BIOCHEMICAL-CHARACTERIZATION OF A PROTEASE INVOLVED IN THE PROCESSINGOF A STREPTOMYCES-RETICULI CELLULASE (AVICELASE)

A 36-kDa protease from Streptomyces reticuli had recently been shown t o be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from S. reticuli to a 42-kDa truncated en zyme. It was induced only in the presence of Avicel, hydroxyethylcellu lose, and xylan. The addition of the nonionic detergent Tween 80 to th e culture medium containing Avicel as the carbon source led to a 10-fo ld increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-i nteraction chromatographies and was characterized biochemically. The e nzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azoca sein, and Azoalbumin. Its highest activity was determined between pH 7 .0 and 7.7 and at 55-degrees-C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the pre sence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory ef fect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cl eavage. Studies with lectins and sugar analysis by mass spectroscopy i ndicated that the cellulase (Avicelase) CeI-1 was neither N nor O glyc osylated. Its processing by the protease occurred at temperatures rang ing from 30 to 55-degrees-C, pH 7.5, in the presence of 2 mM dithiothr eitol.