Double mutation of Saccharomyces cerevisiae for enhanced β-d-fructofuranosidase fructohydrolase productivity and application of growth kinetics for parametric significance analysis.

The kinetics of an extracellular β-d-fructofuranosidase fructohydrolase production by Saccharomyces cerevisiae in a chemically defined medium, i.e., sucrose peptone agar yeast extract at pH 6, was investigated. The wild-type was treated with a chemical mutagen, methyl methane sulfonate. Among the six mutants isolated, methyl methane sulfonate-V was found to be a better enzyme producing strain (52±2.4(a)U/mL). The maximum production (74±3.1(a)U/mL) was accomplished after at 48h (68±2.7(a)mg/mL protein). The mutants were stabilized at low levels of 5-fluoro-cytocine and the viable ones were further processed for optimization of cultural conditions and nutritional requirements. The sucrose concentration, incubation period and pH were optimized to be 30g/L, 28°C, and 6.5, respectively. The methyl methane sulfonate-V exhibited an improvement of over 10 folds in enzyme production when 5g/L ammonium sulfate was used as a nitrogen source. Thin layer chromatography and high-performance liquid chromatography analysis illustrated the optimal enzyme activity supported by the higher rate of hydrolysis of sucrose into monosaccharides, particularly α-d-glucose and β-d-fructose. The values for Qp (2±0.12(c)U/mL/h) and Yp/s (4±1.24(b)U/g) of the mutant were considerably increased in comparison with other yeast strains (both isolates and viable mutants). The mutant could be exploited for enzyme production over a wider temperature range (26-34°C), with significantly high enzyme activity (LSD 0.048, HS) at the optimal temperature.