Purification and characterization of Beauveria bassiana proteinases

Two chymotrypsin-like serine proteinases are produced by B, bassiana 278 wh en grown on different carbon and nitrogen sources. By employing acetone pre cipitation, gel filtration and ion-exchange chromatographies, the enzymes w ere 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 molecu lar mass of approximately 32 kDa hydrolyzed natural protein substrates: cas ein, hide powder azure (HPA), azocoll and much less elastin Congo Red and c ollagen. They differ from each other in the optimum pH value, amino acid co mposition, MICHAELIS constant and susceptibility to natural chymotrypsin in hibitors. Both proteinases hydrolyze sue-Ala-Ala-Pro-Phe-p-NA with an appar ent K-m of 2.03 x 10(-3) M and 1.04 x 10(-4) M, respectively. The turkey ov omukoid (OMTKY) and cathepsin G/chymotrypsin inhibitor inhibit only Protein ase II from the larvae hemolymph of Apis mellifera (AMCI). The association constant of the interaction of this enzyme with AMCI was estimated to be ve ry high (4.11 x 10(9) M-1).