Bioprocess Engineering15 (1996) 253 256 9 Springer-Verlag 1996 Continuous extraction of a recombinant cutinase from Escherichia coli disrupted cells with reversed micelles using a perforated rotating disc contactor M.G. Carneiro-da-Cunha, M.R. Aires-Barros, E.B. Tambourgi, J.M.S. Cabral 253 Abstract This work reports on the continuous extraction of a recombinant cutinase with reversed micelles using a perfor- ated rotating disc contactor. Intracellular cutinase was directly extracted at 20~ with 100 mM AOT in isooctane from complex biological media of Escherichia coli disrupted cells. The optimal conditions for the direct extraction of the enzyme from media containing cell debris led to an extraction yield of 54.4%. 1 Introduction The extraction and purification of proteins using reversed micellar systems have been studied in recent years [1 4]. However, the continuous extraction of the target protein using reversed micellar systems has been limited to few examples in the literature, namely, the extraction of an a-amylase in an aqueous solution by TOMAC reversed micellar phase using two mixer-settlers units [5], the extraction of a pure recombinant cutinase by AOT reversed micelles with a perfor- ated rotating disc contactor [6] and the recovery of intracellu- lar proteins from Candida utilis by reversed micellar extraction in a spray column [7]. Extraction columns with rotary motion, namely the perforated rotating disc contactor with free flow areas have been industrially used as an alternative apparatus to the conventional extraction equipment [8, 9]. With this type of extractors, high mass transfer rates between the phases are obtained leading to higher separation efficiencies [9]. Received: 23 January 1996 M.G. Carneiro-da-Cunha, M.R. Aires-Barros, J.M.S. Cabral Laborat6rio de Engenharia Bioquimica, Instituto Superior T6cnico, Centro de Engenharia Biol6gica e Quimica, 1000 Lisboa, Portugal E.B. Tambourgi UNICAMP/FEQ/DESQ, Caixa Postal 6066, CEP 1381-970, Campinas-SP, Brasil Correspondence to: M.R. Aires-Barros Maria das Gragas Carneiro da Cunha, from ITEP-Instituto Tecnoldgico do Estado de Pernambuco, acknowledges a Ph.D fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and Centro de Pesquisa Aggeu MagalMes, Recife - PE Brasil. This work was partly financed by the BRIDGE Programme (Contract BIOT-CT91-0274(DTEE). This paper describes the continuous extraction of a lipolytic enzyme, a cutinase, from Fusarium solani pisi, which was cloned into the plasmid pMa/Cs-CUF and expressed intracel- lularly in Escherichia coli (strain WK6) [10]. A perforated rotating disc contactor, was used for the continuous extraction of this enzyme with AOT reversed micelles from Escherichia coli disrupted cells media in the presence of cell debris. 2 Materials and methods 2.1 Chemicals Sodium di(2-ethylhexyl) sulfosuccinate (AOT) was purchased from Sigma (St. Louis, USA); Isooctane, analytical grade, and IPTG were from Merck (Darmstadt, Germany). All other chemicals were of analytical grade. 2.2 Experimental apparatus The perforated rotating disc contactor, as shown in Figure 1, was made of Perspex tube 32 mm i.d. and 160 mm high. Seven perforated discs equally separated were mounted on a central shaft which was rotated at a velocity of 220 rpm. The perforated discs were 30 mm in diameter and drilled with 20 holes of 1.5 mm diameter (disc free area for flow 20%). The column was maintained at 20 ~ using water in a refrigerated jacket. 2.3 Enzyme Cutinase was produced by a Escherichia coli recombinant strain (WK6), a kind gift of Corvas International Gent, Belgium. This enzyme is a compact domain protein with 196 aminoacids, a molecular weight of 22KDa and an isoelectric point of 7.0. Its tridimendional structure is known [10] and the catalytic triad Ser 120, Asp 175 and His 188 constitutes the active site. The catalytic serine is not buried under surface loops and is accessible to solvent. 2.4 Cell growth and enzyme production The cells were precultured in Luria-Bertani (LB) medium containing ampicillin (150 btg/ml) in an orbital shaker (250 rpm) at 37 ~ for 8 h. Terrific Broth (TB) medium, supplemented with 20 mM MgSO4 and ampicillin (150 gg/ml),