An Avicel-affinity site in an Avicel-digesting exocellulase from a Trichoderma viride mutant

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Cellulases are crucial enzymes for biomass degradation.
• Understanding cellulase domain function is key to optimizing biofuel production.
• Trichoderma viride is a significant source of industrially relevant cellulases.

Purpose of the Study:

• To elucidate the functional domains of exo-type cellulase (Exo I) from Trichoderma viride.
• To characterize the substrate specificity and enzymatic activity of Exo I and its fragments.
• To determine the role of different domains in Avicel digestion and cellooligosaccharide hydrolysis.

Main Methods:

• Purification of Exo I from Trichoderma viride mutant HK-75.
• Enzymatic digestion assays using Avicel and various cellooligosaccharides.
• Proteolytic cleavage of Exo I using papain.
• Analysis of enzyme fragments (GPExo and Exo I') for adsorption and hydrolysis activity.
• N-terminal and C-terminal amino acid sequencing.

Main Results:

• Exo I hydrolyzed Avicel to cellobiose and shorter oligosaccharides.
• Proteolytic digestion yielded two fragments: GPExo (adsorbs to Avicel) and Exo I' (hydrolyzes cellooligosaccharides).
• GPExo showed high Avicel adsorption but no hydrolysis; Exo I' exhibited high cellooligosaccharide hydrolysis but minimal Avicel activity.
• Sequence analysis confirmed GPExo and Exo I' correspond to distinct domains within Exo I.

Conclusions:

• Exo I possesses two distinct functional domains: an Avicel-binding domain (GPExo) and a catalytic domain (Exo I').
• The adsorption domain (GPExo) facilitates enzyme attachment to the substrate.
• The catalytic domain (Exo I') is responsible for breaking down cellulose chains into smaller sugars.