Journal Article

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity

Hernández-Benítez, L. Joshua, Ramírez-Rodríguez, Miguel A., Rodríguez-Romero, Adela, Hernández-Santoyo, Alejandra

2024

Overview

The red abalone ( Haliotis rufescens ) represents North America’s most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H . rufescens . The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p -nitrophenyl- β -D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% ( w / v ) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a β -sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Algae / Amino Acid Sequence / Amino acids / Animals / Aquaculture / Bioconversion / Biofuels / Biology and Life Sciences / Carboxymethyl cellulose / Carboxymethylcellulose / Cellulase / Cellulase - metabolism / Cellulose / Cellulose - metabolism / Chemical properties / Chromatography / Circular dichroism / Dichroism / Enzymatic activity / Enzyme activity / Enzymes / Gastropoda - genetics / Gel chromatography / Gel filtration / Glycoproteins / Haliotis rufescens / Hepatopancreas / Hydrolases / Identification and classification / Mass spectrometry / Mass spectroscopy / Membrane filters / Oligosaccharides / Physical Sciences / Polysaccharides / Proteins / Reagents / Research and Analysis Methods / Residues / Solvents / Structure-function relationships / Trimers