Multiple enzymatic approaches to hydrolysis of fungal β‐glucans by the soil bacterium Chitinophaga pinensis

Abstract

The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β‐1,3‐glucan and β‐1,6‐glucan. While several fungal β‐1,6‐glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C. pinensis shows strong growth using β‐1,6‐glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C. pinensis secretome to hydrolyse the β‐1,6‐glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β‐1,6‐glucanase activity and which are classified into different GH families. Our data show that C. pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β‐1,6‐glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates.

Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to β-glucans

“…Previously characterised examples of CBM92-containing proteins bound to β−1,3-glucan 11 and carrageenan 6 : both of those domains bind to the same polysaccharide as their appended enzymes can target, suggesting a likely role in enzyme potentiation 2 . Indeed, our phylogenetic analyses show that a number of CBM92 domains are attached to predicted β−1,6-glucanases from enzyme family GH30 (sub-family 3) 55 , and these may be expected to show the same kind of rate potentiation. The natural substrate for these enzymes may be polymeric pustulan as found in lichenous fungi 20 or it may be shorter chains of β-1,6-glucan such as can be found in the cell walls of certain oomycetes 18 .…”

Conversion of β‐1,6‐Glucans to Gentiobiose using an endo‐β‐1,6‐Glucanase PsGly30A from Paenibacillus sp. GKG

“…According to the CAZymes classification, [16] the closest homologues of PsGly30A belong to the GH30_3 subfamily. The closest homologue of PsGly30A for which a crystal structure (PDB ID: 5NGK) has been determined is endo‐β‐1,6‐glucanase BT3312 from Bacteroides thetaiotaomicron , [14] with a sequence similarity of 32 % and the closest characterised member of the GH30 family is Cp Glu30 A from Chitinophaga pinensis , [13] which belongs to the GH30_3 subfamily, with sequence identity of 40 %.…”

“…Analysis of the compounds obtained after hydrolysis of a hydrothermally treated pustulan with PsGly30A showed that gentiobiose was the main product (Figure 6b). In comparison, glucose was the major product produced from pustulan after prolonged hydrolysis using BT3312 from Bacteroides thetaiotaomicron, [14] CpGlu30A from Chitinophaga pinensis, [13] GH30A from Coprinopsis cinerea, [31] and Gly30B from Saccharophagus degradans. [27] As expected, hydrolysis of yeast β-glucan and yeast CW using PsGly30A resulted in significantly lower concentrations of gentiobiose and a similar pattern of additional products besides gentiobiose, compared to pustulan hydrolysis.…”

“…Among all tested substrates, pustulan was the most efficiently hydrolysed substrate, an observation was consistent with the previous reports on GH30 β-1,6glycosidases. [13,27,31,32,40,41] In addition, the specific activity (1262 � 82 U/mg) of PsGly30A towards pustulan was the highest one comparing to previously characterised GH30 family β-1,6glycosidases: 776.50 � 1.02 U/mg for GH30A from Coprinopsis cinerea, [31] 86 U/mg for β-1,6-glucanase from Trichoderma harzianum; [40] 83 U/mg for AfNeg1 from Aspergillus fumigatus; [32] and 14.8 U/mg for LePus30A from Lentinula edodes. [41] The majority of studies did not specify whether hydrothermally treated or untreated pustulan was used, also, the structure of pustulan can vary greatly between the same and different source microorganism species and storage conditions.…”

A First Expression, Purification and Characterization of Endo-β-1,3-Glucanase from Penicillium expansum

“…Similarly, to enhance oligoglucoside hydrolysis, a thermostable β-1,3-glucanase from Trichoderma harzianum was expressed in Pichia pastoris [11,17]. Meanwhile, β-1,3-glucanases from Chitinophaga pinensis hydrolyze β-1,6-glucan pustulan and can be classified into different GH families with different levels of β-1,6-glucanase activity [18]. In the control of fungal pathogens, some biocontrol agents showed interest in beta-1,3-glucanase production.…”