Identification of the gene for beta-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae

AbstractBackgroundβ-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries.ResultsWe identified a β-fructofuranosidase gene (CmINV) from aCeratocystis moniliformisgenome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of theCmINVgene inSaccharomyces cerevisiaeBY4742∆suc2provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of theCmINVgene complemented the inability of the ∆suc2deletion mutant strain ofS. cerevisiaeto grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and aKmandVmaxon sucrose of 7.50 mM and 986 μmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein inS. cerevisiaeas one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme.ConclusionThe properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.