Catalytically enhanced endocellulase Cel5A from Acidothermus cellulolyticus

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• Endocellulase Cel5A from Acidothermus cellulolyticus is crucial for cellulose degradation.
• Product inhibition by cellobiose limits the efficiency of wild-type (WT) enzymes.
• Understanding enzyme-product interactions is key to improving enzymatic activity.

Purpose of the Study:

• To engineer a Cel5A mutant with reduced product inhibition.
• To investigate the structural and kinetic basis for enhanced enzyme activity.
• To improve biomass cellulose hydrolysis through enzyme modification.

Main Methods:

• Site-directed mutagenesis of Tyr245 to Gly (Y245G) in Cel5A.
• Determination of crystal structures for WT and Y245G enzymes.
• Kinetic analysis of enzyme inhibition by cellobiose.
• Density functional theory calculations for enzyme-product interactions.

Main Results:

• The Y245G mutation increased the Ki for cellobiose inhibition by over 1480%.
• The mutant enzyme, with Trichoderma reesei cellobiohydrolase-I, achieved 40% greater soluble sugar release from cellulose.
• Structural analysis revealed loss of a key interaction platform and hydrogen bond in the mutant.
• Reduced binding energy between product and enzyme was observed in the Y245G mutant.

Conclusions:

• Reduced product inhibition is directly linked to increased enzymatic activity in Cel5A.
• The Y245G mutation represents a significant advancement in enzyme engineering for biomass conversion.
• This study provides atomic-level insights into enzyme mechanism and product inhibition.