Search Results (47)

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed. Full article

Biomass from various types of organic waste was tested for possible use in hydrogen production. The composition consisted of lignified samples, green waste, and kitchen scraps such as fruit and vegetable peels and leftover food. For this purpose, the enzymatic pretreatment of organic waste with a combination of five different hydrolytic enzymes (cellulase, amylase, glucoamylase, pectinase and xylase) was investigated to determine its ability to produce hydrogen (H₂) with the hydrolyzate produced here. In course, the anaerobic rod-shaped bacterium T. neapolitana was used for H₂ production. First, the enzymes were investigated using different substrates in preliminary experiments. Subsequently, hydrolyses were carried out using different types of organic waste. In the hydrolysis carried out here for 48 h, an increase in glucose concentration of 481% was measured for waste loads containing starch, corresponding to a glucose concentration at the end of hydrolysis of 7.5 g·L⁻¹. In the subsequent set fermentation in serum bottles, a H₂ yield of 1.26 mmol H₂ was obtained in the overhead space when Terrific Broth Medium with glucose and yeast extract (TBGY medium) was used. When hydrolyzed organic waste was used, even a H₂ yield of 1.37 mmol could be achieved in the overhead space. In addition, a dedicated reactor system for the anaerobic fermentation of T. neapolitana to produce H₂ was developed. The bioreactor developed here can ferment anaerobically with a very low loss of produced gas. Here, after 24 h, a hydrogen concentration of 83% could be measured in the overhead space. Full article

Second-generation biofuels from lignocellulosic biomass remain critical and require several challenges due to lignin compounds’ inefficient degradation and recalcitrate characteristics. In this regard, this study focuses on enzymatic technology as a promising treatment that is beneficial in breaking down the biomass’s hemicellulose and cellulosic parts. Thermostable bacterial species owe thermostable enzymes that are able to degrade complex carbohydrate compounds and produce efficient hydrogen production. The present study investigates the direct utilization of ligninolytic enzymes such as cellulase and xylanase derived from the hyperthermophilic bacteria Thermotoga maritima (ATCC 43589 strain). The results show that xylanase and cellulase enzymes extracted from Thermotoga maritima could depolymerize the lignin bonds of corn stover substrate and release monomers such as Galactose in the media. In conclusion, this study can open a new advanced research window on directly applying a hyperthermophilic consortium of enzymes capable of hydrolyzing lignocellulose material toward hydrogen production. Full article

Microfluidic devices have attracted much attention in the current day owing to the unique advantages they provide. However, their application for industrial use is limited due to manufacturing limitations and high cost. Moreover, the scaling-up process of the microreactor has proven to be difficult. Three-dimensional (3D) printing technology is a promising solution for the above obstacles due to its ability to fabricate complex structures quickly and at a relatively low cost. Hence, combining the advantages of the microscale with 3D printing technology could enhance the applicability of microfluidic devices in the industrial sector. In the present work, a 3D-printed single-channel immobilized enzyme microreactor with a volume capacity of 30 μL was designed and created in one step via the fused deposition modeling (FDM) printing technique, using polylactic acid (PLA) as the printing material. The microreactor underwent surface modification with chitosan, and β-glucosidase from Thermotoga maritima was covalently immobilized. The immobilized biocatalyst retained almost 100% of its initial activity after incubation at different temperatures, while it could be effectively reused for up to 10 successful reaction cycles. Moreover, a multi-channel parallel microreactor incorporating 36 channels was developed, resulting in a significant increase in enzymatic productivity. Full article

Pseudothermotoga hypogea is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding P. hypogea ADH (PhADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other Thermotoga species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of PhADH contains an α/β-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant PhADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant PhADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant PhADH in E. coli significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs. Full article

Starch-like polymers can be created through the use of enzymatic modification with glycogen branching enzymes (GBEs). GBEs are categorized in the glycoside hydrolase (GH) family 13 and 57. Both GH13 and GH57 GBEs exhibit branching and hydrolytic activity. While GH13 GBEs are also capable of α-1,4-transglycosylation, it is yet unknown whether GH57 share this capability. Among the four crystal structures of GH57 GBEs that have been solved, a flexible loop with a conserved tyrosine was identified to play a role in the branching activity. However, it remains unclear whether this flexible loop is also involved in α-1,4-transglycosylation activity. We hypothesize that GH57 GBEs with the flexible loop and tyrosine are also capable of α-1,4-transglycosylation, similar to GH13 GBEs. The aim of the present study was to characterize the activity of GH57 GBEs to investigate a possible α-1,4-transglycosylation activity. Three GH57 GBEs were selected, one from Thermococcus kodakarensis with the flexible loop and two beta-strands; one from Thermotoga maritima, missing the flexible loop and beta-strands; and one from Meiothermus sp., missing the flexible loop but with the two beta-strands. The analysis of chain length distribution over time of modified maltooctadecaose, revealed, for the first time, that all three GH57 GBEs can generate chains longer than the substrate itself, showing that α-1,4-transglycosylation activity is generally present in GH57 GBEs. Full article

The damaging competition between crops and parasitic weeds has a negative impact on agricultural productivity; however, the impact of disturbance on the soil’s microbial community has received less attention. Hence, this study investigates the microbial composition and diversity of the maize rhizosphere infected with Striga hermonthica using a shotgun sequencing approach from two maize-growing fields (Eruwa, Nigeria and Mbuzini, South Africa). The rhizosphere soil DNA was extracted from infested soil using a Nucleospin soil genomic DNA extraction kit and sequenced on an Illumina platform. The dominant phyla were Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Acidobacteria, Chloroflexi, Cyanobacteria, Planctomycetes, Verrucomicrobia, Chlorobi, Proteobacteria, Firmicutes, Nitrospirae, Thermotogae, Synergistetes, Ascomycota, Euryarchaeota, and Crenarchaeota. Bacteria phyla were observed to be of higher proportion in the rhizosphere soil samples obtained from Striga-infested maize field in Eruwa (Es) than those recovered from Mbuzini (Ms). The alpha diversity of microbial communities indicated insignificance differences (p > 0.05) between the five taxonomical groups (phylum, class, order, family, and genus), while the beta diversity produced a significant (p = 0.01, R = 0.52) difference in the microbial diversity of the infested soil. In summary, the study sheds light on the diversity and composition of the microbiome of Striga hermonthica-infested soil, which influences the microbial functions in the management and sustenance of plant health against parasitic weeds. Full article

The basis of MreB research is the study of the MreB protein from the Thermotoga maritima species, since it was the first one whose crystal structure was described. Since MreB proteins from different bacterial species show different polymerisation properties in terms of nucleotide and salt dependence, we conducted our research in this direction. For this, we performed measurements based on tryptophan emission, which were supplemented with temperature-dependent and chemical denaturation experiments. The role of nucleotide binding was studied through the fluorescent analogue TNP-ATP. These experiments show that Thermotoga maritima MreB is stabilised in the presence of low salt buffer and ATP. In the course of our work, we developed a new expression and purification procedure that allows us to obtain a large amount of pure, functional protein. Full article

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO₂-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF. Full article

Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pH_opt of 8.5 and T_opt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pH_opt of 7.0 and T_opt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively. Full article

In the current study, the purified β-mannanase (Man/Cel5B) from Thermotoga maritima was immobilized on glutaraldehyde cross-linked chitosan beads. The immobilization of Man/Cel5B on chitosan beads was confirmed by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. After immobilization, the protein loading efficiency and immobilization yield were found to be 73.3% and 71.8%, respectively. The optimum pH for both free and immobilized enzymes was found to be pH 5.5. However, the optimum temperature of immobilized Man/Cel5B increased by 10 °C, from 85 °C (free Man/Cel5B) to 95 °C (Immobilized). The half-life of free and immobilized enzymes was found to be 7 h and 9 h, respectively, at 85 °C owing to the higher thermostability of immobilized Man/Cel5B. The increase in thermostability was also demonstrated by an increase in the energy of deactivation (209 kJmol⁻¹) for immobilized enzyme compared to its native form (92 kJmol⁻¹), at 85 °C. Furthermore, the immobilized Man/Cel5B displayed good operational stability as it retained 54% of its original activity after 15 repeated catalytic reactions concerning its free form. Full article

In Tengchong County, springs with wide physicochemical diversity provide a multitude of niches for extremophilic microorganisms. In this study, eight middle-low temperature spring sites along two continuous small streams with low water flow and slow speed in the fourth geothermal experience area of Rehai scenic spot were chosen, and geochemical characteristics and HTS of the 16S rRNA V4 region were used to analyze the prokaryotic community structure and diversity in the water and sediment of these sites. The effect of environmental factors on the microbial communities was explored via redundancy analysis (RDA). All sediment samples had higher alpha diversity values than the corresponding water samples. Twenty-five phyla were annotated; Euryarchaeota, Crenarchaeota, Aquificae, Thermotogae and Proteobacteria were the dominant phyla, accounting for 95.31% of all prokaryotes, with relative abundances above 5%. Aquificae dominated in water samples, while Euryarchaeota dominated in sediment samples. RDA indicated that temperature was the main factor influencing the microbial communities in the two streams. The study expands the current understanding of the microbiology of Tengchong hot springs and provides a basis for further mining of hot spring microbial and functional gene resources. Full article

Powdery mildew disease caused by Oidium neolycopersici is one of the major diseases affecting tomato production in South Africa. Interestingly, limited studies exist on how this disease affects the community structure microbial communities associated with tomato plants employing shotgun metagenomics. In this study, we assess how the health status of a tomato plant affects the diversity of the rhizosphere microbial community. We collected soil samples from the rhizosphere of healthy (HR) and diseased (DR; powdery mildew infected) tomatoes, alongside bulk soil (BR), extracted DNA, and did sequencing using shotgun metagenomics. Our results demonstrated that the rhizosphere microbiome alongside some specific functions were abundant in HR followed by DR and bulk soil (BR) in the order HR > DR > BR. We found eighteen (18) bacterial phyla abundant in HR, including Actinobacteria, Acidobacteria, Aquificae, Bacteroidetes, etc. The dominant fungal phyla include; Ascomycota and Basidiomycota, while the prominent archaeal phyla are Thaumarchaeota, Crenarchaeota, and Euryarchaeota. Three (3) bacteria phyla dominated the DR samples; Bacteroidetes, Gemmatimonadetes, and Thermotoga. Our result also employed the SEED subsystem and revealed that the metabolic pathways involved were abundant in HR. The α-diversity demonstrates that there is no significant difference among the rhizosphere microbiomes across the sites, while β-diversity demonstrated a significant difference. Full article

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERA_Tma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERA_Tma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDH_Lx) and DERA_Tma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERA_Tma and MDH_Lx. Full article

The transcriptional repressor Rex plays important roles in regulating the expression of respiratory genes by sensing the reduction–oxidation (redox) state according to the intracellular NAD⁺/NADH balance. Previously, we reported on crystal structures of apo, NAD⁺-bound, and NADH-bound forms of Rex from Thermotoga maritima to analyze the structural basis of transcriptional regulation depending on either NAD⁺ or NADH binding. In this study, the crystal structure of Rex in ternary complex with NAD⁺ and operator DNA revealed that the N-terminal domain of Rex, including the helix-turn-helix motif, forms extensive contacts with DNA in addition to DNA sequence specificity. Structural comparison of the Rex in apo, NAD⁺-bound, NADH-bound, and ternary complex forms provides a comprehensive picture of transcriptional regulation in the Rex. These data demonstrate that the conformational change in Rex when binding with the reduced NADH or oxidized NAD⁺ determines operator DNA binding. The movement of the N-terminal domains toward the operator DNA was blocked upon binding of NADH ligand molecules. The structural results provide insights into the molecular mechanism of Rex binding with operator DNA and cofactor NAD⁺/NADH, which is conserved among Rex family repressors. Structural analysis of Rex from T. maritima also supports the previous hypothesis about the NAD⁺/NADH-specific transcriptional regulation mechanism of Rex homologues. Full article