Visualization of Trichoderma reesei Cellobiohydrolase I and Endoglucanase I on Aspen Cellulose by Using Monoclonal Antibody-Colloidal Gold Conjugates

Abstract

Monoclonal antibodies (MAbs) specific for cellobiohydrolase I (CBH I) and endoglucanase I (EG I) were conjugated to 10-and 15-nm colloidal gold particles, respectively. The binding of CBH I and EG I was visualized by utilizing the MAb-colloidal gold probes. The visualization procedure involved immobilization of cellulose microfibrils on copper electron microscopy grids, incubation of the cellulose-coated grids with celiulase(s), binding of MAb-colloidal gold conjugates to cellulase(s), and visualization via transmission electron microscopy. CBH I was seen bound to apparent crystalline cellulose as well as apparent amorphous cellulose. EG I was seen bound extensively to apparent amorphous cellulose with minimal binding to crystalline cellulose.

Cellulases Dig Deep

“…The types of native and processed celluloses applied in earlier AFM studies, however, were limiting in various ways ( 2 , 7 , 17 – 20 ). The use of highly crystalline cellulose alone, for instance, does not allow the investigation of an entire cellulase system featuring CBHs and endoglucanases (EGs), with the latter ones being known to preferably attack amorphous areas ( 2 , 9 , 11 , 18 , 21 ). In contrast to previous research, we took a distinct approach involving the dissolution of the microcrystalline cellulose Avicel in the ionic liquid 1-butyl-3-methylimidazolium chloride (BMIMCl) at 100 °C, which, upon cooling, resulted in a gel-like material ( 16 ).…”

Fungal Cellulases

“…Several other TEM studies focused on GH7 enzymes, − including those utilizing gold labeling , and labeling with gold-coupled monoclonal antibodies (immuno-EM). , Immuno-EM was utilized to determine that Tr Cel7A and Tr Cel7B preferentially bind to the crystalline and amorphous regions of substrate, respectively …”

Cellulose morphology and enzymatic reactivity: A modified solute exclusion technique

“…On the other hand, Nieves et a1. 22 showed that cellulases adsorb on the cellulose surface as aggregated particles. In such a case, protein multilayers may exist on saturated cellulose, hence, the surface area is overestimated.…”