Impact of Module-X2 and Carbohydrate Binding Module-3 on the catalytic activity of associated glycoside hydrolases towards plant biomass

Abstract

Cellulolytic enzymes capable of hydrolyzing plant biomass are secreted by microbial cells specifically in response to the carbon substrate present in the environment. These enzymes consist of a catalytic domain, generally appended to one or more non-catalytic Carbohydrate Binding Module (CBM), which enhances their activity towards recalcitrant biomass. In the present study, the genome of a cellulolytic microbe Paenibacillus polymyxa A18 was annotated for the presence of CBMs and analyzed their expression in response to the plant biomass and model polysaccharides Avicel, CMC and xylan using quantitative PCR. A gene that encodes X2-CBM3 was found to be maximally induced in response to the biomass and crystalline substrate Avicel. Association of X2-CBM3 with xyloglucanase and endoglucanase led to up to 4.6-fold increase in activity towards insoluble substrates. In the substrate binding study, module X2 showed a higher affinity towards biomass and phosphoric acid swollen cellulose, whereas CBM3 showed a higher affinity towards Avicel. Further structural modeling of X2 also indicated its potential role in substrate binding. Our findings highlighted the role of module X2 along with CBM3 in assisting the enzyme catalysis of agricultural residue and paved the way to engineer glycoside hydrolases for superior activity.

Genome analysis of Paenibacillus polymyxa A18 gives insights into the features associated with its adaptation to the termite gut environment

“…Amongst the cellulose hydrolyzing enzymes, β-1,4-endoglucanases (EGs, EC 3.2.1.4) of GH5 family and β-glucosidases (BGLs, EC 3.2.1.21) of GH1 and GH3 family were represented in the genome. A xyloglucan-specific endoglucanase of GH74 family associated with CBM3 and X2 was also encoded in the genome, as was reported before 28 . Hemicellulose, being a complex polymer, requires the action of several enzymes for its degradation 29 .…”

“…A total of 45 carbohydrate binding modules (CBMs), which are generally associated with GHs for better substrate binding were also found in P. polymyxa A18. A detailed characterization of all CBMs of P. polymyxa A18, including their expression analysis and role in biomass hydrolysis, has been reported in our earlier study 28 . Categorization of CAZymes on the basis of functionality revealed that 76 CAZymes were predicted to be plant cell wall-degrading enzymes (CWDEs).…”

Carbohydrate Binding Modules: Diversity of Domain Architecture in Amylases and Cellulases From Filamentous Microorganisms

“…Based on the enzyme activity studies, endoglucanase with X2-CBM3 domains have a several fold increase in activity toward insoluble substrates. In substrate binding studies, the module X2 showed a higher affinity toward phosphoric acid swollen cellulose, whereas CBM3 showed a higher affinity toward crystalline cellulose (Avicel) (Pasari et al, 2017).…”

In silico Proteomic Analysis Provides Insights Into Phylogenomics and Plant Biomass Deconstruction Potentials of the Tremelalles

“…Although CBMs are most abundant among the soil isolates, majority of the proteins containing these modules do not have CAZYmes modules. CAZYmes without CBMs have been reported to show poor catalytic activities relative to their CBMs associated counterparts (Pasari et al, 2017). To enhance catalytic activity of certain CAZYmes lacking CBMs, the latter modules could be engineered in the enzymes.…”