Abstract
Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme’s commercial utilization. To obtain endoglucanases with substantially improved activity and thermostability, structure-based rational design was carried out based on the Chaetomium thermophilum β-1,4-endoglucanase CTendo45. In this study, five mutant enzymes were constructed by substitution of conserved and noncatalytic residues using site-directed mutagenesis. Mutants were constitutively expressed in Pichia pastoris, purified, and ultimately tested for enzymatic characteristics. Two single mutants, Y30F and Y173F, increased the enzyme’s specific activity 1.35- and 1.87-fold using carboxymethylcellulose sodium (CMC-Na) as a substrate, respectively. Furthermore, CTendo45 and mutants exhibited higher activity towards β-D-glucan than that of CMC-Na, and activities of Y173F and Y30F were also increased obviously against β-D-glucan. In addition, Y173F significantly improved the enzyme’s heat resistance at 80 °C and 90 °C. More interestingly, the double mutant Y30F/Y173F obtained considerably higher stability at elevated temperatures but failed to inherit the increased catalytic efficiency of its single mutant counterparts. This work gives an initial insight into the biological function of conserved and noncatalytic residues of thermostable endoglucanases and proposes a feasible path for the improvement of enzyme redesign proposals.
Highlights
Cellulose, the most abundant renewable carbon resource on earth, is generally considered a sustainable feedstock to replace fossil fuels for biochemical and biotechnological production[1]
The cleft is responsible for bringing the catalytic domain in an appropriate position for cellulose decomposition and gives space for seven glucose units (Fig. 1b)
Aspl[22] is identified as a catalytic acid in the glycosyl group hydrolysis and Aspl[2] acts as the base, enhancing the nucleophilicity of the catalytic water[25]. They are positioned above and to either side of a noncatalytic residue, Y10, which lies at the bottom of the active side groove[6]
Summary
The most abundant renewable carbon resource on earth, is generally considered a sustainable feedstock to replace fossil fuels for biochemical and biotechnological production[1]. The high cost of preparation and low performance in extreme reaction conditions have been perceived as major bottlenecks to industrial applications[8,9,10]. More attention has been drawn to the underlying function of important residues, along with practicality of modifying conserved noncatalytic residues[15,16,17], to generate mutant enzymes with improved properties and to help elucidate structure-function relationships[18]. Thermostable enzymes have excellent tolerance to various harsh conditions, including high salt concentrations and extreme pHs19,20. In order to profoundly improve hydrolysis efficiency at high temperatures while simultaneously reducing microbial contamination in reaction processes, it is important for enzymes to be both thermoactive and thermostable[21,22]. Site-directed mutagenesis of conserved and noncatalytic residues of CTendo[45] was implemented to further enhance specific activity and thermostability, providing a potential biocatalyst for industry
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