Purification and characterization of Beauveria bassiana proteinases

Abstract

Two chymotrypsin-like serine proteinases are produced by B. bassiana 278 when grown on different carbon and nitrogen sources. By employing acetone precipitation, gel filtration and ion-exchange chromatographies, the enzymes were separated from the culture filtrate after propagation of the fungus on medium enriched either with ground larvae of Apis mellifera (Proteinase I) or porcine blood plasma (Proteinase II). The purified enzymes with a molecular mass of approximately 32 kDa hydrolyzed natural protein substrates: casein, hide powder azure (HPA), azocoll and much less elastin Congo Red and collagen. They differ from each other in the optimum pH value, amino acid composition, MICHAELIS constant and susceptibility to natural chymotrypsin inhibitors. Both proteinases hydrolyze suc-Ala-Ala-Pro-Phe-p-NA with an apparent Km of 2.03 × 10-3 M and 1.04 × 10-4 M, respectively. The turkey ovomukoid (OMTKY) and cathepsin G/chymotrypsin inhibitor inhibit only Proteinase II from the larvae hemolymph of Apis mellifera (AMCI). The association constant of the interaction of this enzyme with AMCI was estimated to be very high (4.11 × 109 M-1).