Abstract
Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme’s biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.
Highlights
Arabinofuranosidases (ABFs) (EC 3.2.1.55) are enzymes that are capable of cleaving residues of l-arabinofuranosyl present in various oligosaccharides and in polysaccharides such as hemicellulose; for this reason, they are interesting targets for biotechnological a pplications[1]
T. harzianum IOC-3844 RNA-seq reads and the target protein sequence (MT439956) were obtained. With this target protein sequence, a BLAST search was performed against the CAZy database, where our data suggest that ThABF is more similar to the GH54 family and that this family has several carbohydrate-binding models (CBMs; 1, 2, 6, 13 and 42), as shown in Supplementary Table S1
We bioprospected an arabifuranosidase from the metal-dependent GH54 family from T. harzianum and demonstrated that it showed expanded activity against other substrates
Summary
Arabinofuranosidases (ABFs) (EC 3.2.1.55) are enzymes that are capable of cleaving residues of l-arabinofuranosyl present in various oligosaccharides and in polysaccharides such as hemicellulose; for this reason, they are interesting targets for biotechnological a pplications[1]. Ravanal et al.[4] characterized an α-l-arabinofuranosidase from Penicillium purpurogenum from the GH54 family, demonstrating that this family contains enzymes capable of acting synergistically with others and carrying out hydrolysis of different substrates in addition to arabinose and xylose Based on these characteristics presented by some enzymes in this group, further studies are needed to better understand their enzymatic mechanisms, especially with regard to their application for the conversion of complex substrates such as polysaccharides into bioproducts. It is of interest to apply ABFs for this purpose because in addition, removing l-arabinose, some of these enzymes can act on other sugars involved in s ubstitutions[1] These enzymes are important in the genus Trichoderma, where they play a crucial physiological role[11]. Trichoderma reesei is of particular prominence within the group because of its recognized potential to produce several hydrolytic enzymes; other species of the genus, especially Trichoderma harzianum, have been shown to be as efficient as T. reesei in this regard[13]
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