Thermostabilization of Bacillus circulans xylanase: computational optimization of unstable residues based on thermal fluctuation analysis.
Low thermostability often hampers the applications of xylanases in industrial processes operated at high temperature, such as degradation of biomass or pulp bleaching. Thermostability of enzymes can be improved by the optimization of unstable residues via protein engineering. In this study, computational modeling instead of random mutagenesis was used to optimize unstable residues of Bacillus circulans xylanase (Bcx). The thermal fluctuations of unstable residues known as important to the thermal unfolding of Bcx were investigated by the molecular dynamics (MD) simulations at 300 K and 330 K to identify promising residues. The N52 site in unstable regions showed the highest thermal fluctuations. Subsequently, computational design was conducted to predict the optimal sequences of unstable residues. Five optimal single mutants were predicted by the computational design, and the N52Y mutation showed the thermostabilization effect. The N52 residue is conserved in Bacillus species xylanases and the structure analysis revealed that the N52Y mutation introduced more hydrophobic clusters for thermostability, as well as a more favorable aromatic stacking environment for substrate binding. We confirm that flexible residues at high temperature in unstable regions can be promising targets to improve thermostability of enzymes.
Joo
Jeong Chan
JC
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea.
Pack
Seung Pil
SP
Kim
Yong Hwan
YH
Yoo
Young Je
YJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D000596:Amino Acids; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases