Biochemical Characterization of Two Thermostable Xylanolytic Enzymes Encoded by a Gene Cluster of Caldicellulosiruptor owensensis

Mi, Shuofu; Jia, Xiuyi; Wang, Jinzhi; Qiao, Weibo; Peng, Xiaowei; Han, Young-Tak

doi:10.1371/journal.pone.0105264

Cited by 27 publications

(18 citation statements)

References 40 publications

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“…do not have an extended C-terminus. However, biophysical data showed that GH39 CoXylA (from Caldicellulosiruptor owensensis) forms dimers in solution (Mi et al, 2014), yet supporting oligomerization as an adaptive strategy for extreme environments. It is likely that in the genus Caldicellulosiruptor other molecular mechanisms were evolved for oligomerization and gain of stability; however, the lack of high-resolution structural data does not currently allow us to precisely predict how it occurs.…”

Section: Discussionmentioning

confidence: 99%

Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 β-Xylosidases

Morais

Polo

Domingues

et al. 2020

Front. Bioeng. Biotechnol.

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The glycoside hydrolase family 39 (GH39) is a functionally expanding family with limited understanding about the molecular basis for substrate specificity and extremophilicity. In this work, we demonstrate the key role of the positive-subsite region in modulating substrate affinity and how the lack of a C-terminal extension impacts on oligomerization and structural stability of some GH39 members. The crystallographic and SAXS structures of a new GH39 member from the phytopathogen Xanthomonas citri support the importance of an extended C-terminal to promote oligomerization as a molecular strategy to enhance thermal stability. Comparative structural analysis along with sitedirected mutagenesis showed that two residues located at the positive-subsite region, Lys166 and Asp167, are critical to substrate affinity and catalytic performance, by inducing local changes in the active site for substrate binding. These findings expand the molecular understanding of the mechanisms involved in substrate recognition and structural stability of the GH39 family, which might be instrumental for biological insights, rational enzyme engineering and utilization in biorefineries.

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Section: Discussionmentioning

confidence: 99%

Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 β-Xylosidases

Morais

Polo

Domingues

et al. 2020

Front. Bioeng. Biotechnol.

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“…strain 41M-1 and Xyn10B (Protein Data Bank entry 2W5F; sequence identity, 29%) from Clostridium thermocellum, respectively, as the template. Quaternary structures of XynA and XynB were investigated using size exclusion chromatography with a Superdex 200 column (36,37). Blue dextran 2000 (2,000 kDa), ferritin (400 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) (all obtained from Sigma-Aldrich) were used as reference proteins.…”

Section: Methodsmentioning

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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“…Thermophilic microorganisms are attractive candidates for biomass conversion and overcome a way to break of feedstock process-operating challenges (Mi et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13

Yadav

Maharjan

Korpole

et al. 2018

Front. Bioeng. Biotechnol.

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Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6–9) and temperature (30–65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 μM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

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Biochemical Characterization of Two Thermostable Xylanolytic Enzymes Encoded by a Gene Cluster of Caldicellulosiruptor owensensis

Cited by 27 publications

References 40 publications

Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 β-Xylosidases

Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 β-Xylosidases

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

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13

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