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

Abstract

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 ± 2.4a U/mL). The maximum production (74 ± 3.1a U/mL) was accomplished after at 48 h (68 ± 2.7a 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 30 g/L, 28 °C, and 6.5, respectively. The methyl methane sulfonate-V exhibited an improvement of over 10 folds in enzyme production when 5 g/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 ± 0.12c U/mL/h) and Yp/s (4 ± 1.24b 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.

Biochemical, Molecular, and Transcriptional Highlights of the Biosynthesis of an Effective Biosurfactant Produced by Bacillus safensis PHA3, a Petroleum-Dwelling Bacteria

“…The plates were sprayed thoroughly with 0.2% ninhydrin (Sigma-Aldrich) and allowed to dry at 70°C for 20 min. To confirm the nature of the hexose, the sugar spot was taken from the TLC plate and therefore analyzed by high performance liquid chromatography (HPLC) as described by Sikander et al (2016). The fatty acids profile of the glycolipid was examined as described previously (Kitamoto et al, 1998).…”

Honey Mead Fermentation from Thai Stingless Bee (Tetragonula leaviceps) Honey using Ethanol Tolerant Yeast

“…Haq et al (2005) informed how cultivation conditions can influence the invertase activity of five wild strains of S. cerevisiae isolated from date palm fruit (Phoenix dactylifera) and found invertase activity ranging in 42.02-59.61 U/mL depending on the set cultivation parameters. Ali et al (2016) detected that the wild-type of S. cerevisiae isolated from soil reached only 17 U/mL but its chemically induced mutant reached between 23-74 U/mL of invertase activity again depending on cultivation parameters. On the other hand, Shatif et al (2002) studied the effect of mineral nutrients on the invertase activity of the strain S. cerevisiae GCB-K but the maximal value was only 12.68 U/mL.…”