Efficient activity screening of new glucuronoyl esterases using a pNP-based assay

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• Glucuronoyl esterases (CE15) are crucial for breaking down lignocellulose.
• Identifying novel CE15 enzymes is vital for efficient lignocellulose processing.
• Current screening methods are limited by poor substrate representation.

Purpose of the Study:

• To express and screen fifteen novel fungal CE15 proteins for esterase activity.
• To develop and utilize an improved colorimetric assay for enzyme screening.
• To identify active CE15 candidates for potential industrial applications.

Main Methods:

• Expression and purification of fifteen fungal CE15 proteins.
• Colorimetric screening using methyl ester-UX-β-pNP substrate.
• Activity assays coupled with GH67 and GH43 enzymes.
• Determination of specific activity for active enzymes.

Main Results:

• Seven out of fifteen expressed CE15 proteins exhibited detectable activity.
• Active enzymes were identified from Auricularia subglabra (3), Ganoderma sinensis (2), and Neocallimastix californiae (2).
• CE15 proteins from S. commune, P. anserina, T. versicolor, and C. cinerea were inactive on the tested substrate.

Conclusions:

• A novel colorimetric assay effectively screens for glucuronoyl esterase activity.
• Several active fungal CE15 enzymes were identified, offering potential for lignocellulose valorization.
• Unexpected lack of activity in some known lignocellulose degraders warrants further investigation into substrate specificity.