Terminal Amino Acids Disturb Xylanase Thermostability and Activity

Liu, Liangwei; Zhang, Guoqiang; Zhang, Zhang; Wang, Suya; Chen, Hongge

doi:10.1074/jbc.m111.269753

Cited by 59 publications

(45 citation statements)

References 41 publications

(47 reference statements)

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“…Previous reports indicated that N-terminal region and hydrogen bond network of xylanase played crucial roles in its thermo-stability8915. GH11 xylanases of thermophilic origin generally have longer N-terminal region compared to mesophilic ones, indicating its role on enhancing the thermo-stability3940. Several attempts have been made to enhance the thermo-stability of xylanase by substituting the N-terminal region of various non-thermostable xylanases with those of thermostable xylanases8.…”

Section: Discussionmentioning

confidence: 99%

High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

Fang

Wang

et al. 2016

Sci Rep

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In paper industry, xylanases are used to increase the pulp properties in bleaching process as its eco-friendly nature. The xylanases activity is hindered by high temperature and alkaline conditions with high enzyme production cost in the paper industry. Here, XynHB, an alkaline stable xylanase from Bacillus pumilus HBP8 was mutated at N188A to XynHBN188A. Expressed mutant in E. coli showed 1.5-fold higher xylanase activity than XynHB at 60 °C. The mutant expressed in Pichia pastoris was glycosylated, remained stable for 30 min at 60 °C. XynHBN188A optimized based on codon usage bias for P. pastoris (xynHBN188As) showed an increase of 39.5% enzyme activity. The strain Y16 forming the largest hydrolysis halo in the xylan plate was used in shake flask experiments produced an enzyme activity of 6,403 U/ml. The Y16 strain had 9 copies of the recombinant xynHBN188As gene in the genome revealed by qPCR. The enzymatic activity increased to 48,241 U/ml in a 5 L fermentor. Supplement of 15 U/g xylanase enhanced the brightness of paper products by 2% in bleaching experiment, and thereby improved the tensile strength and burst factor by 13% and 6.5%, respectively. XynHBN188As has a great potential in paper industries.

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Section: Discussionmentioning

confidence: 99%

High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

Fang

Wang

et al. 2016

Sci Rep

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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). In the present study, circular dichroism (CD) spectrum data showed that the substitution of amino acids at the N terminus of XynA and XynATM1 did not cause secondary structure changes in the proteins (data not shown).…”

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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“…This study showed that the deletion of N- and C-terminal (XynΔNC) residues had an opposing effect on Xyn T opt, but had an additive effect on t 1/2 . The analysis of terminal DRs using a model structure of Xyn showed that the new N- and C-terminals can come closer to each other after the deletion of five N-terminal DRs and one C-terminal DR, suggesting that this close contact between the N- and C-terminal can provide additional compactness to the structure thereby making the deletion mutants more stable than native Xyn (88). More recently, the role of N- and C-terminal contact in the family 10 xylanase BSX was investigated through site-directed mutagenesis to assess protein folding and stability of these mutants under more than one extreme condition (12, 13).…”

Section: N- and C-terminal Contacts And Protein Stabilitymentioning

confidence: 99%

EMERGING ROLE OF N- AND C-Terminal INTERACTIONS IN STABILIZING (Β;/Α) 8 FOLD WITH SPECIAL EMPHASIS ON FAMILY 10 XYLANASES

Bhardwaj

Mahanta

Ramakumar

et al. 2012

Computational and Structural Biotechnology Journal

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Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10) xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s) under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

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Terminal Amino Acids Disturb Xylanase Thermostability and Activity

Cited by 59 publications

References 41 publications

High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

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

EMERGING ROLE OF N- AND C-Terminal INTERACTIONS IN STABILIZING (Β;/Α) 8 FOLD WITH SPECIAL EMPHASIS ON FAMILY 10 XYLANASES

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