Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae.
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 BY4742∆suc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the ∆suc2 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.


CONCLUSIONS
The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.
Niël
N
van Wyk
Kim M
KM
Trollope
Emma T
ET
Steenkamp
Brenda D
BD
Wingfield
Department of Microbiology, Stellenbosch University, Room A322, JC Smuts Building, De Beer Street, Private Bag X1, Matieland 7602 Stellenbosch, South Africa. volschenkh@sun.ac.za.
Heinrich
H
Volschenk
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D001711:Biotransformation; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D013395:Sucrose / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D011994:Recombinant Proteins; D013395:Sucrose; D043324:beta-Fructofuranosidase