Xylanase XYL1p from Scytalidium acidophilum: Site-directed mutagenesis and acidophilic adaptation

Balaa, B. Al; Brijs, Kristof; Gebruers, Kurt; Vandenhaute, Jean; Housen, Isabelle

doi:10.1016/j.biortech.2009.06.111

Cited by 22 publications

(10 citation statements)

References 33 publications

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“…Mutating a glycine residue to cysteine in the interior hydrophobic region (You et al 2010) or introduction of arginines by substituting serine and threonine residues on the surface (Sriprang et al 2006) improved the enzyme's thermostability. Comparison of the sequences and structures between the GH11 xylanases with different pH optimum has also allowed the identification of specific residues which is important to the pH stability (Al Balaa et al 2009;De Lemos Esteves et al 2005).…”

Section: Enzyme-substrate Interactions In the Mutantsmentioning

confidence: 99%

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

Cheng

Huang³

et al. 2011

Appl Microbiol Biotechnol

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Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable β-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k (cat) and K (m) values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.

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Section: Enzyme-substrate Interactions In the Mutantsmentioning

confidence: 99%

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

Cheng

Huang³

et al. 2011

Appl Microbiol Biotechnol

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“…The mutations E118A and E118Q of xylanase from Aspergillus niger for the same key position increased the activity by 50 and 16 %, respectively [25]. The same result was shown in XYL1p from S. acidophilum where E141A mutation elevated the specific activity by 50 % [22]. Furthermore, the importance of this residue in the catalytic activity of family 11 xylanases was confirmed by the inverse mutation, the mutation A139E in Xyl1 of Streptomyces sp.…”

Section: Discussionmentioning

confidence: 75%

“…Other studies also proved that the substitution of the residue in other xylanases corresponding to Glu 135 in xylanase Xyn11A-LC could also change the pH activity profile of enzymes. The E141A mutation in XYL1p from Scytalidium acidophilum increased the optimal pH from 3.2 to 4.0 [22]. The E141Q mutation in XynI from Aureobasidium pullulans changed the optimal pH from 2.5 to 3.5 [23].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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BackgroundFamily 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.ResultsA 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.ConclusionsMutants 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0310-9) contains supplementary material, which is available to authorized users.

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“…However, there are others with biotechnological applications such as S. thermophilum, that has been considered as a potential candidate in the biostoning of denim garments, since appreciable amounts of cellulases are produced along with xylanases (Kaur et al, 2006). Another potential species of Scytalidium is S. acidophilum, which grows under extremely acidic and high-temperature conditions, being able to secrete enzymes for processes that require extreme low pH's and/or high temperature (Balaa et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

A NOVEL Scytalidium SPECIES: UNDERSTAND THE CELLULOLYTIC SYSTEM FOR BIOMASS SACCHARIFICATION

Delabona

Silva

Paixão

et al. 2019

Braz. J. Chem. Eng.

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In order to overcome the bottlenecks related to lignocellulosic-derived sugars, the search for more efficient enzymatic cocktails, containing a broad-spectrum of specific activities, relies on an important feature. This paper describes new enzyme activities derived from the novel strain of the Scytalidium genus isolated from the Amazonas rainforest. The production of the enzymatic cocktail was induced by delignifiedhydrothermal bagasse (DHB), and yeast extract was used to improve secretion activities, resulting in a positive influence on total cellulase activity. The enzymatic cocktail produced by this novel strain contains specific activities for biomass degradation, including FPAse, xylanase and β-glucosidase. Moreover, it is capable of hydrolyzing 62% of the alkaline pretreated bagasse, surpassing in 14% the hydrolytic capability achieved by the commercial cocktail Celluclast. To this extent, the strain described here emerges as a reliable alternative to other available enzymes and, consequently, amplification of available specific substrate activities.

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Xylanase XYL1p from Scytalidium acidophilum: Site-directed mutagenesis and acidophilic adaptation

Cited by 22 publications

References 33 publications

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

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

A NOVEL Scytalidium SPECIES: UNDERSTAND THE CELLULOLYTIC SYSTEM FOR BIOMASS SACCHARIFICATION

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