OLIGOPEPTIDASE B FROM <i>Serratia proteamaculans. </i>I. DETERMINATION OF PRIMARY STRUCTURE, ISOLATION AND PURIFICATION OF WILD TYPE AND RECOMBINANT ENZYME VARIANTS
From the psychrotolerant gram-negative microorganism <i>Serratia proteamaculans </i>a novel trypsin-type protease (PSP) was purified by ion-exchange chromatography on Q-sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-sepharose). It was shown that PSP forms a tight complex with GroEL shaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP preparations were obtained with molecular mass 78 kD; the N-terminal amino acid sequence of residues 1-10 was determined and tryptic peptides of PSP were analyzed by mass spectrometry. It was revealed that this enzyme is the previously unknown oligopeptidase B (OpdB). The gene <i>OpdB </i>of <i>S. proteamaculans </i>94 was sequenced and producent <i>E. coli </i>BL-21 (DE3) pOpdB № 22 was constructed. The yield of expressed His<sub>6</sub>-PSP is 150 mg from 100 g of biomass.