Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae
Date
2013-11
Authors
Van Wyk, Niel
Trollope, Kim M.
Steenkamp, Emma T.
Wingfield, Brenda D.
Volschenk, Heinrich
Journal Title
Journal ISSN
Volume Title
Publisher
BioMed Central
Abstract
Background: β-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.
Results: We identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome 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 the CmINV gene in Saccharomyces cerevisiae BY4742Δsuc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the Δsuc2 deletion mutant strain of S. cerevisiae to 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 a Km and Vmax on 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 in
S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme.
Conclusion: The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.
Description
Publication of this article was funded by the Stellenbosch University Open Access Fund.
The original publication is available at :
Van Wyk, N. et al. 2013. Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae. BMC Biotechnology. 13:100, doi:10.1186/1472-6750-13-100.
The original publication is available at http://www.biomedcentral.com/bmcbiotechnol/
The original publication is available at :
Van Wyk, N. et al. 2013. Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae. BMC Biotechnology. 13:100, doi:10.1186/1472-6750-13-100.
The original publication is available at http://www.biomedcentral.com/bmcbiotechnol/
Keywords
β-fructofuranosidase, Short-chain fructooligosaccharides, Ceratocystis moniliformis, Saccharomyces cerevisiae -- Genetics, Heterologous expression
Citation
Van Wyk, N. et al. 2013. Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae. BMC Biotechnology. 13:100, doi:10.1186/1472-6750-13-100.