Síntese e imobilização em resina de troca iônica de β-galactosidase de Kluyveromyces marxianus

Abstract

β-galactosidase is a hydrolase that catalyzes the hydrolysis reaction of lactose. This enzyme has been increasingly highlighted in the industrial field, presents great potential for application in the dairy industry, by its action it is possible to produce lactose-free products, aimed at intolerants, in addition to increasing the digestibility of dairy products and improving their characteristics. In addition, β-galactosidase acts in the production of galacto-oligosaccharides, which have prebiotic effects and help in maintaining the intestinal flora. Lactose, the main carbohydrate in milk, is present in high amounts in whey, designated as a by-product of the dairy industry, in which β-galactosidase presents significant potential for hydrolysis of whey lactose with the possibility of new product developments and technologies. The β-galactosidase enzyme has been immobilized in order to expand and improve its application in the processes. Thus, several methods of immobilization have been applied, one of them is the physical adsorption, simple method involving only weak links. Among the substrates used ion exchange resins have great potential In this work β-galactosidase was produced from yeast Kluyveromyces marxianus and immobilized on Duolite® A-568 ion exchange resin. The production was carried out by submerged fermentation, using lactose present in the permeate of the whey as the carbon source. The enzyme was then characterized as to its thermal stability, pH and storage. After characterization of the free enzyme, it was immobilized on Duolite® A-568 resin. Preliminary tests were performed varying time, enzyme concentration and use of lyophilized enzyme in the immobilization. In the sequence an optimization of the conditions was carried out by means of a Central Composite Planning with the variables pH, buffer concentration and activity offered in the immobilization. For all immobilizations the recovered activity was defined. Glutaraldehyde crosslinking tests were performed at different concentrations. The enzyme also underwent a process with two immobilizations, aiming to increase the recovered activity. As results obtained, the free enzyme presented greater thermal stability at 30 ° C and stability at pH 7.3, besides retaining 80% of the initial activity during seven weeks of storage under refrigeration, both in its soluble and lyophilized form. Preliminary immobilization tests defined the activity offered as 27 U and the one-hour immobilization time, the activity recovered from immobilization with the non-lyophilized and lyophilized enzyme did not present differences. In the optimization, the optimum ranges obtained in the planning were 6.9 to 7.3 for the pH, 0.4 to 0.8 M for the buffer concentration and 24 to 31 U for the offered activity. Crosslinking tests showed that increasing the concentration of the glutaraldehyde solution implied a reduction in the activity of the immobilized biocatalyst. Serial immobilization was important to increase the activity of the immobilized biocatalyst. In general, the enzyme produced was efficient for immobilization in Duolite® A-568 ion exchange resin, taking into account that more variables should be optimized for immobilization improvement.