Abstract: The present study enhanced the Rhizomucor miehei lipase (RML) thermostability through the rational design and the directed evolution, including 2 rounds of error-prone PCR, 2 rounds of site-directed mutagenesis and 4 rounds of saturation mutagenesis totally. Nine enzyme mutants were obtained and all of these mutants could maintain the activities after being the heat treatment at 70 ℃ for 2 h. The activity of the mutant M9(D48V/V67A/S168P/S240A/S311C/D313H) could reach (1 316±31)U/mg after the heat treatment, which was 5.2-fold of wild type at 37 ℃ (the wild type RML has no activity after the heat treatment). With sequencing analysis, the mutant sites D48V and V67A were located on the propeptide of the gene, and the mutant sites of S168P, S240A, S311C and D313H were located on the mature region. After saturation mutation of the two mutants on the propeptide, the thermostability of the mutant changed, which indicated that the mutation of the propeptide could affect the thermostability of the whole RML gene.

Key words: propeptide, gene mutation, Rhizomucor miehei lipase, thermostability