Chemical modification of cellulase from Aspergillus niger

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Cellulase enzymes are crucial for biomass degradation.
• Understanding the active site and essential residues of cellulase is key to optimizing its industrial applications.

Purpose of the Study:

• To identify the specific amino acid residues essential for cellulase activity.
• To elucidate the mechanism of cellulase inhibition by various chemical agents.

Main Methods:

• Chemical modification of cellulase using N-Bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, diazocarbonyl compounds, carbodiimide, and diethyl pyrocarbonate.
• Enzyme activity assays to quantify inhibition.
• Spectroscopic titration experiments to determine residue modification.
• pH-dependence studies of enzyme inactivation.

Main Results:

• Oxidation of one tryptophan residue per cellulase molecule by N-Bromosuccinimide led to complete inactivation.
• CM-cellulose protected the enzyme from N-Bromosuccinimide.
• Reaction with 2-hydroxy-5-nitrobenzyl bromide incorporated 2.3 groups per molecule, with one tryptophan residue essential for activity.
• Inhibition by diazocarbonyl compounds (with Cu2+), carbodiimide, and metal ions (Ag+, Hg2+) was observed.
• pH-dependence suggested carboxyl group involvement in inactivation.
• Diethyl pyrocarbonate modified two histidine residues, retaining 40% activity.
• Metal ion inhibition was attributed to tryptophan interaction, not thiol groups.

Conclusions:

• Tryptophan residues are critical for cellulase catalytic activity.
• Specific chemical modifications can selectively inactivate cellulase, providing insights into its structure-function relationship.
• Cellulase activity is sensitive to various chemical agents and metal ions, with distinct mechanisms of inhibition.