Thermostable Glycoside Hydrolase Research Articles

Unveiling the genomic potential of a novel thermostable glycoside hydrolases producing Neobacillus sedimentimangrovi UE25.

Genetic and enzymatic potential of Neobacillus sedimentimangrovi has not been assembled to date. Here, we report a high-quality genome assembly of thermophilic bacterium Neobacillus sedimentimangrovi UE25 using Illumina Hi-seq 2500. The strain was isolated from a crocodile pond Manghopir, Karachi, Pakistan. QUAST quality parameters showed 37.75% GC content and exhibited the genome into 110 contigs, with a total size of 3,230,777 bases. Genome of N. sedimentimangrovi UE25 harbors phage mediated DNA through horizontal gene exchange from the phages, symbiotic and pathogenic bacteria. Most of the phage genome encodes for hypothetical proteins, protease, and phage assembly proteins. Gene clusters encoding the intrinsic resistance to glycopeptides, isoniazid, rifamycin, elfamycin, macrolide, aminoglycosides, tetracycline and fluoroquinolone were identified into the genome. Since, the strain has been reported for the production of many industrially important thermostable enzymes, therefore, the genomic data of thermostable enzymes might be helpful to employ this species in commercial sectors. Probing genes of multiple thermostable glycoside hydrolase enzymes especially xylanases of N. sedimentimangrovi UE25 showed genetic diversity among the genes and confer the industrial importance of this microorganism. Furthermore, the genome of N. sedimentimangrovi will greatly improve our understanding of its genetics and evolution.

Heterologous expression and characterization of glycoside hydrolase with its potential applications in hyperthermic environment

With the progressive focus on renewable energy via biofuels production from lignocellulosic biomass, cellulases are the key enzymes that play a fundamental role in this regard. This study aims to unravel the characteristics of Thermotoga maritima MSB8 (Tma) (a hyperthermophile from hot springs) thermostable glycoside hydrolase enzyme. Here, a glycoside hydrolase gene of Thermotoga maritima (Tma) was heterologously expressed and characterized. The gene was placed in the pQE-30 expression vector under the T5 promotor, and the construct pQE-30-Gh was then successfully integrated into Escherichia coli BL21 (DH5α) genome by transformation. Sequence of the glycoside hydrolase contained an open reading frame of 2.124 kbp, encoded a polypeptide of 721 amino acid residues. The molecular weight of the recombinant protein estimated was 79 kDa. The glycoside hydrolase was purified by Ni+2-NTA affinity chromatography and its enzymatic activity was investigated. The recombinant enzyme is highly stable within an extreme pH range (2.0–7.0) and highly thermostable at 80 °C for 72 h indicating its viability in hyperthermic environment and acidic nature. Moreover, the Ca2+ and Mn2+ introduction stimulated the residual activity of recombinant enzyme. Conclusively, the thermostable glycoside hydrolase possesses potential to be exploited for industrial applications at hyperthermic environment.

Microbial diversity of thermophiles with biomass deconstruction potential in a foliage-rich hot spring.

The ability of thermophilic microorganisms and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability, and decreased risk of contamination. Exploitation of efficient thermostable glycoside hydrolases (GHs) could accelerate the industrialization of biofuels and biochemicals. However, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y‐shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y‐shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus, Desulfurobacterium, Fervidobacterium, Geobacillus, Meiothermus, Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes.

Molecular Modelling of a Thermostable Glycoside Hydrolase from <i>Caldivirga </i><i>m</i><i>aquilingensis</i> and Its Substrate Docking Mechanism for Galactooligosaccharides Synthesis

Glycoside hydrolases (GHs) are very important enzymes that can catalyze the breakdown of the glycosidic bonds between carbohydrates and non-carbohydrates to synthesize GOS prebiotic sugars and hydrolyze lactose in the dairy industry. GOS can stimulate the growth of gut microbiota to have beneficial health effects in the host. The current work investigated molecular modelling and lactose substrate docking of a thermostable GH family 1 from Caldivirga maquilingensis strain IC-167 and determined its mechanism for GOS synthesis. The 3D model structure obtained from the Swiss model analysis tool revealed that GH 1 from Caldivirga maquilingensis was a tetramer with a catalytic pocket at the center of each monomer. The overall quality of the model was 93%. When lactose was docked in the catalytic site using AutoDock Vina software, the catalytic amino acid residues were identified to be Glu 387 and Glu 209 which acted as nucleophile and acid/base residues respectively. Other amino acid residues like His 153, Gln 19, Glu 432, Ala 266, Asn 267, Ser 268 and Trp 433 were also found to be surrounding the catalytic site and playing an essential role in ligand binding and recognition of the lactose substrate for GOS synthesis. These findings offer an understanding of how enzyme protein structure determines catalytic specificity, which serves as new knowledge basis to engineer GH 1 from C. maquilingensis for the biosynthesis of GOS with a broad or narrow degree of polymerization range.

Extraction of Glucuronoarabinoxylan from Quinoa Stalks (Chenopodium quinoa Willd.) and Evaluation of Xylooligosaccharides Produced by GH10 and GH11 Xylanases

Byproducts from quinoa are not yet well explored sources of hemicellulose or products thereof. In this work, xylan from milled quinoa stalks was retrieved to 66% recovery by akaline extraction using 0.5 M NaOH at 80 °C, followed by ethanol precipitation. The isolated polymer eluted as a single peak in size-exclusion chromatography with a molecular weight of >700 kDa. Analysis by Fourier transform infrared spectroscopy and nuclear magnetic resonance (NMR) combined with acid hydrolysis to monomers showed that the polymer was built of a backbone of β(1 → 4)-linked xylose residues that were substituted by 4-O-methylglucuronic acids, arabinose, and galactose in an approximate molar ratio of 114:23:5:1. NMR analysis also indicated the presence of α(1 → 5)-linked arabinose substituents in dimeric or oligomeric forms. The main xylooligosaccharides (XOs) produced after hydrolysis of the extracted glucuronoarabinoxylan polymer by thermostable glycoside hydrolases (GHs) from families 10 and 11 were xylobiose and xylotriose, followed by peaks of putative substituted XOs. Quantification of the unsubstituted XOs using standards showed that the highest yield from the soluble glucuronoarabinoxylan fraction was 1.26 g/100 g of xylan fraction, only slightly higher than the yield (1.00 g/100 g of xylan fraction) from the insoluble fraction (p < 0.05). No difference in yield was found between reactions in buffer or water (p > 0.05). This study shows that quinoa stalks represent a novel source of glucuronoarabinoxylan, with a substituent structure that allowed for limited production of XOs by GH10 or GH11 enzymes.

Lactulose production by a thermostable glycoside hydrolase from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167.

Lactulose has various uses in the food and pharmaceutical fields. Thermostable enzymes have many advantages for industrial exploitation, including high substrate solubilities as well as reduced risk of process contamination. Enzymatic synthesis of lactulose employing a transgalactosylation reaction by a recombinant thermostable glycoside hydrolase (GH1) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was investigated. The optimal pH for lactulose production was found to be 4.5, while the optimal temperature was 85 °C, before it dropped moderately to 83% at 90 °C. However, the relative activity for lactulose synthesis dropped sharply to 35% at 95 °C. At optimal reaction conditions of 70% (w/w) initial sugar substrates with molar ratio of lactose to fructose of 1:4, 15 U mL-1 enzyme concentration and 85 °C, the time course reaction produced a maximum lactulose concentration of 108 g L-1 at 4 h, corresponding to a lactulose yield of 14% and 27 g L-1 h-1 productivity with 84% lactose conversion. The transgalactosylation reaction for lactulose synthesis was greatly influenced by the ratio of galactose donor to acceptor. This novel GH1 may be useful for process applications owing to its high activity in very concentrated substrate reaction media and promising thermostability. © 2017 Society of Chemical Industry.

Characterization of a thermostable glycoside hydrolase family 36 α-galactosidase from Caldicellulosiruptor bescii

The putative gene cluster involved in the degradation of the raffinose family oligosaccharides (RFO) was identified in Caldicellulosiruptor bescii. Within the cluster, the gene encoding a putative α-galactosidase (CbAga36) was cloned and expressed in Escherichia coli. Size exclusion chromatography of the purified rCbAga36 indicated that the native form was a tetramer. Its primary sequence was similar to the family of glycoside hydrolase 36. The purified recombinant CbAga36 (rCbAga36) was optimally active at pH 5.0 and 70°C and had a half-life of 15h and 10h at 70°C and 80°C, respectively. rCbAga36 showed high activity with the artificial substrate (p-nitrophenyl α-d-galactopyranoside, pNPαGal) exhibiting lower Km and higher kcat than natural substrates such as melibiose and raffinose. Although rCbAga36 demonstrated preferential activity toward the hydrolysis of RFO such as raffinose and stachyose, it did not degrade the polymeric galactomannans. Our results imply that CbAga36 may play a role in the degradation of RFO, transported into the cytoplasm via a transporter into galactose, which is further utilized as an energy source in C.bescii. Furthermore, its ability to synthesize novel oligosaccharides by transglycosylation renders this enzyme potentially useful for the production of dietary oligosaccharides with novel function.

Characterization and crystal structure of a thermostable glycoside hydrolase family 45 1,4-β-endoglucanase from Thielavia terrestris.

1,4-β-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris. The recombinant protein shows optimal activity at 60°C, pH 4-5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80°C for 2.5h. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1.36-1.58Å. The protein folds into two overall regions: one is a six-stranded β-barrel, and the other one consists of several extended loops. Between the two regions lies the substrate-binding channel, which is an open cleft spanning across the protein surface. A continuous substrate-binding cleft from subsite -4 to +3 were clearly identified in the complex structures. Notably, the flexible V-VI loop (113Gly-114Gly-115Asp-116Leu-117Gly-118Ser) is found to open in the presence of -1 sugar, with D115 and L116 swung away to yield a space to accommodate the catalytic acid D122 and the 2,5B boat conformation of -1 sugar during transition state. Collectively, we characterized the enzyme properties of P. pastoris-expressed TtCel45A and solved high-resolution crystal structures of the enzyme. These results are of great interests in industrial applications and provide new insights into the fundamental understanding of enzyme catalytic mechanism of GH45 endoglucanases.

Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity.

The thermostable β-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric three-domain representative from glycoside hydrolase family 3. By using chemical reactivation with exogenous nucleophiles in previous studies with TnBg13B, the catalytic nucleophile (D242) and corresponding acid/base residue (E458) were determined. Identifying these residues led to the attempt of converting TnBgl3B into a β-glucosynthase, where three nucleophilic variants were created (TnBgl3B_D242G, TnBgl3B_D242A, TnBgl3B_D242S) and all of them failed to exhibit glucosynthase activity. A deeper analysis of the TnBgl3B active site led to the generation of three additional variants, each of which received a single-point mutation. Two of these variants were altered at the -1 subsite (Y210F, W243F) and the third received a substitution near the binding site's aglycone region (N248R). Kinetic evaluation of these three variants revealed that W243F substitution reduced hydrolytic turnover while maintaining KM This key W243F mutation was then introduced into the original nucleophile variants and the resulting double mutants were successfully converted into β-glucosynthases that were assayed using two separate biosynthetic methods. The first reaction used an α-glucosyl fluoride donor with a 4-nitrophenyl-β-d-glucopyranoside (4NPGlc) acceptor, and the second used 4NPGlc as both the donor and acceptor in the presence of the exogenous nucleophile formate. The primary specificity observed was a β-1,3-linked disaccharide product, while a secondary β-1,4-linked disaccharide product was observed with increased incubation times. Additional analysis revealed that substituting quercetin-3-glycoside for the second reaction's acceptor molecule resulted in the successful production of quercetin-3,4'-diglycosides with yields up to 40%.

Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester.

In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme “cocktail”, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60 to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

Contribution of a family 1 carbohydrate-binding module in thermostable glycoside hydrolase 10 xylanase from Talaromyces cellulolyticus toward synergistic enzymatic hydrolysis of lignocellulose.

BackgroundEnzymatic removal of hemicellulose components such as xylan is an important factor for maintaining high glucose conversion from lignocelluloses subjected to low-severity pretreatment. Supplementation of xylanase in the cellulase mixture enhances glucose release from pretreated lignocellulose. Filamentous fungi produce multiple xylanases in their cellulase system, and some of them have modular structures consisting of a catalytic domain and a family 1 carbohydrate-binding module (CBM1). However, the role of CBM1 in xylanase in the synergistic hydrolysis of lignocellulose has not been investigated in depth.ResultsThermostable endo-β-1,4-xylanase (Xyl10A) from Talaromyces cellulolyticus, which is recognized as one of the core enzymes in the fungal cellulase system, has a modular structure consisting of a glycoside hydrolase family 10 catalytic domain and CBM1 at the C-terminus separated by a linker region. Three recombinant Xyl10A variants, that is, intact Xyl10A (Xyl10Awt), CBM1-deleted Xyl10A (Xyl10AdC), and CBM1 and linker region-deleted Xyl10A (Xyl10AdLC), were constructed and overexpressed in T. cellulolyticus. Cellulose-binding ability of Xyl10A CBM1 was demonstrated using quartz crystal microbalance with dissipation monitoring. Xyl10AdC and Xyl10AdLC showed relatively high catalytic activities for soluble and insoluble xylan substrates, whereas Xyl10Awt was more effective in xylan hydrolysis of wet disc-mill treated rice straw (WDM-RS). The enzyme mixture of cellulase monocomponents and intact or mutant Xyl10A enhanced the hydrolysis of WDM-RS glucan, with the most efficient synergism found in the interactions with Xyl10Awt. The increased glucan hydrolysis yield exhibited a linear relationship with the xylan hydrolysis yield by each enzyme. This relationship revealed significant hydrolysis of WDM-RS glucan with lower supplementation of Xyl10Awt.ConclusionsOur results suggest that Xyl10A CBM1 has the following two roles in synergistic hydrolysis of lignocellulose by Xyl10A and cellulases: enhancement of lignocellulosic xylan hydrolysis by binding to cellulose, and the efficient removal of xylan obstacles that interrupt the cellulase activity (because of similar binding target of CBM1). The combination of CBM-containing cellulases and xylanases in a fugal cellulase system could contribute to reduction of the enzyme loading in the hydrolysis of pretreated lignocelluloses.

Characterization of a highly thermostable glycoside hydrolase family 10 xylanase from Malbranchea cinnamomea

A thermostable xylanase (McXyn10) from the thermophilic fungus Malbranchea cinnamomea strain S168 was purified and biochemically characterized. The enzyme was purified to homogeneity with a molecular mass of 43.5kDa on SDS–PAGE. The optimal pH and temperature of the purified enzyme were pH 6.5 and 80°C, respectively. The enzyme showed a broad range of pH stability (pH 4.0–10.5), and was stable up to 70°C with a thermal denaturing half life of 76.0min. The enzyme exhibited strict specificity for various xylans as substrates, but displayed no activity toward other tested polysaccharides. McXyn10 hydrolyzed birchwood xylan, beechwood xylan and oat-spelt xylan, yielded mainly xylobiose, xylotriose and xylooligosaccharides with degree of polymerization (DP) above 5, while yielded xylobiose from xylotriose and xylotetraose. The xylanase gene was further cloned. It had an open reading frame of 1191bp with two introns. The deduced amino acid sequence of the gene showed highest identity (58%) with a glycoside hydrolase family 10 xylanase from Aureobasidium pullulans.

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08.

The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-beta-xylanase and beta-xylosidase. beta-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-beta-xylanase into xylose monomers. The beta-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4(3)2(1)2, with unit-cell parameters a = b = 62.53, c = 277.4 A diffracted to 1.55 A resolution. The rectangular crystals belonged to space group P2(1), with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 A, beta = 90.5 degrees , and diffracted to 1.80 A resolution.

The methylotrophic yeast as a host for the expression and production of thermostable xylanase from the bacterium

A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3gl(-1) xylanase with a maximum activity of 3130Ul(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T(m)) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.

Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3).

Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-beta- D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l(-1)), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.