S cerevisiae hexokinase

Sugar analyses were performed on 2500-fold diluted GJ and GJ + _tfosEp in 100 mM potassium phosphate, pH 7.0, containing 10 mM MgSO₄, 1 mM NAD⁺, 1.5 mM ATP and 20 U mL⁻¹Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Worthington Biochemical Corporation). Concentrations of glucose, fructose, sucrose and fructan were determined from the changes in absorbance at 340 nm following sequential addition of 20 U mL⁻¹ 
S. cerevisiae hexokinase (Worthington Biochemical Corporation), 20 U mL⁻¹E. coli phosphoglucose isomerase (Megazyme International Ireland Ltd), 1.5 U mL⁻¹ 
S. cerevisiae sucrase/maltase (Megazyme International Ireland Ltd) and 150 μg _tfosEp (purified as previously described Martel et al. [2010 ]) respectively. Standards of glucose, fructose, sucrose and chicory inulin were used to calibrate the assay.

At specific time intervals (t₀h, t₄₈h, t₇₂h and t₉₆h) Bioscreen measurements were suspended and a 10-μL volume of culture supernatant removed from representative experimental wells for ethanol and sugar analyses.
Sugar analyses were performed on suitably diluted (typically 2,500-fold) culture medium in 100 mM potassium phosphate, pH 7.0, containing 10 mM MgSO₄, 1 mM NAD⁺, 1.5 mM ATP and 20 U mL⁻¹Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Worthington Biochemical Corporation). Concentrations of glucose, fructose, sucrose and fructan were determined from the changes in absorbance at 340 nm following sequential addition of 20 U mL⁻¹ 
S. cerevisiae hexokinase (Worthington Biochemical Corporation), 20 U mL⁻¹E. coli phosphoglucose isomerase (Megazyme International Ireland Ltd), 1.5 U mL⁻¹ 
S. cerevisiae sucrase/maltase (Megazyme International Ireland Ltd) and 10 U mL⁻¹ fructanase from Aspergillus niger (Megazyme International Ireland Ltd), respectively. Standards of glucose, fructose, sucrose and chicory inulin were used to calibrate the assay.
Ethanol determinations were made using a spectrophotometric ethanol assay kit (K-ETOH 11/06; Megazyme Ltd) according to manufacturer’s instructions. All samples were diluted 1,000-fold with distilled water prior to analysis.