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

Cheng, Ya‐Shan; Chen, Chun‐Chi; Huang, Chun‐Hsiang; Ko, Tzu‐Ping; Luo, Wenjun; Huang, Jian‐Wen; Liu, Je-Ruei; Guo, Rey‐Ting

doi:10.1074/jbc.m114.550905

Cited by 69 publications

(43 citation statements)

References 48 publications

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“…The difference in thermostability between free and GL-1-immoblilized Xyl2 and multipoint immobilized Xyl2 (AM-1/GLY-3) amounted to as much as 18 °C. These T50 % values compare well with the optimal temperatures measured for thermophilic xylanases; among them are the enzymes from bacteria such as Dictyoglomus thermophilum XynB (optimal temperature 75 °C) [60], Thermobifida fusca NTU22 Xyl11 (70 °C) [61], and Thermopolyspora flexuosa XynA (80 °C) [62], and fungal xylanases from Neocallimastix patriciarum XynCDBFV (optimal temperature 65 °C) [63] and Chaetomium thermophilum Xyn11A (optimal temperature 80 °C) [48]. Therefore, these comparisons qualify Xyl2 and, especially, AM-1 and GLY-3 immobilized Xyl2, as thermophilic/thermostable enzyme preparations.…”

Section: Resultssupporting

confidence: 53%

Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst

Gómez

Payne

Savko

et al. 2016

Biotechnol Biofuels

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BackgroundReplacing fossil fuel with renewable sources such as lignocellulosic biomass is currently a promising alternative for obtaining biofuel and for fighting against the consequences of climate change. However, the recalcitrant structure of lignocellulosic biomass residues constitutes a major limitation for its widespread use in industry. The efficient hydrolysis of lignocellulosic materials requires the complementary action of multiple enzymes including xylanases and β-xylosidases, which are responsible for cleaving exo- and endoxylan linkages, that release oligocarbohydrates that can be further processed by other enzymes.ResultsWe have identified the endo-β-1,4-xylanase Xyl2 from Fusarium oxysporum as a promising glycoside hydrolase family 11 enzyme for the industrial degradation of xylan. To characterize Xyl2, we have cloned the synthetic optimized gene and expressed and purified recombinant Xyl2 to homogeneity, finally obtaining 10 mg pure Xyl2 per liter of culture. The crystal structure of Xyl2 at 1.56 Å resolution and the structure of a methyl-xylopyranoside Xyl2 complex at 2.84 Å resolution cast a highly detailed view of the active site of the enzyme, revealing the molecular basis for the high catalytic efficiency of Xyl2. The kinetic analysis of Xyl2 demonstrates high xylanase activity and non-negligible β-xylosidase activity under a variety of experimental conditions including alkaline pH and elevated temperature. Immobilizing Xyl2 on a variety of solid supports enhances the enzymatic properties that render Xyl2 a promising industrial biocatalyst, which, together with the detailed structural data, may establish Xyl2 as a platform for future developments of industrially relevant xylanases.Conclusions F. oxysporum Xyl2 is a GH11 xylanase which is highly active in free form and immobilized onto a variety of solid supports in a wide pH range. Furthermore, immobilization of Xyl2 on certain supports significantly increases its thermal stability. A mechanistic rationale for Xyl2's remarkable catalytic efficiency at alkaline pH is proposed on the basis of two crystallographic structures. Together, these properties render Xyl2 an attractive biocatalyst for the sustainable industrial degradation of xylan.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0605-z) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 53%

Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst

Gómez

Payne

Savko

et al. 2016

Biotechnol Biofuels

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“…Xylanases from fungi or mesophilic bacteria generally share a low optimum temperature and poor thermal stability (5). Thermostable xylanases have been characterized from some thermophilic microorganisms, such as Clostridium thermocellum (6), Rhodothermus marinus (7), Thermotoga species (8), Sulfolobus solfataricus (9), Neocallimastix patriciarum (10), and Thermococcus zilligii (11), which exhibit increased specific activity and higher stability.…”

mentioning

confidence: 99%

Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

Meng

Chen

et al. 2015

Appl Environ Microbiol

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cXylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol ؊1 versus 94.7 U nmol ؊1 ) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (T m ), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions. X ylanases (endo-1,4-␤-xylanase; EC 3.2.1.8) hydrolyze the ␤-1,4 bond in the xylan backbone of hemicellulose, which yields short xylooligosaccharides or xylose. They are distributed in several glycoside hydrolase families (GH), such as GH5, GH7, GH8, GH10, GH11, and GH43, and most of them belong to GH10 and GH11 according to the CAZy (Carbohydrate Active Enzymes) database (1). Xylanase families differ in their physicochemical properties (molecular mass and isoelectric point [pI]), three-dimensional structures, and catalytic mechanisms (2). Enzymes from GH family 11 have a relatively low molecular weight and a high pI, and they possess a ␤-jelly roll secondary structure, a double displacement catalytic mechanism, and two glutamates acting as catalytic residues (3). In contrast, members of GH10 typically have a high molecular mass and a low pI and display an (␣/␤) 8 barrel fold. Xylanases have been found in many organisms, such as bacteria, fungi, algae, and protozoa (4). Xylanases from fungi or mesophilic bacteria generally share a low optimum temperature and poor thermal stability (5). Thermostable xylanases have been characterized from some thermophilic microorganisms, such as Clostridium thermocellum (6...

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“…The Cys40–Cys67 bond located near the N‐terminal region may more significantly contribute to the stabilization of LC‐CelA than Cys100–Cys105, because the number of the residues in the loop formed by Cys40–Cys67 is higher than that formed by Cys100–Cys105. It has been reported for GH family 11 xylanase that a unique N‐terminal 11‐residue extension contributes to protein stability by forming hydrogen bonds with the core region [44]. It has also been reported for this enzyme that mutations in the N‐terminal region [45] and engineering of an N‐terminal disulfide bond [46–49] stabilize the protein.…”

Section: Resultsmentioning

confidence: 96%

Structure and stability of metagenome‐derived glycoside hydrolase family 12 cellulase (LC‐CelA) a homolog of Cel12A from Rhodothermus marinus

et al. 2014

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

Cited by 69 publications

References 48 publications

Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst

Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst

Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

Structure and stability of metagenome‐derived glycoside hydrolase family 12 cellulase (LC‐CelA) a homolog of Cel12A from Rhodothermus marinus

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