title: 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±2.4(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.
authors.affiliation: Institute of Industrial Biotechnology (IIB), GC University Lahore, Pakistan. Electronic address: alisbiotech@yahoo.com.
authors.forename: Sikander
authors.initials: S
authors.lastname: Ali
authors.affiliation: Department of Botany, Kinnaird College for Women (KCW), Lahore, Pakistan.
authors.forename: Aafia
authors.initials: A
authors.lastname: Aslam
authors.affiliation: Sustainable Development Study Centre (SDSC), GC University Lahore, Pakistan.
authors.forename: Muhammad Umar
authors.initials: MU
authors.lastname: Hayyat
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003470:Culture Media / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D016296:Mutagenesis; D009153:Mutagens / Q000378:metabolism; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D012705:Serratia; D013395:Sucrose / Q000378:metabolism; D013441:Sulfinic Acids / Q000378:metabolism; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000096:biosynthesis*
chemical_list: D003470:Culture Media; D009153:Mutagens; D013441:Sulfinic Acids; C082399:methyl methanethiosulfinate; D013395:Sucrose; D043324:beta-Fructofuranosidase
keywords: Entropy and enthalpy; Kinetics and thermodynamics; Saccharomyces cerevisiae; β-d-Fructofuranosidase fructohydrolase