Melibiase in Immobilized Cells of Watermelon

Abstract

Cells of suspension culture Citrullus vulgaris cv. "Samara" were permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest melibiase activity was at pH 5.4 and 60°C. The hydrolysis of substrate was linear for 3.5 h, reaching 65-70% conversion of the substrate. The cells, characterized by high enzyme activity and stability in long-term storage, showed convenient physico-mechanical properties (physical protection from shear forces and easy separation of product from biocatalysts).Plant cells were first immobilized by Brodelius et al. 1979 [1]. In the last decades several methods for fixation of biocatalysts have been developed. Enzymes, living or nonliving microorganisms and animal and plant cells, as well as combined systems, have been bound within or to carrier materials [2][3][4][5]. Immobilization of cells or enzymes represents an effective way to obtain highly efficient enzyme catalysts important for biotransformation processes [6]. Many matrices from synthetic polymers or biological materials (e.g., agar, agarose, kappa-carragneenan, collagen, chitosan, polyacrylamide, polyurethane, cellulose) have been used for the immobilization of cells [7,8]. The spontaneous adhesion or covalent binding of cells to the surface of insoluble carriers was also examined [9, 10]. Recently, polyvinylalcohol and glutaraldehyde [11,12] or Tween 80 and glutaraldehyde [13] have been used for cell immobilization.Melibiase (α-D-galactoside galactohydrolase EC 3.2.1.22) α-galactosidase is an exoglycosidase removing terminal galactose residues from the galactosaccharides and carbohydrate moiety of glycoproteins. Melibiase is of particular interest in view of its biotechnological applications. The enzyme has been employed for the hydrolysis of raffinose to aid in the crystallization of sucrose. The studied enzyme is also used to convert the galactooligosaccharides (stachyose, verbascose, ajugose) in soybean meal to food and feed materials [14].Microrgamisms are the preferred sources of melibiase [15]. Although melibiase is generally present also in plants, this source has not been used previously. In this paper, the enzymatic hydrolysis of the terminal α-galactosidic linkage of glycosides (raffinose, stachyose) by free, as well as glutaraldehyde-immobilized, cells of Citrullus vulgaris cv. "Samara" was studied.Cells immmobilized by cell entrapment are cultivated in a similar way as cell suspension cultures [7,8,16]. The microscopic observation of watermelon cells immobilized with glutaraldehyde showed small morphological changes in comparison with cells in suspension. A thinning of the cell walls after permeabilization by Tween 80 was observed. Important was also the appearance of cell plasmolysis and a small aggregation of cells occurring after immobilization. According to the respiration rate and vital staining (fluorescein or 2,3,5-triphenyltetrazolium chloride), cells immobilized by glutaraldehyde were not viable. Also glucose was utilized only by cells in suspension, but not by immobilized ones (Fig. 1).The permeabil...