Non-Structured Amino-Acid Impact on GH11 Differs from GH10 Xylanase

Liu, Liangwei; Sun, Xiaofeng; Yan, Ping; Wang, Limin; Chen, Hongge

doi:10.1371/journal.pone.0045762

Cited by 12 publications

(12 citation statements)

References 24 publications

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“…Usually thermophilic GH11 xylanases possess a β-strand in the N-terminal region, while mesophilic GH11 xylanases have a long irregular loop at the same position [30]. It is more likely that the reason PjxA was unstable at elevated temperature is the looseness of the irregular loop.…”

Section: Discussionmentioning

confidence: 99%

Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase

et al. 2019

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Background: A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30 L), combined with an additional disulfide bridge in the N-terminal region. Results: The T m value of mutant PTxA-DB was improved from 21.3°C to 76.6°C, and its half-life was found to be 53.6 min at 60°C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the β-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30 L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values. Conclusions: These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.

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Section: Discussionmentioning

confidence: 99%

Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase

et al. 2019

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“…Previous studies have reported that terminal amino acid residues are crucial for the entire enzyme (42,54). Liu et al have reported that disordered terminal amino acid deletions increased regular secondary structural contents, which led to slow decreases in Gibbs free energy in the thermal denaturation process and ultimately enhanced enzyme thermostability (55).…”

Section: Discussionmentioning

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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“…In addition, terminal sequences also influence enzymes in different ways (23). For example, the C-terminal region of Trichoderma reesei endo-1,4-xylanase changed its half-life (24), deletion of the N-and C-terminal sequences of Aspergillus niger xylanase resulted in a lower temperature optimum and decreased thermostability (25), and disruption of the terminal interaction of a GH10 xylanase resulted in decreased stability (26). The reason might be that removal of these terminal sequences changed the secondary structure contents, aromatic cluster formation, in vivo folding, and Gibbs free energy.…”

Section: Discussionmentioning

confidence: 99%

A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases

Xue

Zhao

et al. 2014

Appl Environ Microbiol

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c Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more ␣ helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

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Non-Structured Amino-Acid Impact on GH11 Differs from GH10 Xylanase

Cited by 12 publications

References 24 publications

Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase

Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase

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

A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases

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