Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases

This study describes the isolation and characterization of esterase genes from the anaerobic ruminal fungus Neocallimastix patriciarum. Researchers screened a cDNA expression library using β-naphthyl acetate as a substrate and identified four different esterase genes (bnaA–D). Complete sequences were determined for three of these genes. Two enzymes (BnaA and BnaC) demonstrated acetylxylan esterase activity, capable of removing acetyl groups from xylan, an important feature for plant fiber degradation in ruminants. None of the enzymes showed cinnamoyl ester hydrolase activity despite initial screening attempts. Sequence analysis revealed that the catalytic domains of BnaA, BnaB, and BnaC belong to a previously undescribed family of Ser/His active site hydrolases. BnaA and BnaC possess carboxy-terminal protein-docking domains homologous to similar domains in other anaerobic fungal enzymes, suggesting they may function as components of cellulosome-like complexes. The study identifies additional members of this new hydrolase family across diverse organisms, including fungi and bacteria, indicating this represents an important class of enzymes involved in complex polysaccharide degradation.

Key findings

• Two of four cloned esterases (BnaA and BnaC) from N. patriciarum possess acetylxylan esterase activity that removes acetyl groups from xylan
• BnaA, BnaB, and BnaC catalytic domains constitute a new family of Ser/His hydrolases with three conserved amino acid sequence motifs
• BnaA and BnaC contain carboxy-terminal protein-docking domains similar to those in other anaerobic fungal enzymes, suggesting involvement in cellulosome-like complexes
• Members of this new hydrolase family are present across diverse organisms including fungi, bacteria, and mammals, indicating broad evolutionary distribution
• None of the cloned enzymes exhibited cinnamoyl ester hydrolase activity despite presence of this activity in the parent organism