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Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2.

ABSTRACT: Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure's stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.

SUBMITTER: Miao H

PROVIDER: S-EPMC8745443 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundXylanases have been widely employed in many industrial processes, and thermophilic xylanases are in great demand for meeting the high-temperature requirements of biotechnological treatments. In this work, we aim to improve the thermostability of XynCDBFV, a glycoside hydrolase (GH) family 11 xylanase from the ruminal fungus Neocallimastix patriciarum, by site-directed mutagenesis. We report favorable mutations at the C-terminus from B-factor comparison and multiple sequence alignment.ResultsC-terminal residues 207-NGGA-210 in XynCDBFV were discovered to exhibit pronounced flexibility based on comparison of normalized B-factors. Multiple sequence alignment revealed that beneficial residues 207-SSGS-210 are highly conserved in GH11 xylanases. Thus, a recombinant xylanase, Xyn-MUT, was constructed by substituting three residues (N207S, G208S, A210S) at the C-terminus of XynCDBFV. Xyn-MUT exhibited higher thermostability than XynCDBFV at ≥70 °C. Xyn-MUT showed promising improvement in residual activity with a thermal retention of 14% compared to that of XynCDBFV after 1 h incubation at 80 °C; Xyn-MUT maintained around 50% of the maximal activity after incubation at 95 °C for 1 h. Kinetic measurements showed that the recombinant Xyn-MUT had greater kinetic efficiency than XynCDBFV (Km, 0.22 and 0.59 µM, respectively). Catalytic efficiency values (kcat/Km) of Xyn-MUT also increased (1.64-fold) compared to that of XynCDBFV. Molecular dynamics simulations were performed to explore the improved catalytic efficiency and thermostability: (1) the substrate-binding cleft of Xyn-MUT prefers to open to a larger extent to allow substrate access to the active site residues, and (2) hydrogen bond pairs S208-N205 and S210-A55 in Xyn-MUT contribute significantly to the improved thermostability. In addition, three xylanases with single point mutations were tested, and temperature assays verified that the substituted residues S208 and S210 give rise to the improved thermostability.ConclusionsThis is the first report for GH11 recombinant with improved thermostability based on C-terminus replacement. The resulting Xyn-MUT will be an attractive candidate for industrial applications.

Dataset Information

Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2.

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