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bioRxiv · 10.1101/2020.07.02.183293

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

Nemmaru, B.·Ramirez, N.·Farino, C. J.·Yarbrough, J. M.·Kravhcenko, N.·Chundawat, S. P. S.
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Abstract

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

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Nemmaru, B., Ramirez, N., Farino, C. J., Yarbrough, J. M., Kravhcenko, N., Chundawat, S. P. S.. 2020-07-03. Reduced type-A carbohydrate-binding module interactions to cellulose leads to improved endocellulase activity. https://doi.org/10.1101/2020.07.02.183293

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