Figure 1.

Endoglucanase activity in culture supernatants obtained during tube-scale screening of transformants. Culture supernatants were collected from the AOX1 strains 48 hours after induction, and from the GAP strains after 48 hours of incubation (see Methods for more details). A) Endoglucanase activity was measured on filter paper by incubating 10 μl culture supernatant with 40 μl of a suspension of phosphoric acid swollen filter paper (1% w/v) and 10 μl 50 mM Na-citrate buffer (pH 5.0) for 1 hour at 50°C. B) Endoglucanase activity was measured on carboxymethylcellulose by incubating 50 μl appropriately diluted culture supernatant with 450 μl 1% (w/v) carboxymethylcellulose in 50 mM Na-citrate buffer (pH 5.0) for 10 min at 50°C. Enzyme activities were assessed by measuring formation of reducing sugars. Filter paper activity is expressed as percentage of substrate conversion; CMCase activity is expressed as nkat/ml. The activity values were background corrected by measuring reducing sugars present in the culture supernatants. Since culture supernatants of PichiaPink^TM transformed with the empty vector, lacking endoglucanases, showed no activity, all activity can be attributed to the recombinant endoglucanases. The data are means from triplicate experiments; error bars reflect standard deviations. The clone number is indicated in the bars.