Abstract
The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.
Highlights
Based on amino acid sequence and in consequence structural similarity, glycoside hydrolase (GH) enzymes are grouped into GH families, as comprehensively documented in the carbohydrate-active enzymes (CAZy) database
Two non-contiguous segments within the 905 residue EngU primary structure displayed partial similarity to the Pfam seed representing GH family 42 and to the β-glycanases family in the Superfamily database, which suggested EngU belongs to the GH-A clan of glycoside hydrolases (Fig. 1)
A carbohydrate-binding module CBM4 was found to be inserted between the two β-glycanase parts which results in a large distance of the key catalytic residues along the primary structure of EngU
Summary
Based on amino acid sequence and in consequence structural similarity, glycoside hydrolase (GH) enzymes are grouped into GH families, as comprehensively documented in the carbohydrate-active enzymes (CAZy) database (http://www.cazy.org/). Different branches of the industry such as the chemical, biofuel, food, feed and pharmaceutical industry have a large interest in hydrolases that cleave the glycosidic bonds of plant-derived polysaccharides such as mixed-linkage β-glucans, cellulose, or xylan[1,2]. The degradation of these polysaccharides usually requires the action of multiple enzyme activities[3]. In the last few years, functional screening has helped to discover novel enzymes from various environmental starting materials such as from gut microbiome, biogas plant or soil samples[18,19,20,21]
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