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 O2 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 kcat= 0.40 ± 0.05 s−1, much slower than kcat= 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 → O2, kcat= 0.057 ± 0.004 s−1. Half maximal rates corresponded to Km(Avicel) = 8 ± 2 gl−1 with ferricyanide, and 3.3 ± 0.5 gl−1 with O2 as acceptor.