Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability.

Methylotrophic yeasts have widely been used as model organisms for understanding cellular functions and biochemical activities in lower eukaryotes. The gene encoding an aspartic protease (MCAP) from Mucor circinelloides DSM 2183 was cloned and expressed into Pichia pastoris using both the native M. circinelloides signal peptide (mcSP) and α-factor secretion signal from Saccharomyces cerevisiae (α-MF).

Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris.

Background: Extracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme.