Abstract The main goal of this work was to study the stereoselective behavior of immobilized Geobacillus thermocatenulatus lipase (BTL2) in a sequential batch reactor using the partial and asymmetric hydrolysis of dimethyl-3 phenylglutarate (DMFG) as a model reaction. To reach this goal, BTL2 lipase was immobilized on Sepharose and silica supports with cyanogen bromide and octyl groups (monofunctional supports) and undecanol-glyoxyl and octyl-epoxides groups (heterofunctional supports), to determine the effect of the enzyme orientation during the immobilization process on their catalytic properties. In the hydrolysis of DMFG, the biocatalyst obtained with undecanol-glyoxyl Sepharose proved to be the most stereolective with an enantiomeric excess (e.e.) value of 90% in aqueous media. This behavior can be attributed to differences in the orientation of the lipase on the support. In sequential batch reactor operation, the e.e. remained constant in the first two batches; however, from the third batch on the e.e. decreased slightly maybe due to a change in the conformation of the enzyme at the reaction conditions. Finally, the high purity S-methyl-3-phenyl glutarate produced in sequential batch reactor operation shows that the biocatalyst can be reused at least twice without losing stereoselectivity, favoring a reduction in the process cost.