beta-Galactosidase from Aspergillus oryzae was immobilized by different methods, being the enzyme immobilized in glyoxyl-agarose selected for the synthesis of galacto-oligosaccharides from lactose at high concentrations. The synthesis was optimized with respect to lactose concentration and enzyme to substrate ratio using lactose conversion into galacto-oligosaccharides as objective function. Optimum values were 54.6% (w/v) lactose and 33 IU g(-1) lactose and at such conditions conversion was close to 30%, similar to the one obtained with soluble beta-galactosidase and comparable with the best reported values for immobilized beta-galactosidases. In the sequential batch production of galacto-oligosaccharides, biocatalyst efficiency was increased by 190% with respect to the free enzyme in solution, and 8500g of galacto-oligosaccharides per gram of enzyme preparation were produced after 10 batches. This amount can be further increased by considering additional batches up to the point of biocatalyst replacement and is significantly higher than the maximum attainable by using the soluble enzyme. (C) 2010 Elsevier Ltd. All rights reserved.