Themolecular mass cut-off of ultrafiltration membrane was 10 kDa(Millipore, regenerated cellulose). Ten milliliters of top phase con-taining the enzyme and 20 mL (two diavolumes) of tris–HCl buffer(pH 7.0, 100 mM) were added at the membrane module. Approxi-mately 20 mL were permeated at each cycle; then, the process wasstopped and a volume buffer was added at the same rate as perme-ate was generated, until 5 cycles have been completed.