Back to Contents

Pak. J. Bot., 48(1): 357-262, 2015.

FREE FULL TEXT PDF

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

SIKANDER ALI^1* AND MUHAMMAD UMAR HAYYAT²

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

Key words: Yarrowia lipolytica, Invertase, Kinetic study, Microbial fermentation, Batch culture, EMS mutagenesis.

¹Institute of Industrial Biotechnology, GC University Lahore, Pakistan

²Sustainable Development Study Centre, GC University Lahore, Pakistan

^*Corresponding author e-mail: alisbiotech@yahoo.com