Abstract
The cellulose binding domain (CBD) of cellulase binding to cellulosic materials is the initiation of a synergistic action on the enzymatic hydrolysis of the most abundant renewable biomass resources in nature. The binding of the CBD domain to cellulosic substrates generally relies on the interaction between the aromatic amino acids structurally located on the flat face of the CBD domain and the glucose rings of cellulose. In this study, we found the CBD domain of a newly cloned Penicillium crustosum endoglucanase EGL1, which was phylogenetically related to Aspergillus, Fusarium and Rhizopus, and divergent from the well-characterized Trichoderma reeseis cellulase CBD domain, contain two conserved aromatic amino acid-rich regions, Y451-Y452 and Y477-Y478-Y479, among which three amino acids Y451, Y477, and Y478 structurally sited on a flat face of this domain. Cellulose binding assays with green fluorescence protein as the marker, adsorption isotherm assays and an isothermal titration calorimetry assays revealed that although these three amino acids participated in this process, the Y451-Y452 appears to contribute more to the cellulose binding than Y477-Y478-Y479. Further glycine scanning mutagenesis and structural modelling revealed that the binding between CBD domain and cellulosic materials might be multi-amino-acids that participated in this process. The flexible poly-glucose molecule could contact Y451, Y477, and Y478 which form the contacting flat face of CBD domain as the typical model, some other amino acids in or outside the flat face might also participate in the interaction. Thus, it is possible that the conserved Y451-Y452 of CBD might have a higher chance of contacting the cellulosic substrates, contributing more to the affinity of CBD than the other amino acids.
Highlights
Cellulosic materials are the most abundant renewable biomass resources in nature
The catalytic domain (CD) domain is responsible for the hydrolysis of cellulose, and the cellulose binding domains (CBD) mediates the binding of the enzyme to cellulose; cellulase has evolved as a modular enzyme
We systematically studied the role of aromatic amino acids in CBD binding affinity by the methods of glycine scanning mutagenesis, cellulose-binding assays with green fluorescence protein as the marker, adsorption isotherm assays, and isothermal titration calorimetry assays, and found these amino acids differentially contributed to CBD binding on the cellulosic substrates
Summary
Cellulosic materials are the most abundant renewable biomass resources in nature. These cellulosic materials can be enzymatically hydrolysed into sugars by cellulases and fermented into cellulosic ethanol and other cellulose-based biofuels, which are important alternative energy sources for reducing environmental pollution and ensuring the security of energy. Aromatic amino acids in the CBD domain of endoglucanase sources [1, 2]. Due to their critical function in the bioconversion of cellulosic materials, cellulases have deeply allured the interest of researchers. The CD domain is responsible for the hydrolysis of cellulose, and the CBD mediates the binding of the enzyme to cellulose; cellulase has evolved as a modular enzyme. The hydrolysis activity of cellulose is generally dependent on the CBD domain, which could adsorb to the matrix cellulose and endorse the catalytic domain to exert its cellulose degradation capacity [4,5,6,7,8]
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