. Hydrolysis of methyl mandelate
A mass of 200 mg of the immobilized preparations was added to 2 mL of 10 mM
substrate in different concentrations of buffer (25–500 mM of sodium phosphate
buffer) at pH 7 and 25 ◦C under continuous stirring. The conversion degree was
analyzed by RP-HPLC (Spectra Physic SP 100 coupled with an UV detector SpectraPhysic
SP 8450) using a Kromasil C18 (15 cm × 0.46 cm) column. Samples (20 L)
were injected and eluted at a flow rate of 2.0 mL/min using acetonitrile/10 mM
ammonium acetate (20:80, v/v) at pH 3.2 as mobile phase and UV detection was
performed at 230 nm. The acid has a retention time of 2.5 min while the ester has a
retention time of 6 min. One unit of enzyme activity was defined as the amount of
enzyme necessary to produce 1 mol of mandelic acid per minute under the conditions
described above. Activity was determined by triplicate with a maximum
conversion of 10–15%, and data are given as average values.