BETA-GALACTOSIDASE ENCAPSULATED IN LIPOSOMES: PROTECTION AGAINST INACTIVATION IN AGRESSIVE ENVIRONMENTS.

J.M.SANCHEZ; M.A.PERILLO

UNQ-Bs. As. Argentina

Workshop; First Mercosur workshop on biomembranes; 2000

UNQ-SAB-SBBF-SOBLA-USP

Introduction       Beta-galactosidase is an enzyme widely distributed, capable of catalyzing the hydrolysis of terminal b-glycosidic bonds present in carbohydrates, glycolipids, glycoproteins and glycosamineglicans (Tanaka et al., 1995)       This enzymatic activity has been widely studied having  medical (Mian, 1998), nutritional (Hoskava, 1980), biotechnological (Alton et al., 1998; Jost et al., 1999) and therapeutic (Jacob et al., 1961) interests. Particularly, the b-galactosidase from Escherichia coli was instrumental in the development of the operon model (Bagnis et al., 1999), and today is one of the most commonly used enzymes in molecular biology (Sanchez and Perillo, 2000)       In previous reports (Sanchez and Perillo, 2000) we showed that b-galactosidase was able to interact with lipidic surfaces and its activity could be modulated according to the enzyme possibility to penetrate or just be adsorbed to that dimensionality restricted space. In the presence of phospholipid liposomes, at a relative high concentration (1.2 mM), we described an increased in maximal reaction rate  values (Vmax).       In the present work we investigated the effect of b-galactosidase-lipid interaction on the enzymatic activity in a wide range of pH and temperature values of the media  and in the presence of proteolytic enzymes. Methods       For determining enzymatic activity the method applied was essentially that of Wallenfels (1961) using ortho ? nitrophenol-b-D-galactopiranoside (ONPG) as substrate at a concentración corresponding to a pseudo-first orden regime and high enough to reach maximal reaction rate (Vmax).  After an incubation period of 15 min, the reaction was stopped by the addition of 0.18 M sodium carbonate, pH 10.  The absorbance of the ortho nitro phenoxide formed (ONPx) was determined measuring the absorbance at 420 NM (A420), after adding 10 mM SDS (final concentración) in order to micellized the vesicles and avoid possible artifacts due to light scattering in the presence of liposomes (this and other correction methods were previously thoroughly studied and applied with identical results). The reaction rate was calculated as the amount of orthonitrophenol produced per minute. Enzyme encapsulation Dried lipid (29.4 mg soybean phosphatidylcholine, PC) was dispersed in 0.5 ml of water containing the enzyme and vortexing for 5 min. at room temperature. Effects of temperature and pH Samples consisting of enzyme encapsulated or not, were submitted to a 30 min. incubation period at pH 6.8 and at different temperatures ranging from 15 to 55 ºC or at a fixed temperature of 37 ºC and in media of  different  pH values within 2 and 12. Then, after adjusting the pH of the media to a value of 6.8, ONPG was added and the reaction rate was measured at 37 ºC. Proteolytic treatment Beta-galactosidase, encapsulated or not, was incubated for 30 min at room temperature in the absence or in the presence of a solution of Protease type IV form Sigma which had or had not been previously inactivated through a high temperature treatment. Results and Discussion Maximal b-galactosidase activity in an homogeneous and aqueous media was found at pH 6.8 (with measurable activities between pH 5.5 and 9) and between 40 and 45ºC (no shown). Pre-incubation a pH between 2 and 3.5 as well as at 12 caused irreversible inactivation of non encapsulated enzyme activity (Fig.1a).       Encapsulation protected the enzyme form inactivation due to pre-incubation within a pH range between 3.5 and 12. Pre-incubation at temperature above 40 ºC caused irreversible inactivation of the enzymatic activity if the enzyme was in an homogeneous aqueous media. Encapsulation prevented that inactivation up to 52 ºC (Fig.1b). The activating effects due to lipid-enzyme interaction could be observed independently of the pre-incubation treatment applied (Fig.1a and 1b).       As shown in Fig.2 protease (not heated) abolished enzyme activity in aqueous homogeneous media. Proteolytic inactivation of b-galactosidase was avoided by encapsulation.       Concluding, b-galactosidase encapsulation in phospholipid multilamellar vesicles protects the enzyme from inactivation in aggresive media.