Effects of Propeptide on the Expression and Enzyme Function of Aspergillus pseudoglaucus Aspartic Protease App

Abstract:

The gene coding aspartic protease App was cloned from the genome of Aspergillus pseudoglaucus. Its self propeptide gene pro，the propeptide gene CP from Candida tropicalis candidapepsin，the gene PP from Saccharomyces cerevisiae proteinase A，the gene RP from Rhizomucor miehei proteinase，and the gene SP from Candida albicans aspartic proteinase 2 were cloned at the 5′-terminal of App gene. The different gene fragments including the different propeptides were obtained，and expressed in Escherichia coli. SDS-PAGE analysis showed that App could not expressed in E. coli without any propeptides at 5'-terminal App gene. When casein was used as substate，proApp with its self propeptide showed the highest specific activity of 903 U/mg at 55 ℃ and pH 2.8. Although the App with propeptides of the other proteases were expressed at a high level in E. coli，they did not present enzyme activity towards casein. The results of circular dichroism showed that the different propeptides changed the constituents of the secondary structure of protease，and thus the protein were not correctly folded. With milk protein as the substrate，proApp showed the highest hyhrolytic activity at 55 ℃ and pH 2.2. This work shows the important of the specific propeptide on heterologous expression and hydrolytic function of protease，and provides a good research basis for aspartic protease catalyzing the hydrolysis of milk protein.