Abstract
Penicillium camembertii (PCL), a mono- and di-acylglycerol lipase (DGL), has the vital potential in the oil chemistry for food industry. However, known DGLs are mesophilic enzymes which restricts its application in the industry. To improve thermostability of PCL, we used amino acid substitution by comparison of amino acids compositions of PCL and protein sequences from typical thermophilic bacteria. Then, some conservative residues around active center were avoided to mutate according to homologous alignment analyses. Furthermore, the list was narrowed down to 28 candidate mutational sites of PCL by analyzing the hydrophobic interaction of amino acids in the structure. And among them only the mutant PCL-D25R had formed an additional salt bridge between R25-D32 and increased more hydrogen bonds interaction. Therefore, mutant PCL-D25R were constructed and expressed. Thermal inactivation assay showed that the half-life of mutant PCL-D25R at 45 °C increased 4-fold compared to that of PCL-WT. Melting temperature of mutant PCL-D25R increased to 49.5 °C from 46.5 °C by fluorescence-based thermal stability assay. This study provides a valuable strategy for engineering DGL thermostability.
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