Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

Abstract

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.

Introduction

β-Galactosidases have been obtained from a variety of microorganisms and sources including fungi, bacteria, yeasts, plants, etc. β-Galactosidase is indeed an important enzyme in food industry and has found significant applications in enhancing sweetness, solubility, flavor and digestibility of dairy products [1]. A major application of β-galactosidase is lactose hydrolysis, a process that results in the formation of glucose and galactose. Lactose is the major sugar (4–5%) present in milk and its hydrolysis makes milk fit for consumption of lactose intolerant people [2]. Apart from lactose hydrolysis, β-galactosidase also finds application in galacto-oligosaccharide (GOS) formation via transgalactosylation reaction. During enzymatic hydrolysis of lactose into glucose and galactose, using β-galactosidase, the enzyme is also able to transfer galactose to the hydroxyl groups of galactose or glucose in a process called “transgalactosylation” and, thus, produces galacto-oligosaccharides (GOS) [3], [4]. GOS are non-digestible oligosaccharides which are recognized as prebiotics thus stimulating the growth of Bifidobacteria in the lower part of the human intestine [5].

In case of β-galactosidase, enzyme parameters and price eventually determine the cost of lactose hydrolysis or GOS formation processes. The use of immobilized β-galactosidase for lactose hydrolysis in case of whey, has become economically feasible in spite of the cost of the enzyme and of the immobilization process. This has been mainly attributed to the facts that immobilized enzymes can be reused several times, and there is a possibility of developing a continuous hydrolysis process [6]. In general, the immobilization of enzymes on various supports is driven by the benefits such as continuous process, enzyme reuse, enhanced enzyme stability, i.e. resistance against extreme pH, temperature, high ionic strengths, etc. [1], [2]. Moreover, it is believed that the possible disadvantages such as low retained activity as a consequence of the immobilization procedure are largely compensated by the possibility of the reuse of the enzyme [7].

β-Galactosidase has been immobilized on various supports including liquid aphrons (surfactant-stabilized solvent droplets), anion exchangers, magnetic beads, cotton cloth, polyethylene films, chitosan particles, etc. The resulting changes in activity and pH optimum, as a function of support material and immobilization methods, have been reported. These studies have been reviewed in detail by Husain [1]. Apart from conventional supports, in recent years membranes have been used for β-galactosidase immobilization [8], [9]. Membranes have several advantages in enzyme immobilization such as the absence of mass transfer limitations and the convenient scale-up by adaptation of the available membrane surface area [10], [11]. Furthermore, some level of product separation is also achieved along with biocatalytic conversion in enzyme membrane reactors (EMRs) [12]. Typically, membrane separations have the advantage of requiring only a limited amount of energy, because there is no phase change involved in such processes [12].

Immobilization of β-galactosidase on nylon and polysulphone membranes via grafting/crosslinking with epoxy groups have been investigated and changes in activity and stability were observed [7], [13]. In terms of application, the immobilization of β-galactosidase on tubular ceramic membranes with Al₂O₃ support layer and TiO₂ separation layer were investigated for GOS formation thus aiming at a simultaneous conversion and separation process [3]. The benefits associated with application of immobilized enzymes in biotechnological processes have stimulated the interest of the researchers towards the research addressed to improve the performance of the biocatalytic membranes [13]. Therefore, finding an appropriate membrane for enzyme immobilization is a matter of great interest among researchers. Though the inherent properties of membranes such as hydrophobicity, hyrophilicity and molecular weight cut-off (MWCO) are of significant importance in enzyme immobilization, however, membrane's interaction with enzyme, permeability to substrates and products, chemical and biological stability, high mechanical strength and presence of certain chemical groups are often the prerequisites [8].

The aim of the present study is to immobilize β-galactosidase on a mixed-matrix membrane via adsorption. The focus is to estimate the enzyme immobilizing capacity of the membrane under optimal conditions of enzyme concentration, temperature, contact time and pH. The characteristics of immobilized enzymes were studied and compared to that of free enzyme to highlight the significant impacts of immobilization. This article presents the first results of β-galactosidase on an in-house prepared membrane. These results are being used to further tailor the membrane specifically for optimal β-galactosidase immobilization.