; D004220:Disulfides; D043326:Glucan 1,3-beta-Glucosidase; C117648:beta-1,3-exoglucanase
keywords: cell walls; glucanase; glycoside hydrolases; laminarin; polyphenols; wine