Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus

Abstract

The GH11 xylanase XynA from Streptomyces rameus L2001 has favorable hydrolytic properties. However, its poor thermal stability hinders its widespread application in industry. In this study, mutants Mut1 and Mut2 were constructed by rationally combining the mutations 11 YHDGYF 16 , 23 AP 24 / 23 SP 24 , and 32 GP 33 . The residual enzyme activity of these combinational mutants was more than 85% when incubated at 80 and 90 °C for 12 h, and thus are the most thermotolerant xylanases known to date. The reduced flexibility of the N-terminus, increased overall rigidity, as well as the surface net charge of Mut1 and Mut2 may be partially responsible for the improved thermal stability. In addition, the specific activity and catalytic efficiency of Mut1 and Mut2 were improved compared with those of wild-type XynA. The broader catalytic cleft and enhanced flexibility of the "thumb" of Mut1 and Mut2 may be partially responsible for the improved specific activity and catalytic efficiency.

Deciphering the Crucial Roles of the Quorum-Sensing Transcription Factor SdiA in NADPH Metabolism and (S)-Equol Production in Escherichia coli Nissle 1917

“…The molecular-docking findings were validated through an additional 100 ns of molecular-dynamics simulations. The root-mean-square deviation (RMSD), a pivotal parameter for assessing conformational fluctuations within the protein–ligand complex, inversely correlates with complex stability [ 46 ]. Notably, during the 60–100 ns period, the RMSD values of C10-HSL-SdiA_EcN exhibited lower values compared with C10-HSL-SdiA_K12, implying heightened thermal stability within the C10-HSL-SdiA_EcN complex ( Figure 5 B).…”

Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design

“…In fact, it was reported that a larger substrate-binding channel leads to higher enzyme activity . In a previous study, mutation in the GH11 xylanase XynA causes crack propagation near the catalytic pocket and results in enhanced enzyme activity . In summary, a more open catalytic cleft appeared to facilitate substrate access to the active pocket and led to a higher catalytic efficiency.…”

Combining Flexible Region Design and Automatic Design to Enhance the Thermal Stability and Catalytic Efficiency of Leucine Dehydrogenase

“…Subsequently, simulations of 100 ps each are performed in the NVT and NPT ensembles, followed by MD simulations for 50 ns at 300, 330, and 360 K, respectively. By analyzing atomic trajectories, data on root-mean-square deviation (RMSD), solvent-accessible surface area (SASA), root-mean-square fluctuation (RMSF), and radius of gyration (RG) are generated. , …”