Kinetics of improved extracellular ?-d-fructofuranosidase fructohydrolase production by a derepressed Saccharomyces cerevisiae

Abstract

Aims: β‐d‐fructofuranosidase fructohydrolase (FFH, EC 3·2·1·26) is an enzyme which hydrolyses the α‐1,4 glycosidic bonds of sucrose and releases monosaccharides. The present study deals with the kinetics of improved extracellular FFH production by Saccharomyces cerevisiae in batch culture. Materials and Results: Strains of S. cerevisiae can show increased FFH activity when grown on chemically defined medium. In the present study, wild‐culture S. cerevisiae GCB‐IV was mutated by treatment with ethyl methane sulfonate (EMS). Among six yeast mutants, EMS‐II was found to be the highest FFH‐producing strain (51·46 ± 2·4 U ml−1). Maximum FFH production (78·46 ± 3·2 U ml−1) was obtained 48 h after incubation by this 2‐deoxy‐d‐glucose (2dg)‐resistant mutant (76·20 mg ml−1 protein). The optimal concentration of sucrose, incubation period and initial pH were 30·0 g l−1, 28°C and 6·5, respectively. The mutant EMS‐II showed improvement in FFH production when 5·0 g l−1 urea was added as a sole nitrogen source into SAPY medium. Values for Qp (1·802 ± 0·2 U ml−1 h−1) and Yp/s (3·460 ± 1·1 U g−1) of EMS‐II were significantly improved over the other yeast strains. Conclusion: The Ea value (40·28 ± 3·5 kJ mol−1) of EMS‐II was significant (P ≤ 0·05) when compared with its wild‐culture progenitor GCB‐IV. In addition, thermodynamic studies revealed that the cell system exerts protection against thermal inactivation of FFH (33·55 ± 4·2 kJ mol−1). Significance and Impact of the Study: The EMS‐II mutant can be exploited for FFH production over a wide range of incubation temperature (26–34°C).