Rational protein design for thermostabilization of glycoside hydrolases based on structural analysis

Abstract

Glycosidases are used in the food, chemical, and energy industries. These proteins are some of the most frequently used such enzymes, and their thermostability is essential for long-term and/or repeated use. In addition to thermostability, modification of the substrate selectivity and improvement of the glycosidase activities are also important. Thermostabilization of enzymes can be performed by directed evolution via random mutagenesis or by rational design via site-directed mutagenesis; each approach has advantages and disadvantages. In this paper, we introduce thermostabilization of glycoside hydrolases by rational protein design using site-directed mutagenesis along with X-ray crystallography and simulation modeling. We focus on the methods of thermostabilization of glycoside hydrolases by linking the N- and C-terminal ends, introducing disulfide bridges, and optimizing β-turn structures to promote hydrophobic interactions.

Recent Advances in β-Glucosidase Sequence and Structure Engineering: A Brief Review

“…BGL plays a key role in consolidated bioprocessing; however, high temperature causes BGL denaturation [ 11 , 99 ]. A comparative analysis on the enzymatic properties and amino acid composition of mesophilic, thermophilic, and hyperthermophilic BGLs revealed a number of factors that contribute to the thermal stability of proteins, such as hydrophobic effects [ 100 , 101 ], hydrogen-bonding and electrostatic interactions [ 100 , 102 ], aromatic interactions [ 100 , 103 ], protein structural densification [ 100 ], reduction in unfolding entropy [ 99 , 101 ], etc.…”

Recent Advances in β-glucosidase Engineering: A Brief Review

“…BGL plays a key role in consolidated bioprocessing; however, high temperature cause BGL denaturation [11,97]. A comparative analysis on the enzymatic properties and amino acid composition of mesophilic, thermophilic, and hyperthermophilic BGLs revealed a number of factors that contribute to the thermal stability of proteins, such as hydrophobic effects [98,99], hydrogenbonding and electrostatic interactions [98,100], aromatic interactions [98,101], protein structural densification [98], reduction of unfolding entropy [97,99], etc.…”

Structure and activity of a thermally stable mutant of Acanthamoeba actophorin

“…We created a series of 20 actophorin mutants (Table 5) based primarily on assessing the structural aspects of actophorin (for similar approaches, see Watanabe et al, 2018). The thermal stability of a protein can often be increased by filling internal cavities (Chen & Stites, 2001; for example, replacing a valine residue with a phenylalanine residue).…”