Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii

Abstract

We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction.

Screening of alginate lyase-excreting microorganisms from the surface of brown algae

“…Among them, novel alginolytic activity in Paenibacillus , Leclercia and Planomicrobium has not been previously reported. Paenibacillus produces many kinds of extracellular enzymes such as cellulose, proteases, amylase and other polysaccharide-degrading enzymes, which can be used in a wide range of industrial fields (Adlakha et al 2015; Budi et al 2000; Das et al 2016; Dong et al 2016; Lan Pham et al 1998; Mathews et al 2016). However, production of alginate lyase by Paenibacillus has never been reported.…”

Role of Natural Binding Proteins in Therapy and Diagnostics

“…The chitosan-binding domain is a specific region within proteins or peptides that binds to chitosan, such as chitosanases [72]. Classified as carbohydrate-binding modules (CBMs), these domains are part of the carbohydrate-active enzymes, and their binding often depends on chitosan's physical and chemical properties [72,145]. Chitosan-binding domains interact with chitosan through electrostatic interactions, hydrogen bonding, and hydrophobic effects.…”

Paenibacillus algicola sp. nov., a novel alginate lyase-producing marine bacterium

“…Marine sediments derived from cage farm [8], tidal-flat [9] and abyssal region [10] were also the important habitats. Paenibacillus produces many kinds of extracellular enzymes, for instance, chitosanase [11], glucanase [12], multifunctional cellulase/mannanase/xylanase [13] and other enzymes, which can be used in a wide range of industrial fields. However, only several species in the genus were reported to possess alginate lyase [14–16].…”