Improving the activity of Trichoderma reesei cel7B through stabilizing the transition state

Area of Science:

• Biotechnology
• Enzyme Engineering
• Biofuels

Background:

• Trichoderma reesei (Tr.) cellulases are crucial for converting cellulose into sugars for biofuel production.
• Lignocellulose recalcitrance necessitates improved enzyme activity and substrate promiscuity for efficient biofuel conversion.
• Enhancing endoglucanase activity against xylan, a component of lignocellulose, is a key challenge.

Purpose of the Study:

• To improve the catalytic activity of the endoglucanase Tr. Cel7B against xylan.
• To enhance the enzyme's promiscuity for hydrolyzing substrates beyond cellulose.
• To demonstrate the effectiveness of computational methods in enzyme engineering.

Main Methods:

• Utilized quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations to identify the transition state of xylo-oligosaccharide hydrolysis.
• Employed free energy calculations to rank the effect of introduced mutations on transition state stabilization.
• Experimentally evaluated seven top-ranked mutants for their enzymatic activity.

Main Results:

• Three engineered mutants (A208Q, A222D, and G230R) exhibited significantly higher activity against xylan (up to 47%) and filter paper (up to 50%) compared to the wild-type Tr. Cel7B.
• Combining single mutations further enhanced enzyme activity.
• The free energy method proved effective for engineering Tr. Cel7B activity.

Conclusions:

• Computational free energy calculations are a powerful tool for engineering enzyme activity, specifically for Tr. Cel7B.
• The developed mutants show potential for improving lignocellulosic biofuel production by enhancing cellulose and xylan hydrolysis.
• This approach can be extended to engineer other Trichoderma reesei cellulases for broader applications.