Malonate diesters with highly branched side chains containing a preexisting chiral center were prepared from optically pure amino alcohols and subjected to asymmetric enzymatic hydrolysis by Porcine Liver Esterase (PLE). Recombinant PLE isoenzymes have been utilized in this work to synthesize diastereomerically enriched malonate half‐esters from enantiopure malonate diesters. The diastereomeric excess of the product half‐esters was further improved in the later steps of synthesis either by simple recrystallization or flash column chromatography. The diastereomerically enriched half‐ester was transformed into a novel 5‐substituted Cα‐methyl‐β‐proline analogue (3R,5S)‐1c, in high optical purity employing a stereoselective cyclization methodology. This β‐proline analogue was tested for activity as a catalyst of the Mannich reaction. The β‐proline analogue derived from the hydrolysis reaction by the crude PLE appeared to catalyze the Mannich reaction between an α‐imino ester and an aldehyde providing decent to good diastereoselectivities. However, the enantioselectivities in the reaction was low. The second diastereomer of the 5‐benzyl‐substituted Cα‐methyl‐β‐proline, (3S,5S)‐1c was prepared by enzymatic hydrolysis using PLE isoenzyme 3 and tested for its catalytic activity in the Mannich reaction. Amino acid, (3S,5S)‐1c catalyzed the Mannich reaction between isovaleraldehyde and an α‐imino ester yielding the “anti” selective product with an optical purity of 99 %ee.