Enhanced thermostability of a mesophilic xylanase by<i>N</i>-terminal replacement designed by molecular dynamics simulation

Yin, Xin; Li, Jianfang; Wang, Junqing; Tang, Cun-Duo; Wu, Minchen

doi:10.1002/jsfa.6134

Cited by 32 publications

(16 citation statements)

References 30 publications

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“…Until now, rational design of improved GH11 xylanases have typically focused on three aspects: (1) replacement of the NTR with corresponding parts from thermostable enzymes [17–19]; (2) stabilization of α-helices by introducing disulfide bridges or electrostatic interactions [21, 31]; (3) modification of surface characteristics to form a tighter packing enzyme with fewer cavities [16, 32]. In this work, we found a dominant sequence 207-SSGS-210 at the C-terminus and replaced 207-NGGA-210 in XynCDBFV with this dominant sequence.…”

Section: Discussionmentioning

confidence: 99%

“…These advantageous properties make GH11 more suited for industrial applications. Thus, the determinants for the improved properties of GH11 have been widely explored [13–17]. Among them, understanding the heat-resistance mechanism for GH11 has become an intense research area owing to the high-temperature requirement in various industrial conditions.…”

Section: Introductionmentioning

confidence: 99%

“…It has been widely accepted that N-terminal region (NTR) of GH11 is of great importance in maintaining xylanase thermostability, and several heat-resistant recombinants have been developed by replacing the NTR with the corresponding parts from thermostable xylanases [17–19]. In 2014, the Rey-Ting Guo group solved the crystal structure of a GH11 xylanase (XynCDBFV) from the ruminal fungus Neocallimastix patriciarum [20].…”

Section: Introductionmentioning

confidence: 99%

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Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis

Han

Miao

Ding

et al. 2017

Biotechnol Biofuels

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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 (K m, 0.22 and 0.59 µM, respectively). Catalytic efficiency values (k cat /K m) 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0824-y) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis

Han

Miao

Ding

et al. 2017

Biotechnol Biofuels

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“…The 3D structure of the xylanases were viewed and analyzed by DS ViewerPro6.0. The disulfide bridge was predicted by the DiANNA 1.1 web server (http://bioinformatics.bc.edu/clotelab/DiANNA/) (Yin et al, 2013b). The active center of the xylanases was predicted by PROSITE (http://prosite.expasy.org) (Jeong et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Exploration of a N-terminal disulfide bridge to improve the thermostability of a GH11 xylanase from <i>Aspergillus niger</i>

Zhou

Wang

et al. 2016

J. Gen. Appl. Microbiol.

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“…a) Curves of RMSD values of SyXyn11 (1) and SyXyn11 C5T (2) after MD simulation at 500 K for 10 ns. b) The distributions of RMSD values of SyXyn11 (1) and SyXyn11 C5T(2).…”

mentioning

confidence: 99%

A unique disulfide bridge of the thermophilic xylanase syxyn11 plays a key role in its thermostability

Yin

Yao

et al. 2014

Biochemistry Moscow

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Based on the hyperthermostable family 11 xylanase (EvXyn11(TS)) gene sequence (EU591743), the gene Syxyn11 encoding a thermophilic xylanase SyXyn11 was synthesized with synonymous codons biasing towards Pichia pastoris. The homology alignment of primary structures among family 11 xylanases revealed that, at their N-termini, only SyXyn11 contains a disulfide bridge (Cys5-Cys32). This to some extent implied the significance of the disulfide bridge of SyXyn11 to its thermostability. To confirm the correlation between the N-terminal disulfide bridge and thermostability, a SyXyn11(C5T)-encoding gene, Syxyn11(C5T), was constructed by mutating the Cys5 codon of Syxyn11 to Thr5. Then, the genes for the recombinant xylanases, reSyXyn11 and reSyXyn11(C5T), were expressed in P. pastoris GS115, yielding xylanase activity of about 35 U per ml cell culture. Both xylanases were purified to homogeneity with specific activities of 363 and 344 U/mg, respectively. The temperature optimum and stability of reSyXyn11(C5T) decreased to 70 and 50°C from 85 and 80°C of reSyXyn11, respectively. There was no obvious change in pH characteristics.

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Enhanced thermostability of a mesophilic xylanase byN-terminal replacement designed by molecular dynamics simulation

Abstract: This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn11⁵⁷ a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes.

Cited by 32 publications

References 30 publications

Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis

Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis

Exploration of a N-terminal disulfide bridge to improve the thermostability of a GH11 xylanase from <i>Aspergillus niger</i>

A unique disulfide bridge of the thermophilic xylanase syxyn11 plays a key role in its thermostability

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