Signi®cance of Location of Enzymes on Their Release During Microbial Cell Disruption B. Balasundaram, A.B. Pandit Department of Chemical Engineering University Department of Chemical Technology, Matunga, Mumbai ± 400 019 India, telephone: +91 22 4145616; fax: +91 22 4145614; e-mail: abp@udct.ernet.in Received 5 September 2000; accepted 1 August 2001 Abstract: The release kinetics of the enzyme invertase and alcohol dehydrogenase from yeast and penicillin ac- ylase from E. coli during disruption using various tech- niques has been investigated. The disruption techniques used were sonication, high-pressure homogenization, and hydrodynamic cavitation. The ®rst-order-release ki- netics was applied for the determination of release rate of these enzymes and total soluble proteins. Location factor LF) values were calculated using these release rates. The location of the enzymes as given by the values of location factor coincided well with those reported in the literature. Varying values of location factor for the same enzyme by different disruption techniques gave some indications about the selectivity of release of a target enzyme by dif- ferent disruption techniques. Varying values of location factor for the same enzyme with the use of a particular equipment or disruption technique at different conditions reveals the degree to which the cell is disrupted. Few plausible applications of this location factor concept have been predicted and these speculations have been exam- ined. This location factor concept has been used for monitoring the heat-induced translocation of ADH and location of penicillin acylase during the growth period of E. coli cells. ã 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 607±614, 2001. Keywords: cell disruption; location factor; release kinet- ics; cavitation INTRODUCTION A large proportion of the potentially useful microbial products are retained within the cells. The microbial cell can be genetically manipulated to make the product ex- tracellular or to make the cell leaky with respect to the target microbial product. The genetic manipulation of microbial cells to make them leaky is limited in scope Chisti and Moo-Young, 1986). Most recombinant gene products have been produced as intracellular products Sauer et al., 1988). The secretion of recombinant protein to the periplasmic space has numerous advantages over its expression in the cytoplasm French et al., 1996). Hence, disruption of the cell becomes inevitable Feliu et al., 1988). Of the methods available, physical methods are often favored due to the economic and operational limi- tations of the chemical and enzymatic methods. The cell- disruption process is dependent on cell properties such as thephysicalstrengthofthecellwallofthemicroorganism Sauer et al., 1988) and on the intracellular location of enzymeinthemicrobialcellFollowsetal.,1971:Hettwer and Wang, 1989: Kuboi et al., 1995: Kula and Schutte, 1987: Melendres et al., 1993; Schutte et al., 1983). Thus, location of the enzyme can in¯uence its release rate dur- ing the process of disruption. The release kinetics of a particular enzyme and total soluble proteins has been used by Kuboi et al., 1995) for identifying the location of an enzyme. This concept of identi®cation of location of enzymebasedonthereleasekineticswasgiventhetermas release selectivity. Later, it was renamed as location factor by Umakoshi et al. l998). This location factor value depends on the release rate of the enzyme and total soluble proteins. The location factor values for the same enzyme could probably vary with different equipment or even within the use of the same equipment at different disruption operating) conditions because the cells get disrupted to a different degree and might result in the variation of the release rate of enzyme and protein. The objective of the present work was to disrupt the microbial cells using different disruption equipment and determine the location factor values. The enzymes whose location are well-known were selected for the study. In the present study, yeast was disrupted for the release of Alcohol dehydrogenase ADH) and invertase and E. coli cells were disrupted for the release of pen- icillin acylase by various disruption equipments. The disruption equipments included a sonicator, high-pres- sure homogenizer, and hydrodynamic cavitation set-up. The release kinetics of these enzymes and total soluble proteins have been studied and the location factor val- ues have been calculated. MATERIALS AND METHODS Microorganism Bakers yeast Tower brand) was obtained from a local supplier as a source of protein and enzymes invertase and alcohol dehydrogenase). Correspondence to: A.B. Pandit ã 2001 John Wiley & Sons, Inc.