Detection of major xylanase-containing cellulose-binding domain from Penicillium verruculosum by combination of chromatofocusing and limited proteolysis

Adsorption on microcrystalline cellulose of enzyme components of cellulase complex from Penicillium verruculosum was studied by chromatofocusing on a Mono P column. The most strongly adsorbed and major component was identifie d as xylanase (XYN) with MW 65 kDa and pi 4.5. The high adsorption degree o f XYN on cellulose indicated the possible presence of a cellulose-binding d omain in the molecular structure. Limited proteolysis of XYN with papain wa s carried out. Kinetics of proteolysis was monitored by sodium dodecyl sulf ate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and measuring ac tivities toward insoluble xylan and 4-methyl-umbelliferyl-beta-D-lactoside (MUF-LAC). During the proteolysis, formation of two polypeptides with MW 51 and 14 kDa was observed. No loss of activity toward the soluble substrate was observed, whereas the activity toward xylan decreased rapidly. Adsorpti on distribution coefficient (K-d) of the core protein separated by gel-filt ration was found to be 15 times lower than the K-d, for the initial nondige sted XYN (0.02 and 0.29 L/g, respectively). The activity of core protein to ward insoluble xylan was close to zero, whereas the activity toward MUF-LAC was close to that exhibited by the original enzyme. The results presented indicate a bifunctional organization of XYN, where one domain acts as a bin ding anchor for insoluble substrates and the other, localized in the core p rotein, contains the active site.