Nearly 65% of the surface of a lipase, from Bacillus subtilis, is occupied by the loops. Since the loops are dynamic components of a protein, located on the surface and are tolerant to substitutions, we subjected all 91 amino acids of the loops to site saturation mutagenesis to identify mutations that improve the stability and activity of lipase in dimethyl sulfoxide (DMSO). Based on a novel screening system, we have identified six positions in the lipase, from a population of 18,000 transformants that contributed to higher activity in DMSO. We combined all the six mutations into one lipase gene (6SR), purified the protein to study its activity and structural properties. 6SR has shown eight times higher catalytic turnover in 60% DMSO and showed a marginal shift in DMSO tolerance. 6SR showed a similar secondary structure with little alteration in tertiary structure. The melting temperature of 6SR is lower than the wild type and binds the least to hydrophobic fluorescent probes, indicating that the surface has become more polar in nature. This study provides clues to the role of loop amino acids in modulating the activity in organic solvents.