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

Abstract

Backgroundβ-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 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.ConclusionThe properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.

Structural Properties, Production, and Commercialisation of Invertase

“…This mechanism uses aspartate from NDPN motif as a nucleophile, while glutamate is used as an acid/base catalyst. Furthermore, aspartate residue from RDP motif is used as a transition state stabiliser (Van Wyk et al 2013). This process involves the protonation of the glycosidic oxygen by the acid/base catalyst, followed by a nucleophile attack on the anomeric carbon of the sugar substrate by a carboxylate group (Figure 2(a)).…”

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

“…Many recombinant pathways (like with p-coumaric acid and raspberry ketone) employed Arabidopsis thaliana genes, mainly due its sequence availability as it is a model organism and hardly because it is the 'best' enzyme candidates. Related to this is the mystery of how certain recombinant genes are expressed in high levels whereas similar ones are expressed poorly [94]. This underlines the utility of still employing a trial-and-error approach and exploring many recombinant genes to assess their ability to be expressed in yeast.…”

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

“…Many recombinant pathways (like with p-coumaric acid and raspberry ketone) employed Arabidopsis thaliana genes, mainly due its sequence availability as it is a model organism and hardly because it is the 'best' enzyme candidates. Related to this is the mystery of how certain recombinant genes are expressed in high levels whereas similar ones are expressed poorly [93]. This underlines the utility of still employing a trial-and-error approach and exploring many recombinant genes to assess their ability to be expressed in yeast.…”