Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline optimum pH and broad substrate specificity. The activity and stability of these enzymes for a limited temperature range (30-50 A degrees C) need attention for its industrial application. In the present study, Bacillus subtilis LipJ was rationally designed for low-temperature adaptation. Small amino acids with lower volume and without side chain branches have high occurrence among psychrophilic proteins. Met residue is reported to be preferred for cold adaptation as it is thermolabile in nature and undergoes oxidation at high temperature. Therefore, the Ile137 residue, three residues downstream the catalytic residue Asp133, was substituted by Met. Biochemical study demonstrated that variant Ile137Met was optimally active at 20 A degrees C whereas parent enzyme was most active at 37 A degrees C. The variant retained 70-80 % relative activity at 10 A degrees C where parent enzyme demonstrated low activity. Ile137Met was observed to be unstable at and above 30 A degrees C. Kinetic study demonstrated increased K (m) and k (cat) values for variant referring improved catalytic efficiency but poor substrate affinity. Homolog modelling predicted lowered number of weak interactions by substituted Met137 as molecular basis of increased flexibility of variant. Hence, increased structure flexibility might be responsible for poor substrate affinity but increased molecular motion for higher catalysis at cold.