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The main goal of this study was to investigate the immobilization of commercial ß-galactosidase from Kluyveromyces lactis (Lactozym®) on Eupergit® C. A Plackett-Burman design was proposed. The ionic strength and pH were the variables that presented significant effect (p<0.1) on immobilization. The increase in the ionic strength from 0.1 to 1.5 M and the increase in pH from 6.6 to 7.4 represented an increase of 28.56% and a reduction of 18.19% in the immobilization yield, respectively. At 25°C, pH 6.6, ionic strength of 1.5 M, immobilization for 8 h, 1 mM of divalent magnesium ion and 0.4 mL of enzyme added, reached 85% immobilization yield. The free and immobilized enzymes were characterized. pH and temperature profiles showed maximum activity at pH 6.6 and 45°C, for both free and immobilized enzymes. There was a gain in thermal stability with enzyme immobilization and there was an increase of about four times in the half-life of the immobilized derivative at 45°C (from 0.43 h to 1.78 h). This greater thermal stability was also made clear through the calculation of thermodynamic parameters (ΔH, ΔG and ΔS). Km values, 30.33 mM and 104.00 mM for free and immobilized enzymes, respectively, represented a reduction in substrate affinity after immobilization, possibly owing to stereo-conformational factors. In a batch reactor for lactose hydrolysis from cheese whey, an increase in lactose conversion with immobilization was observed at 40°C and 45°C (90.43% and 65.36%, respectively) in relation to the free enzyme (84.17% and 39.58%, respectively).

Many food, cosmetic and pharmaceutical industries have increased their interest in short-chain esters due to their flavor properties. From the industrial standpoint, enzyme reactions are the most economical strategy to reach green products with neither toxicity nor damage to human health. Isoamyl butyrate (pear flavor) was synthesized by isoamyl alcohol (a byproduct of alcohol production) and butyric acid with the use of the immobilized lipase Lipozyme TL IM and hexane as solvents. Reaction variables (temperature, butyric acid concentration, isoamyl alcohol:butyric acid molar ratio and enzyme concentration) were investigated in ester conversion (%), concentration (mol L-1) and productivity (mmol ester g-1 mixture . h), by applying a sequential strategy of the Fractional Factorial Design (FFD) and the Central Composite Rotatable Design (CCRD). High isoamyl butyrate conversion of 95.8% was achieved at 24 hours. At 3 hours, the highest isoamyl butyrate concentration (1.64 mol L-1) and productivity (0.19 mmol ester g-1 mixture . h) were obtained under different reaction conditions. Due to high specificity and selectivity of lipases, process parameters of this study and their interaction with the Lipozyme TL IM are fundamental to understand and optimize the system so as to achieve maximum yield to scale up. Results show that fusel oil may be recycled by the green chemistry process proposed by this study.

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