Imobilização de βeta-galactosidase comercial e produzida por K. marxianus em sílica e avaliação da síntese enzimática de galacto-oligossacarídeos

Abstract

The β-galactosidase enzyme is widely applied in industry. It performs lactose hydrolysis and also transgalactosylation, producing galactooligosaccharides (GOS), which have prebiotic effects in the human body. The use of β-galactosidase may present limitations due to its characteristics, such as restriction in temperatures, pHs, and impossibility of reuse, among others. Kluyveromyces lactis and Kluyveromyces marxianus are potential yeasts for obtaining β-galactosidase and interfering in its characteristics. In order to circumvent these limitations in the processes, enzyme immobilization methods are used. Covalent bonding is one of these processes, it promotes resistance of immobilized enzymes under different conditions and reduces leaching losses. Silica is a widely used support, as it promotes good enzyme binding to the support. Therefore, the aim of the current work was to evaluate the immobilization of commercial β-galactosidase from K. lactis and β-galactosidase produced by K. marxianus in silica with controlled porosity and subsequently carry out the synthesis of galactooligosaccharides. An evaluation of the best immobilization conditions for commercial β-galactosidase was performed with a Central Composite Design (CCD). The immobilized biocatalyst was characterized in relation to the influence of pH and temperature on enzyme activity, thermal stability, pH and storage, reuse, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The hydrolysis of lactose with the immobilized enzyme was also evaluated in a fixed bed. The β-galactosidase produced by submerged fermentation of K. marxianus was evaluated for immobilization on silica and for some partial purification techniques. The synthesis of galactooligosaccharides was performed with the commercial enzyme and produced in its free and immobilized form. The ionic strength of the buffer was evaluated in the transgalactosylation and a Fractional Factorial Design was also proposed to evaluate the interference of the initial lactose concentration, agitation, and temperature in the synthesis of GOS. Magnetic and mechanical stirring and the fixed bed reactor configuration were also evaluated in the synthesis of GOS. The best immobilization conditions were at 1,0% γ-aminopropyltriethoxysilane (APTES) concentration, initial enzyme activity offered at 21 U.mL-1, and 6,86% glutaraldehyde concentration. The immobilized biocatalyst showed optimal pH at 7.0, the temperature at 30 °C, and stability at pH 7.5. Thermal stability was better at a milder temperature of 20 °C. In 4 cycles of reuse, the enzyme maintained ≈70% of the initial activity and stored (8 °C) maintained its activity in 44% after 105 days. The FTIR allowed visualization of enzyme groups and supported enzyme binding. The images obtained from SEM showed the structure of the silica. With the fixed bed, a lactose conversion of ≈47% was obtained. The immobilization of the β-galactosidase produced with K. marxianus was limited and among the partial purification techniques the dialysis process stood out. The GOS were produced in high concentrations of lactose (400 g.L-1), with a shorter time (3 h) for free enzyme and longer for immobilized enzyme (6 h). The effects of the variables evaluated in the Fractional Factorial Design were positive, the most effective influence was agitation, the highest concentration of GOS was 67.70 g.L-1. Both in magnetic and mechanical agitation it was possible to synthesize the GOS with the immobilized enzyme, the GOS concentrations were 70.81 and 90.37 g.L-1, respectively. In a fixed bed, the highest GOS concentrations were obtained for the column packed with immobilized silica, reaching a value of 100.18 g.L-1. The enzyme produced by K. marxianus synthesized GOS in its free form (106.97 g.L-1). Silica immobilized β-galactosidase shows promise in both lactose hydrolysis and GOS synthesis.