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Nemmaru, B.

Publications and source records attributed to Nemmaru, B..

2 recordsLinked to original sources
bioRxiv2020

Reduced type-A carbohydrate-binding module interactions to cellulose leads to improved endocellulase activity

Dissociation of non-productively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling non-productive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three model Type-A CBMs (families 1, 3a, and 64) tethered to a multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a small-library of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring cellulose hydrolysis activity of CelE-CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipation (QCM-D) were employed to measure CBM adsorption and desorption rate constants Kon and Koff, respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates (Koff) and reduced effective adsorption rates (nKon), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in non-productive enzyme binding and develop improved cellulolytic enzymes for industrial applications.Competing Interest StatementShishir P.S. Chundawat declares a conflict of interest and competing financial interest(s) having filed two patent applications on pretreatment processes to produce cellulose-III enriched cellulosic biomass for biofuels production (US20130244293A1and WO2011133571A2). All other authors declare that they have no competing interests.View Full Text

biochemistry↗
bioRxiv2020

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have non-catalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two well-studied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond rupture assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the cautious use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods. Significance StatementMultimodal biomolecular binding interactions involving carbohydrate polymers (e.g., cellulose, starch, chitin, glycosaminoglycans) are fundamental molecular processes relevant to the recognition, biosynthesis, and degradation of all major terrestrial and aquatic biomass. Protein-carbohydrate binding interactions are also critical to industrial biotechnology operations such as enzymatically-catalyzed bioconversion of starch and lignocellulose into biochemicals like ethanol. However, despite the ubiquitous importance of such interfacial processes, we have a poor molecular-level understanding of protein-polysaccharide binding interactions. Here, we provide a comprehensive experimental and theoretical analysis of bulk ensemble versus single-molecule binding interactions of enzyme motors and associated non-catalytic binding domains with cellulosic polysaccharides to highlight the critical limitations of applying classical biochemical assay techniques alone to understanding protein adsorption or biological activity at solid-liquid interfaces.

biochemistry↗