β-galactosidase from Kluyveromyces lactis has been immobilized on chitosan-glutaraldehyde particles employing different enzyme loadings (in a range of 0.7–7 mg/g of support). The biocatalysts had been later utilized to produce lactulose by transglycosylation using milk whey supplemented with fructose. The amount of produced lactulose decreased when the enzyme loading increased, from around 18 mg/mL using the biocatalysts with only 0.7 mg/g to around 7 mg/mL using the biocatalyst with a loading of 7 mg/mL. The results showed in this paper shows the great effect of the substrates and product concentration gradients inside the biocatalyst particle, not only lactulose, but also glucose and galactose. That is, the use of a excessive enzyme loading can lead to a significant worsening of the β-galactosidase biocatalyst performance in kinetically controlled synthesis.