Impact of a conserved tyrosine residue on binding of family 1 carbohydrate binding modules to cellulose allomorphs
Creators: name: Dagia, Akash
Publisher: name: No Publisher Supplied
Year: 2018

description: The recalcitrance of cellulose, coupled with non-productive binding of cellulases, is considered to be a major bottleneck in the deconstruction of biomass into biofuels (Jeoh, T., Cardona, M. J., Karuna, N., Mudinoor, A. R. &amp; Nill, J. Mechanistic kinetic models of enzymatic cellulose hydrolysis—A review. Biotechnol. Bioeng. 114, 1369–1385 (2017). doi:10.1002/bit.26277). Past research to address the recalcitrance of cellulose, has led the development of pre-treatment technologies like the Extractive Ammonia process (Sousa, L. et al. Next-generation ammonia pretreatment enhances cellulosic biofuel production. Energy Environ. Sci. 9, 1215–1223 (2016). doi:10.1039/c5ee03051j), which can modify the ultrastructure of native crystalline cellulose-I to cellulose-III allomorph (Chundawat, S. P. S. et al. Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate. J. Am. Chem. Soc. 133, 11163–11174 (2011). doi:10.1021/ja2011115). Surprisingly, it was found previsouly that some full-length cellulases bind with lower apparent affinity to crystalline cellulose-III, while the enzymatic hydrolysis rate for this modified cellulose-III allomorph was between two to five-folds higher by fungal cellulase enzyme cocktails (Gao, D. et al. Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis. Proc. Natl. Acad. Sci. 110, 10922–10927 (2013). doi:10.1073/pnas.1213426110). Our results attest to better understanding the role of carbohydrate-binding modules (CBMs) on the reduced binding affinity of full-length fungal cellulases seen towards cellulose-III. Here, we closely explore the role of key amino acid residues of a Family 1 CBM that are likely to impact protein binding interactions with the surface of cellulose-III. Single-site saturation mutagenesis libraries were generated at such key positions to better understand impact on CBM-1 adsorption to both cellulose allomorphs. We report results here relating to the: (i) Expression and purification of green fluorescent protein (GFP) tagged wild-type CBM-1 protein construct and its single-site saturation mutagenesis protein library for subsequent binding/structural characterization, (ii) Effect of pH and salt concentration on the apparent binding affinity or partition coefficient of wild-type CBM-1 protein and its saturation mutagenesis mutants library towards cellulose allomorphs. We also report regression correlations between the experimentally measured binding parameters and various in silico sequence/structural modeling derived Rosetta software estimated parameters that are indicative of protein function. We also discuss a roadmap for future studies that include analysis of full scale binding isotherm of wild type and some of the key mutants to native and pre-treated cellulose, cloning and testing of overall cellulase activity with mutant CBM1-cellulases to understand the correlation between CBM-mediated overall binding affinity and cellulolytic activity on different cellulose allomorphs. This work has important implications for creation of more efficient cellulase enzymes, which can pave the way towards sustainable production of biofuels from ammonia-pretreated lignocellulosic biomass.