Improved biocatalysts based on Bacillus circulans β-galactosidase immobilized onto epoxy-activated acrylic supports: Applications in whey processing
Graphical abstract
Highlights
► β-Galactosidase from Bacillus circulans was immobilized onto Eupergit C and Sepabeads EP. ► Thermostability was improved by alkaline treatment and blocking with glycine. ► Eupergit-based derivatives showed half-life 10-fold higher than Sepabeads derivatives. ► Improved derivatives were used for lactose hydrolysis in cheese whey at 50 °C. ► The enzymatic derivatives could be reused batchwise for at least 6 cycles.
Introduction
β-Galactosidase (EC 3.2.1.23) is one of the most frequently used enzymes in the food industry. It hydrolyses lactose, releasing glucose and galactose, and makes it possible for people with lactose intolerance to consume milk and other dairy products [1]. Another application related to food technology is in the elaboration of products such as ice-cream, condensed milk and milk sweet, where it reduces lactose concentration thus avoiding its crystallization. Besides, this enzyme in immobilized form has a great potential for the processing of whey and whey permeates, the most representative by-products of cheese industries [1]. The dairy industry generates worldwide an amount of 4.7 × 106 tons cheese whey per year [2]. A great part of this by-product is discarded and cause environmental problems. In addition to its use for lactose removal, β-galactosidase can also catalyze transgalactosylation reactions to produce galactosides and oligosaccharides [3], [4]. The enzyme is produced by animals, plants, and microorganisms [5].
Commercial β-galactosidase from Bacillus circulans (Biolacta N-5, Daiwa Kasei) presents at least four isoforms, but the so-called β-galactosidase-I is the most abundant. This has a monomeric structure with molecular weight of 212 kDa, displays optimal activity at 44 °C and its optimum pH is in the range 5.5–6.5 [6], similar to the pH of sweet whey [7], [8]. The enzyme also catalyzes the production of a large amount of oligosaccharides and shows a preferential regioselectivity toward formation of β (1–4) linkages [9], [10], [11], [12], [13]. These properties make this enzyme very suitable for use both for the hydrolysis of lactose in milk and whey, and for oligosaccharide production.
In a previous paper, we analyzed the immobilization process of the enzyme onto supports of Sepabeads type (Sepabeads EP and Sepabeads HFA) and the effects of stabilizing treatments [14]. However, the stability of these biocatalysts was not high enough for useful application, so that further improvements were required.
Sepabeads® EP, as well as the more widely known Eupergit® C, are commercial epoxy (oxirane)-activated acrylic resins, formed by spherical macroporous particles possessing large internal surfaces, and characterized by a low water uptake [15]. The epoxy groups in these supports are attached to their surface through short spacer arms, forming a very dense monolayer of reactive moieties. They have very high loading capacities (e.g. 100 mg of protein/mL of packed gel can be achieved), competing with any other available supports [16]. These non-biodegradable matrices have high storage and chemical stabilities in neutral aqueous media, so they can be easily and long-term handled before and during immobilization procedures. They are non-toxic and can be used both in pharmaceutical and food industries [15]. Eupergit C and Sepabeads EP differ in their internal morphology and hydrophobicity, which determine important differences in their properties [14]. The internal geometry of Sepabeads matrices is composed by cylindrical pores surrounded by convex and planar surfaces with high geometrical congruence for reaction with proteins [15]. On the other hand, Eupergit® C geometry possesses convex rod-like surfaces and to a lesser extent, planar surfaces [14], [16]. Furthermore, commercial Eupergit® C offers two carriers with the same chemical structure, Eupergit C and Eupergit C 250 L, the only differences between them being the larger pores in Eupergit C 250 L and the greater number of oxirane groups in Eupergit C [15], [17].
Protein immobilization onto Sepabeads EP or Eupergit C follows a two-step mechanism. The primary event is a rapid physical adsorption of the protein onto the resin; in a second step, a covalent reaction takes place between the adsorbed protein and the support. Epoxy groups react with amino groups in proteins under very mild experimental conditions (e.g. pH 7.5), with minimal chemical modification of the protein and formation of very stable secondary amine bonds [15].
In this work, we analyzed and compared the performances of three acrylic carriers (Eupergit C, Eupergit C 250 L and Sepabeads EP) for the covalent immobilization of β-galactosidase from B. circulans. The immobilization processes were evaluated in terms of applied enzyme load, pH, ionic strength, and incubation time. Stabilization of the derivatives by post-immobilization treatments was performed, and the improved biocatalysts were assayed for the hydrolysis of lactose from cheese wheys, with the aim of assessing their performances.
Section snippets
Materials and methods
The enzyme β-galactosidase from B. circulans (Biolacta N-5) was kindly provided by Daiwa Kasei (Osaka, Japan). Eupergit C and Eupergit C 250 L were a gift from Röhm Pharma (Darmstadt, Germany), while Sepabeads EC-EP was donated by Resindion (Milano, Italy). o-Nitrophenyl-β-d-galactopyranoside (ONPG) and lactose were purchased from Sigma (St. Louis, MO), bicinchoninic acid (BCA) from Pierce (Rockford, IL), and all other chemicals were of analytical grade. The enzymatic kit for glucose
Preparation of β-galactosidase derivatives
In this study, an evaluation is done on the performance of Eupergit® carriers (Eupergit C, Eupergit C 250 L) for the immobilization of β-galactosidase from B. circulans, in comparison with Sepabeads EP.
The effects of pH, ionic strength, time of incubation, and applied enzyme load on the immobilization process onto Sepabeads EP at 25 °C have been previously evaluated [14]. Results of comparative studies reported in the present article show that immobilization of this enzyme onto Eupergit C differs
Conclusions
Comparison of different conditions for the immobilization of β-galactosidase from B. circulans showed that Eupergit C and Eupergit C 250 L required the use of milder conditions (1.0 M potassium phosphate pH 7.5, 24 h incubation) than Sepabeads EP (1.4 M, pH 8.5, 48 h) to achieve maximal yields.
The stabilization achieved by either the blocking process alone or the combined strategy (incubation during 24 h at pH 8.5, followed by blocking of excess oxirane groups) was much more effective for
Acknowledgements
The authors would like to thank Röhm Pharma (Darmstadt, Germany), Resindion (Milano, Italy), and Daiwa Kasei (Osaka, Japan), for their generous supply of epoxy-activated resins and enzyme. P.T. was supported by the Programa de Desarrollo de las Ciencias Básicas (PEDECIBA-Química) and the Agencia Nacional de Investigación e Innovación (ANII). We thank Dr. Valerie Dee for linguistic revision of the manuscript.
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