Increasing kinetic stability of industrial enzymes is a key objective of protein engineering. In this study, a cold-active Candida albicans lipase Lip5 was selected as a model to improve its stability. With no 3D structure available, multiple-sequence alignment of homologous lipases with different kinetic stabilities in the same superfamily was used to identify the mostly likely positions relevant to protein stability; these positions (V39, I52, and I290) were selected for proline incorporation in Lip5 as a strategy to increase the protein stability. Of the mutants generated, two of them (V39P and I290P) displayed a twofold increase in kinetic stability and improved thermodynamic stability as well. None of the mutants exhibited secondary or tertiary structure changes as shown by circular dichroism and fluorescence spectroscopy analyses. These results demonstrate that in the absence of 3D structure, multiple-sequence alignment could be a useful tool to direct proline incorporation for lipase stability engineering.Practical applications: In the absence of a 3D structure, multiple-sequence alignment-based proline incorporation is a useful tool to engineer lipase towards improved stability.