© The Royal Society of Chemistry 2017. Lipase of Rhizopus oryze expressed in Pichia pastoris (ROLpp) was selectively immobilized on octyl-Sepharose, fixing the open and active conformations. This enzyme was compared to the commercial one (ROLsigma) and a unique enzyme of 32 kDa was selectively adsorbed in both cases. Small differences in the N-terminal peptide sequence of both lipases seem to be involved in the enzyme fixing on the solid support, affecting the active site structure. This phenomenon resulted in a strong difference in catalytic properties between immobilized enzymes, with ROLpp being the most active, specific and regioselective heterogeneous biocatalyst in the hydrolysis of lactal hexaacetate. Immobilized ROLpp was 8 times more active, and more specific than immobilized ROLsigma, with excellent regioselectivity (monodeprotection in 3-OH, >99% yield). Solid-phase chemical modification of the N-terminus of immobilized ROLpp was attempted because of the moderate results obtained in the hydrolysis of glucal triacetate. Different biomolecules were introduced and the enzyme catalytic properties in this reaction were assessed. The modification of ROLpp with a polycarboxylated peptide (pA) improved the activity, specificity and regioselectivity of the enzyme, producing mainly the 3-OH monodeprotected glucal. The presence of acetonitrile 3% (v/v) in the reaction medium negatively affected ROLpp, being completely unspecific, whereas ROLpp modified with p1 conserved the specificity and regioselectivity shown in fully aqueous medium. The presence of dioxane improved the specificity and varied the regiopreference of the immobilized lipase (from C-3 to C-6 and C-4 monohydrolyzed products). The posterior modification with pA improved the specificity and the regio-preference of ROLpp towards C-6 monohydrolyzed product.