Continuous monitoring of cellulose oxidation by cellobiose oxidase fromPhanerochaete chrysosporium

Cellobiose oxidase activity from Phanerochaete chrysosporium was monitored continuously by two techniques that are independent of the presence of a solid substrate, measuring either ferricyanide by the electrode potential of anaerobic ferricyanide plus ferrocyanide, or O 2 with a specific electrode. Both methods demonstrated that microcrystalline cellulose (Avicel) is a substrate for oxidation. All experiments were at pH 4.0 and 30°C. Rates were evaluated for two-electron transfer. For Avicel → ferricyanide, a study of initial rates gave k cat = 0.40 ± 0.05 s −1 , much slower than k cat = 3.6 ± 0.3 s −1 with cellobiose as substrate. With Avicel plus 0.2 μM cellobiose oxidase, the rate fell to half its initial value in 3–8 min, indicating a heterogeneity of reducing ends. For Avicel → O 2 , k cat = 0.057 ± 0.004 s −1 . Half maximal rates corresponded to K m ( Avicel ) = 8 ± 2 gl −1 with ferricyanide, and 3.3 ± 0.5 gl −1 with O 2 as acceptor.