Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Abstract

BackgroundThe non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited.ResultsEight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1.ConclusionsOur results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1333-3) contains supplementary material, which is available to authorized users.

Metal Affinity Immobilization of the Processive Endoglucanase EG5C-1 from Bacillus subtilis on a Recyclable pH-Responsive Polymer

“…Due to their dual functions, processive endoglucanases, coupled with a β-glucosidase, may be sufficient for the degradation of cellulose . To date, several studies were focused on structural features and biochemical characteristics of processive endoglucanases. − Efforts to immobilize such an enzyme for improved stability and reusability are, however, still scarce.…”

Processive binding mechanism of Cel9G from Clostridium cellulovorans: molecular dynamics and free energy landscape investigations

“…In general, many cellulases are typical multimodule enzymes and contain a catalytic domain (CD) and a carbohydrate-binding module (CBM) connected by a linker peptide. 30,67,68 The flexibility of the linker peptide has a significant effect on the catalytic mechanism. 30,[69][70][71][72] For example, Boisset et al proposed that Cel45 from Humicola insolens, which features a flexible linker, may exhibit an inchworm-like mechanism for hydrolysis.…”

“…30,67,68 The flexibility of the linker peptide has a significant effect on the catalytic mechanism. 30,[69][70][71][72] For example, Boisset et al proposed that Cel45 from Humicola insolens, which features a flexible linker, may exhibit an inchworm-like mechanism for hydrolysis. The linker region (residues 441-457) in Cel9G shows some rigidity because of a lack of glycine residues.…”

“…14,22,27 Although CBM3c was shown to exhibit weak interactions with cellulose owing to a lack of significant aromatic residues, 24,28 it is likely that it still facilitates the processive mechanism by feeding a sugar chain into the catalytic domain. 29,30 Notably, the removal of the CBM3c domain results in the complete loss of the catalytic activity and the blocking of the processive binding process. Using computational analysis, we had previously shown that the entire binding cleft of Cel9G can accommodate a polysaccharide with a degree of polymerization as high as 18 (G18).…”

Improving the Catalytic Efficiency of a GH5 Processive Endoglucanase by a Combinatorial Strategy Using Consensus Mutagenesis and Loop Engineering

“…9,12,13 Furthermore, the bulk of research has primarily concentrated on exploring the contributions and mechanisms of various carbohydrate-binding modules concerning the processivity of such enzymes. 7,11,13,20 Currently, engineering studies of processive EGs mainly focus on specific spatial regions within the protein such as the active site or the substrate binding pocket. Several studies reported that specific aromatic amino acids in the substrate binding pocket may improve binding affinity and facilitate processivity via carbohydrate-π interactions.…”