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Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase.

ABSTRACT: BACKGROUND:A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30?L), combined with an additional disulfide bridge in the N-terminal region. RESULTS:The Tm value of mutant PTxA-DB was improved from 21.3?°C to 76.6?°C, and its half-life was found to be 53.6?min at 60?°C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the ?-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30?L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values. CONCLUSIONS:These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.

SUBMITTER: Xiong K

PROVIDER: S-EPMC6659274 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase.

Xiong Ke K Hou Jie J Jiang Yuefeng Y Li Xiuting X Teng Chao C Li Qin Q Fan Guangsen G Yang Ran R Zhang Chengnan C

BMC biotechnology 20190725 1

<h4>Background</h4>A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residue ...[more]

PMID: 31345213

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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

Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase.

Publications

Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase.

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