LAUSR

Abstract β-galactosidases from Lactobacillus leichmannii 313 (LL 313) were immobilized for the first time by cross-linked enzyme aggregates (CLEA) technology. By using three precipitants (saturated ammonium sulfate (ASF), acetone (ACT), and isopropanol (IPA) in CLEA preparation and assessing performance in comparison with crude enzyme, recovered activities of 23% (ASF-CLEA), 18% (ACT-CLEA), 13.6% (IPA-CLEA) were obtained. Immobilization did not change the optimal pH (7.0) and temperature (55 °C), except for ACT-CLEA (60 °C). CLEA retained activity (∼ 90% for ASF-CLEA) after 2 weeks storage at 4 °C in buffer; and for ACT-CLEA and IPA-CLEA, ∼ 50% of initial activity was retained after 10 cycles of use. Plackett–Burman (PB) and Response Surface Methodology (RSM) optimization with ASF as precipitant gave 23.5 mM of glutaraldehyde, reaction pH 6.9 and reaction time of 3.4 h as the optimum conditions giving highest recovered activity of 37.7%. The kinetics (Vmax and Km) were 0.06 mmol g−1 min−1 and 4.95 mM respectively for ASF-CLEA, compared with 0.07 mmol g−1 min−1 and 4.08 mM for crude enzyme. All CLEA types exhibited the capability to hydrolyze lactose and generate GOS. This study shows that CLEA is a suitable technique to immobilize β-galactosidase from LL313, and the resulting enzyme system has promising applications in the food industry.