Abstract
The propeptide is a short sequence that facilitates protein folding. In this study, four highly active Rhizomucor miehei lipase (RML) mutants were obtained through saturation mutagenesis at three propeptide positions: Ser8, Pro35, and Pro47. The enzyme activities of mutants P35 N, P47 G, P47 N, and S8E/P35S/P47A observed at 40 °C, and pH 8.0 were 10.19, 7.53, 6.15, and 8.24 times of that wild-type RML, respectively. The S8E/P35S/P47A mutant showed good thermostability. After incubation at 40 °C for 1 h, 98.98 % of its initial activity remained, whereas wild-type RML retained only 78.76 %. This result indicated that the enhancement of hydrophilicity of 35- and 47- amino-acid residues could promote the interaction between the propeptide and the mature peptide and the enzyme activity and expression level. Highly conserved sites had a more significant impact on enzyme performance than did other sites, similar to the Pro35 and Pro47 mutants showed in this study. This study provides a new idea for protein modification: enzyme performance can be improved through propeptide regulation.
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