Clonaggio ed espressione di nuove serin proteasi per il mangime dei polli da carne

The aim of my project was to clone, express and test the acid stability of three different proteases belonging to the same family of S1 serine proteases. The enzymes named Aurantica, Bingchenggensis, and Scabiei came respectively from three strains: Stigmatella aurantica, Streptomyces bingchenggensis and Streptomyces scabiei. Based on Basic Local Alignment Search Tool (BLAST), three proteins, which presented a high identity value with a S1 serine protease, patented as an acid stable protease from Nocardiopsis prasina, were selected. These enzymes could be utilized in the poultry feed industry to increase the absorption of the protein content in the broiler chickens fed with soybeans and other kinds of feed. The acid stability feature is necessary to preserve the activity of the enzymes at the low pH in stomach as far as the intestine where they have to be active. The wild type genes encoding for these three proteases were optimized and synthesized by GeneArt for the transformation of Bacillus subtilis strain. After the subcloning in B. subtilis the expression and the activity at pH 7 were tested. All the enzymes gave high expression yield and high activity at pH= 5.0, 6.0, 7.0 and 8.0. Furthermore 3-carboxypropionyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) (Sigma) was used for testing α-chymotrypsin activity. Only one protease, Bingchenggensis, retained high activity after two hours incubation at pH 2.0 and 3.0. This enzyme may be relevant as feed additive in the poultry feed industry.