KINETIC EVIDENCE OF A NOVEL INVERTASE IN AN ETHYL METHANE SULPLONATE DEREPRESSED MUTANT OF YARROWIA LIPOLYTICA

摘要

In the present study, we report on the kinetics of an extracellular invertase production by Yarrowia lipolytica in definedmedium i.e., sucrose peptone agar yeast extract, pH 6 (SAPY). The wild-type IIB-II was treated with ethyl methanesulphonate (EMS) as a chemical mutagen. Among the six mutants isolated, EMS-IV was found to be the best enzymeproducing mutant strain (51±2.4a U/ml). The maximum enzyme production (73±3.1a U/ml) occurred at 48 h of incubation(67±2.7a mg/ml protein). The potential mutant was stabilized at low levels of 2-deoxy-D-glucose (2dg) and the viablemutants were further optimized both culturally and nutritionally. The sucrose concentration, incubation period and pH wereoptimized to be 30 g/l, 28°C and 6.5, respectively. EMS-IV exhibited an improvement of over 10 folds in enzymeproduction when 5 g/l ammonium sulphate was used as a nitrogen source. Thin layer chromatography (TLC) and highperformance liquid chromatography (HPLC) analysis showed that optimal enzyme activity caused a higher hydrolysis rateof sucrose into monosaccharides (α-D-glucose and β-D-fructose). The values for Qp (1.7±0.12c U/ml/h) and Yp/s (3.7±1.24bU/g) of the mutant were considerably higher in comparison to the wild-type or all other yeast strains. The mutant could beused for enzyme production over the temperature range of 26-34°C, which is highly significant (LSD 0.048, HS).