(290d) Desorption of CBH1 From BMCC Substrate Is a Function of Enzymatic Activity

Ye, Z., University of Louisville
Berson, R. E., University of Louisville
Lane, A. N., University of Louisville

Desorption of the cellulase CBH1 from its substrate bacterial microcrystalline cellulose (BMCC) was studied as a function of intrinsic enzymatic activity. Intrinsic enzymatic activity was altered by varying the temperature, shear stress, or by titration with inhibitors. CBH1 was first purified from Spezyme CP cellulases by ion exchange chromatography. The maximum adsorption capacity of CBH1 was about 4 µmol/g BMCC. At an enzyme loading of 2.1 µmol/g BMCC, almost all CBH1 was adsorbed onto the substrate. No desorption of CBH1 was detected during a 1-hour incubation at 0 oC after a ten-fold dilution. The CBH1 and BMCC were incubated together for two days during which free enzyme was measured in the solution. Desorption was quantified by measuring the CBH1 content in the supernatant buffer solution using the Bradford Assay. The CBH1 showed different desorption behavior depending on enzymatic activity. About 30% desorption was observed at 0 oC (with end-over-end mixing), nearly 100% desorption of CBH1 occurred at 50 oC at a shear rate of 150 rpm on a shaker table whereas only 35% desorption of CBH1 occurred at 50 oC at a shear rate of 300 rpm where the enzymatic activity was lower than at 150 rpm. The half life of desorption increased from 2.35 to 6.95 hour on addition of the inhibitor GdnHCl, With the addition of K2PdCl6,, which denatured the catalytic domain of the CBH1, desorption decreased from ~100% to ~40% after two-days incubation at 50 oC and 150 rpm. The intrinsic enzymatic activity of desorbed enzyme was determined using the p-nitrophenylcellobiose hydrolysis assay. The amount of CBH1 desorption was seen to decrease as a function of decreasing enzymatic activity, whether the reduced activity was due to the denaturing agent, low incubation temperature, or difference in the shear rate. This suggests that the release of CBH1 is mainly due to product formation while release due to reversible binding is negligible.