d-Tagatose, a potential low calorific substitute for sucrose, can be produced by bioconversion of d-galactose catalysed by l-arabinose isomerase. l-Arabinose isomerase from Shewanella sp. ANA-3 is unique for its ability to catalyse bioconversion reactions under mesophilic conditions. However, d-galactose not being a natural substrate for l-arabinose isomerase is catalysed at a slower rate. We attempted to increase the biocatalytic efficiency of Shewanella sp. l-arabinose isomerase by rational design to enhance galactose isomerisation activity. In silico molecular docking, analysis has revealed that F279 is sterically hindering the binding of d-galactose at the C6 position. Substitution of bulky Phe residue with smaller hydrophilic residues such as Asn and Thr increased the galactose isomerase activity by 86 % and 12 % respectively. At mesophilic conditions, F279N mutant catalysed the bioconversion of d-galactose more efficiently than l-arabinose, indicating a shift in substrate preference.