Structural insights into the acidophilic pH adaptation of a novel endo-1,4-β-xylanase from Scytalidium acidophilum

Michaux, Catherine; Pouyez, Jenny; Mayard, Aurélie; Vandurm, Pierre; Housen, Isabelle

doi:10.1016/j.biochi.2010.07.003

Cited by 31 publications

(14 citation statements)

References 56 publications

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“…4, the optimum pH of mutant G116C-Q120C shifted from 9.5 to 10.0, and the stable pH range of mutant P35C-G426C extended from 7.0 to 11.0 (wild type) to 6.0 to 12.0. Previous studies have shown that salt bridges close to catalytic groups play critical roles in determining the optimum pH of enzymes (28,29). In this study, the new salt bridge around the active sites in mutant G116C-Q120C (Fig.…”

Section: Figmentioning

confidence: 65%

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Liu

Deng

Yang

et al. 2014

Appl Environ Microbiol

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High thermostability is required for alkaline ␣-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline ␣-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants-P35C-G426C, G116C-Q120C, and R436C-M480C-of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (k cat /K m ) increased from 1.8 ؋ 10 4 to 2.4 ؋ 10 4 liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

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Section: Figmentioning

confidence: 65%

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Liu

Deng

Yang

et al. 2014

Appl Environ Microbiol

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“…Usually, the enzyme optimal pH was affected by the protein ionizable groups [ 55 ] and surface charge [ 56 ]. Previous researches showed that acidophilic enzyme tended to have more acidic surface [ 57 , 58 ]. Therefore, the more negative charge in the P102C-N125C region in the double mutant might be the reason for the shift of optimal pH.…”

Section: Discussionmentioning

confidence: 99%

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis

Niu

Zhu

et al. 2016

PLoS ONE

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1,3–1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C) were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm) compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

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“…Three of them are in an equivalent position that connects the cord and strand ␤12, including the xylanases from Aspergillus kawachii (36), Aspergillus niger (37), and Scytalidium acidophilum (38). For xylanases from Paecilomyces varioti Bainier (30) and Thermomyces lanuginosus (12), the disulfide bond is located between helix ␣4 and strand ␤11.…”

Section: Discussionmentioning

confidence: 99%

Structural Analysis of a Glycoside Hydrolase Family 11 Xylanase from Neocallimastix patriciarum

Cheng

Chen

Huang

et al. 2014

Journal of Biological Chemistry

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Background: Thermophilic xylanases are valuable in many industrial applications. Results: The structures of a xylanase XynCDBFV and its complex with xylooligosaccharides were determined, and its N-terminal region (NTR) contributes to thermostability. Conclusion: NTR may stabilize the overall protein folding of XynCDBFV. Significance: The structural and functional investigation of unprecedented NTR of XynCDBFV provides a new insight into the molecular basis of thermophilic xylanases.

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Structural insights into the acidophilic pH adaptation of a novel endo-1,4-β-xylanase from Scytalidium acidophilum

Cited by 31 publications

References 56 publications

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis

Structural Analysis of a Glycoside Hydrolase Family 11 Xylanase from Neocallimastix patriciarum

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