High yield electroextraction of proteins from yeast by a flow process V. Ganeva, a B. Galutzov, a and J. Teissi  e b, * a Faculty of Biology, University of Sofia, 8 blvd Dragan Tzankov, 1164 Sofia, Bulgaria b IPBS CNRS UMR5089, 205 Route de Narbonne, 31077 Toulouse c edex 4, France Received 27 September 2002 Abstract High yields of intracellular enzymes from yeast can be obtained by application of a series of electric field pulses with a flow process. Up to 80–90% of the total activity can be liberated without any further or previous treatment of cells. The method is based on electroinduced changes in the cell envelope leading to a leakage of part of the intracellular proteins without formation of debris and permits treatment of large volumes. Field parameters require a limited electrical power. Treatment of at least 20% wet weight suspensions is possible. The optimal field conditions must be adjusted to the suspension concentration. Maximal yield is obtained within4hat30 °C for enzymes from Saccharomyces cerevisiae such as hexokinase, 3-phosphoglycerate kinase, and glyceraldehyde- 3-phosphate dehydrogenase. The extraction of b-D-galactosidase from Kluyveromyces lactis lasts 10h but can be accelerated by adding dithiothreitol in the postpulse medium. The specific activities of the electroextracted enzymes are higher than those obtained by mechanical disintegration or enzymatic lysis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Pulsed electric field; Electropulsation; Flow system; Extraction; Intracellular enzymes; Saccharomyces cerevisiae; Kluyveromyces lactis Yeasts are widely used for industrial production of homologous proteins, most of them with intracellular locations. Nowadays Saccharomyces cerevisiae and yeasts from genera Kluyveromyces, Pichia, and Han- senula have become a suitable host for production of recombinant proteins with biotechnological and phar- macological applications [1,2]. Their secretion from the cell is the optimal way for isolation, but often this is impossible or of low efficiency. Thus, the newly syn- thesized heterologous proteins remain accumulated in the cell cytoplasm. The main methods utilized for liberation of intracel- lular enzymes are mechanical disintegration and chem- icalextraction[3,4].Althoughapplicableonalargescale these are relatively drastic procedures, which affect the stability of the proteins or introduce additional impu- rities to be removed in the associated downstream pro- cesses. So, alternative methods for enzyme extraction are badly needed to ensure a higher selectivity of release and mild experimental conditions to preserve the en- zyme activity at a maximal level. Cell treatment with high intensity electric field pulses provokes a change in the membrane structure leading to a loss of its barrier function—a phenomenon indicated as electropermeabilization or ‘‘electroporation’’ [5–7]. By a proper choice of the parameters of the applied electric field this change in the membrane permeability can be reversible or irreversible, causing leakage of cy- toplasmic content and cell death [8]. Introduced in the 1980s [9–11] electric field treatment using a flow system seems nowadays a very promising technique for sterili- zation of foods [12]. Despite numerous papers on the electropermeabili- zation and its multiple applications, one aspect of the irreversible electropermeabilization—the release of macromolecules from cells possessing a cell wall and its potential industrial application—was totally neglected up to now. Recentlyweshowedthattheapplicationofaseriesof electric pulses could provoke an important release of different cytoplasmic enzymes from yeast by a batch process [13,14]. The efficiency of this process was Analytical Biochemistry 315 (2003) 77–84 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +33-5-61-17-59-94. E-mail address: justin.teissie@ipbs.fr (J. Teissi  e). 0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(02)00699-1