SOLVENT EXTRACTION OF β-GALACTOSIDASE FROM KLUYVEROMYCES LACTIS YIELDS A STABLE AND HIGHLY ACTIVE ENZYME PREPARATION

Abstract

ABSTRACT The yeast Kluyveromyces lactis was cultured in cheese whey and β-galactosidase was extracted from cells by an organic solvent procedure. Under optimum conditions (pH 8.5, 25C), approximately 90% of the intracellular β-galactosidase activity was released into 0.1 M potassium phosphate solution with 2% chloroform after 9 h. The cell-free extract containing β-galactosidase activity was concentrated and the enzyme partially characterized. This enzyme has exhibited an optimum activity at a pH range of 6.2–6.6 and temperature of 40–45C on O-nitrophenyl-β-D-galctopyranoside. The maximum enzyme activity in milk was found at 45C and the concentrated enzymatic extract was found to have 19 U/mg protein. A feasible preparation for commercial application containing 20% glycerol maintained 89% of activity after 2 months at 4C, demonstrating stability. This preparation analyzed on 5% nondenaturing polyacrylamide gel electrophoresis exhibited two bands suggesting two aggregation forms with β-galactosidase activity. PRACTICAL APPLICATIONS Kluyveromyces lactis yeast cells are a safe source of β−galactosidase for food and pharmaceutical industries. Their application to prior hydrolysis of lactose in milk products has become commonplace in alleviating the symptoms of lactose intolerance. It is also of great interest to prevent lactose crystallization in concentrated or frozen dairy products such as condensedmilk, ice cream and “dulce de leche,” thereby increasing consumers' acceptability. In addition, treatment of cheese whey with β-galactosidase increases its fermentability to convert cheese whey lactose into ethanol helping to reduce the pollution caused by large volumes of this by-product released in the environment. Solvent extraction of β-galactosidase from K. lactis is a low-cost process and yields a stable and highly active enzyme preparation.