A two-stage aqueous phase extraction of the enzyme fumarase from baker's yeast is described with respect to the recycling of the upper effluent phase from the secondary extraction. It was found that the binodal curve position was only slightly altered by the presence of protein at different load levels and that enzyme specific activity in the product was improved at higher total protein concentrations, which simplified the recycling strategy. On-line enzyme and protein assays showed that disturbances during start-up and desludging operations were reflected in variations in the protein concentrations in the various streams, but that the enzyme activities remained relatively stable. Recycling reduced the polyethylene requirements by 50% and that for potassium phosphate by 12%. A maximum saving of PEG (polyethylene glycol) of approximately 70 YO was achieved at optimum conditions of extraction, compared to a value of 90% predicted by a theoretical description of the system. An economic analysis of the recycled process showed significant advantages compared with a non-recycled system, even though the capital outlay requirements for an automated, continuous system are much higher than for a manually run process.

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A B S T R A C TAutomated assay techniques are described for on-line measurements of fumarase activity and total protein concentration, including in-line sample dilution and sample multiplexing during continuous aqueous phase extraction. Fumarase was determined by following the conversion of L-malate to fumurate at a wavelength of 250 nm, while the protein assay was based on the Biuret reaction. Actual assay times of 2and 4 min were achieved for the fumarase and protein measurements, respectively, with an effective measurement cycle time of 2 min. Standard deviations of c. 3.2 and 2% of the measured values were calculated for the enzyme and protein values, respectively. The assay system was coupled to a computer to allow on-line data visualization and storage. K e y words: enzyme (fumarase), protein, automated assay, Biuret, aqueous phase extraction.