Purification and characterization of two exo-cellobiohydrolases from the brown-rot fungus Coniophora puteana (Schum ex Fr) Karst.

Two extracellular exo-cellobiohydrolases (EC 3.2.1.91) were purified to homogeneity from the culture filtrate of the brown-rot fungus Coniophora puteana (Schum ex Fr) Karsten, strain EMPA 62. The purification scheme involved three successive chromatographic steps, namely Q Sepharose fast flow, Superose 12, and Fractogel TSK DEAE-650S. The two enzymes, named cellobiohydrolase (CBH) I and CBH II, were purified by a factor of 4.6 and 3.9, respectively, with an activity recovery of 9 and 19% of total, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both enzymes migrated as single bands according to a M(r) of 52,000 for CBH I and 50,000 for CBH II; by FPLC gel filtration (TSK G3000 SW) the M(r)'s were higher (65,000 and 60,000). Both enzymes were glycosylated, had similar isoelectric points (pI 3.6 and 3.55) and nearly identical pH optima for activity close to 5. Endoglycosidase H digestion gave two distinct polypeptides where the molecular weight was lowered by 6.5 kDa for CBH I and by 2.5 kDa for CBH II. The specific activities for the hydrolysis of p-nitrophenyllactoside (pNPL) were nearly identical for both enzymes (0.46 versus 0.40 mumol mg-1 min-1 at 40 degrees C) and the Km values (6.8 and 4.3 mM at 30 degrees C) were also very close. Both enzymes were competitively inhibited by cellobiose: with pNPL as substrate, Ki values of 1.2 mM for CBH I and 2.4 mM for CBH II were determined. The two enzymes acted in an identical fashion on cellulose (either amorphous or crystalline) and on cellodextrins, liberating mainly cellobiose, but were inactive on dyed carboxymethylcellulose. Cellobiose was not hydrolyzed whereas cellotriitol was hydrolyzed to equimolar amounts of cellobiose and glucitol: these results support the interpretation that these enzymes are exo-cellobiohydrolases. Their presence in a brown-rot fungus is a new fact.