BIOTECHNOLOGY LETTERS Volume 17 No.8 (August 199.5)pp.851-852 Recieved 14th June CONCENTRATING ALKALINE SERINE PROTEASE, SUBTILISIN, USING A TEMPERATURE-SENSITIVE HYDROGEL Jun Han, Chang-Ho Park* and Roger Ruan Dept. of Biosystems and Agricultural Engineering, University of Minnesota, St. Paul, MN 55108, U.S.A. *Dept. of Chemical Engineering, Kyung Hee University, Kyunggi-do, 449-701, Korea SUMMARY The alkaline serine protease, subtilisin, produced by Bacillus licheniformis was concentrated using hydrogel ultrafiltration. Separation efficiency at 15° and 200C was 84 % but decreased above 25°C. INTRODUCTION A hydrogel, poly (N-isopropylacrylamide), changes its volume reversibly upon temperature swings and was used for enzyme concentration. However, the hydrogel ultrafiltration process did not appear useful for concentrating cellu!ase enzymes because separation efficiency was only 30 - 40% (Park and Orozco-Avila, 1992). This study showed that enzyme separation efficiency could be increased to 84% if the gel was allowed to swell at a lower temperature of 20"C or below. MATERIALS AND METHODS Subtilisin Fermentation. A seed culture of Bacillus licheniformis (ATCC 21418) was grown for 48 h at 35°C in 250 mL flasks containing 25 mL of the fermentation medium (1% glucose, 5% soluble starch, 1% soytone, 0.5% ammonium phosphate and 0.05% MgSO4 7H20). The bacteria were removed from the broth by centrifugation to stop further production of subtilisin during storage and concentration experiments. Preparation of the Hydrogel Beads. Beads of a temperature-sensitive hydrogel, poly (N-isopropylacrylamide) were prepared using inverse suspension polymerization with paraffin oil as the continuous phase and pluronic L-81 (BASF) as a nonionic polymeric surfactant. The details of preparation procedure is described in Park and Hoffman (1994). Enzyme Concentrating Experiments. Fermented broth (25 mL) with specific enzyme activity of 1.2 units/mL (total activity 30 units) was added to test tubes with 2.2 g of dry gel. The gel was allowed to swell for 9 hrs in an environmental chamber controllcd at 15, 20, 25, 30 and 350C before the gel was removed by filtration. Raffinate volumc was measured using a graduated cylinder. Separation Efficiency. Separation efficiency (rl) defined by Frcitas and Cussler (1987) was used. measured concentration change (in the raffinate) v I- x 100- x I(X) (1) that expected from gel volume change ar/af- 1 mf/mr - 1 851