Dataset Information

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis.

ABSTRACT: Family 11 alkaline xylanases have great potential economic applications in the pulp and paper industry. In this study, we would improve the alkalophilicity of family 11 alkaline xylanase Xyn11A-LC from Bacillus sp. SN5, for the better application in this field.A random mutation library of Xyn11A-LC with about 10,000 clones was constructed by error-prone PCR. One mutant, M52-C10 (V116A and E135V), with improved alkalophilicity was obtained from the library. Site-directed mutation showed that the mutation E135V was responsible for the alkalophilicity of the mutant. The variant E135V shifted the optimum pH of the wild-type enzyme from 7.5 to 8.0. Compared to the relative activities of the wild type enzyme, those of the mutant E135V increased by 17.5, 18.9, 14.3 and 9.5 % at pH 8.5, 9.0, 9.5 and 10.0, respectively. Furthermore, Glu135 saturation mutagenesis showed that the only mutant to have better alkalophilicity than E135V was E135R. The optimal pH of the mutant E135R was 8.5, 1.0 pH units higher than that of the wild-type. In addition, compared to the wild-type enzyme, the mutations E135V and E135R increased the catalytic efficiency (k cat/K m) by 57 and 37 %, respectively. Structural analysis showed that the residue at position 135, located in the eight-residue loop on the protein surface, might improve the alkalophilicity and catalytic activity by the elimination of the negative charge and the formation of salt-bridge.Mutants E135V and E135R with improved alkalophilicity were obtained by directed evolution and site saturation mutagenesis. The residue at position 135 in the eight-residue loop on the protein surface was found to play an important role in the pH activity profile of family 11 xylanases. This study provided alkalophilic mutants for application in bleaching process, and it was also helpful to understand the alkaline adaptation mechanism of family 11 xylanases.

SUBMITTER: Bai W

PROVIDER: S-EPMC5101721 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis.

Bai Wenqin W Cao Yufan Y Liu Jun J Wang Qinhong Q Jia Zhenhu Z

BMC biotechnology 20161108 1

<h4>Background</h4>Family 11 alkaline xylanases have great potential economic applications in the pulp and paper industry. In this study, we would improve the alkalophilicity of family 11 alkaline xylanase Xyn11A-LC from Bacillus sp. SN5, for the better application in this field.<h4>Results</h4>A random mutation library of Xyn11A-LC with about 10,000 clones was constructed by error-prone PCR. One mutant, M52-C10 (V116A and E135V), with improved alkalophilicity was obtained from the library. Site ...[more]

PMID: 27825339

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Project description:BackgroundImproving the hydrolytic performance of hemicellulases to degrade lignocellulosic biomass is of considerable importance for second-generation biorefinery. Xylanase, as the crucial hemicellulase, must be thermostable and have high activity for its potential use in the bioethanol industry. To obtain excellent xylanase candidates, it is necessary to understand the structure-function relationships to provide a meaningful reference to improve the enzyme properties. This study aimed to investigate the catalytic mechanism of a highly active hyperthermophilic xylanase variant, XYL10C-ΔN, for hemicellulose degradation.ResultsBy removing the N-terminal 66 amino acids, the variant XYL10C-ΔN showed a 1.8-fold improvement in catalytic efficiency and could hydrolyze corn stover more efficiently in hydrolysis of corn stover; however, it retained similar thermostability to the wild-type XYL10C. Based on the crystal structures of XYL10C-ΔN and its complex with xylobiose, Glu175 located on loop 3 was found to be specific to GH10 xylanases and probably accounts for the excellent enzyme properties by interacting with Lys135 and Met137 on loop 2. Site-saturation mutagenesis confirmed that XYL10C-ΔN with glutamate acid at position 175 had the highest catalytic efficiency, specific activity, and the broadest pH-activity profile. The functional roles of Glu175 were also verified in the mutants of another two GH10 xylanases, XylE and XynE2, which showed increased catalytic efficiencies and wider pH-activity profiles.ConclusionsXYL10C-ΔN, with excellent thermostability, high catalytic efficiency, and great lignocellulose-degrading capability, is a valuable candidate xylanase for the biofuel industry. The mechanism underlying improved activity of XYN10C-ΔN was thus investigated through structural analysis and functional verification, and Glu175 was identified to play the key role in the improved catalytic efficiency. This study revealed the importance of a key residue (Glu175) in XYN10C-ΔN and provides a reference to modify GH10 xylanases for improved catalytic performance.

Project description:Significant progress has been made in isolating novel alkaline ?-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these ?-mannanases. We report the catalytic domain structure of an industrially important ?-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (?/?)(8)-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 ?-mannanases. It was revealed that BSP165 MAN and other subfamily 8 ?-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to ?-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline ?-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (?/?)(8)-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK(a) calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 ?-mannanases and will help to further understanding of alkaline adaptation mechanism.

Dataset Information

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis.

Publications

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis.

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