Biochemical Characterization of a Protease Involved in the Processing of a Streptomyces reticuli Cellulase (Avicelase)

Abstract

A 36-kDa protease from Streptomyces reticuli had recently been shown to 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 enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold 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-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55°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 presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor 0 glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55°C, pH 7.5, in the presence of 2 mM dithiothreitol. * Corresponding author. lase) Cel-1. Additional studies suggest that this enzyme is not covalently glycosylated.

The complex extracellular biology ofStreptomyces

“…The binding domain targets the enzyme to Avicel during early stages of growth. After this, the C-terminal 42-kDa region is released by a 36-kDa metalloprotease (Moormann et al, 1993). The 42 kDa form hydrolyses Avicel to oligomers that are further processed to cellobiose and cellotriose by a glucosidase (Schlochtermeier et al, 1992a).…”

Binding and Substrate Specificities of a Streptomyces olivaceoviridis Chitinase in Comparison with Its Proteolytically Processed Form

“…Earlier we had shown that, in the course of cultivation, one extracellular S. reticuli protease specifically cleaves the cellulase (Avicelase). Thus a truncated cellulase not containing the binding domain is released to the culture filtrate (Schlochtermeier et al, 1992a,b;Moormann et al, 1993). Similarly the substrate-bound 59-kDa chitinase is specifically proteolytically processed to a 47-kDa truncated form which diffuses into the culture filtrate.…”

Physiological Studies of Cellulase (Avicelase) Synthesis in Streptomyces reticuli

“…Avicelase consists of a Cterminal catalytic domain belonging to the family E (4,11), one central domain of an as-yet-unknown function, and an N-terminal substrate-binding domain. In the course of cultivation, the strain produces an extracellular 36-kDa protease (9) which processes Avicelase to a truncated, catalytically active 42-kDa enzyme lacking the cellulose-binding domain and the central part of the progenitor enzyme (11). Several Streptomyces strains acquire the ability to degrade crystalline cellulose if they are transformed with the plasmid pZV1.…”