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Catalases, heme or non-heme, are catalysts that decompose hydrogen peroxide. Among them, non-heme or manganese-catalases have been studied from limited organisms. We report here heterologous production of a manganese-catalase, Cat-IIGt, previously annotated as a hypothetical protein, from a thermophilic bacterium Geobacillus thermopakistaniensis. Recombinant Cat-IIGt, produced as inactive inclusion bodies in Escherichia coli, was solubilized and refolded into a soluble and highly active form. Sequence homology, absorption spectra, resistance to sodium azide inhibition and activation by Mn2+ indicated that it was a manganese-catalase. Metal analysis revealed the presence of ∼2 Mn2+ and ∼2 Ca2+ per subunit of Cat-IIGt. Recombinant Cat-IIGt exhibited highest activity at pH 10.0 and 70°C. The enzyme was highly active with a specific activity of 40,529 μmol min−1 mg−1. The apparent Km and kcat values were 75 mM and 1.5 × 104 s−1 subunit−1, respectively. Recombinant Cat-IIGt was highly thermostable with a half-life of 30 min at 100°C. The structural attributes of Cat-IIGt, including the metal and substrate binding residues, were predicted by homology modeling and molecular docking studies. High activity and thermostability and alkaline nature make Cat-IIGt a potential candidate for textile and paper processing industries.

Surface of oil-contaminated soil from Industrial Estate of Al-Mafraq city, Jordan, was investigated for the presence of aerobic oil-degrading moderately thermophilic bacteria. A pure culture of spore forming aerobic chemoorganogeterotrophic rod shaped bacterial isolate, designated as strain j3n, was obtained. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain j3n is closely related to gram-positive bacteria of kaustophilus thermoleovorans cluster of Geobacillus genus. Strain j3n grew aerobically with oil, hexadecane, benzoate and acetate. Growth data indicated that utilization of hexadecane but not of oil and benzoate might be under catabolite repression control. Possibility of a regulation of alkane degradation by acetate in aerobic thermophilic gram-positive bacteria of Geobacillus spp. was shown for the first time.

Superoxide dismutases are the enzymes involved in dismutation of superoxide radicals into oxygen and hydrogen peroxide. The present work reports a thermostable Fe/Mn SOD of Geobacillus sp. strain PCH100 (GsSOD) isolated from glacial soil. Purified recombinant GsSOD is a dimeric protein of ~57 kDa that exhibited highest activity at a temperature of 10 °C and pH of 7.8. Maximum enzyme velocity and Michaelis constant of the GsSOD were 1098.90 units/mg and 0.62 μM, respectively. At 80 °C, thermal inactivation rate constant and half-life of GsSOD were 3.33 × 10−3 min−1 and 208 min, respectively. Interestingly, GsSOD tolerated a temperature of 100 °C and 130 °C up to 15 min and 5 min, respectively. Circular dichroism and differential scanning calorimetry confirmed thermostable nature of GsSOD. Apoenzyme of GsSOD regained enzymatic activity in the presence of Fe2+ and Mn2+ as metal ion cofactors. GsSOD was stable under varying concentrations of chemicals, namely ethylenediaminetetraacetic acid, potassium cyanide, hydrogen peroxide, chloroform-ethanol, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, Tween-20, Triton X-100, urea, and guanidine hydrochloride. The enzyme exhibited >70% activity in presence of 10 mM metal ions. Owing to its thermostable nature and resistance to chemical inhibitors, GsSOD is a potential enzyme for industrial applications.

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the γ-class of CA (termed as Cag). Only a limited number of γ-class CA’s have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar γ-CA’s the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized γ-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO2 to HCO3− when compared with Cam. Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES) and 25Mg and 67Zn NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme.

The production, characterization and bioactivities of exopolysaccharides (EPSs) from a thermophilic bacterium Geobacillus sp. strain WSUCF1 were investigated. Using glucose as a carbon source 525.7 mg/L of exoproduct were produced in a 40-L bioreactor at 60 °C. Two purified EPSs were obtained: EPS-1 was a glucomannan containing mannose and glucose in a molar ratio of 1:0.21, while EPS-2 was composed of mannan only. The molecular weights of both EPSs were estimated to be approximately 1000 kDa, their FTIR and NMR spectra indicated the presence of α-type glycosidic bonds in a linear structure, and XRD analysis indicated a low degree of crystallinity of 0.11 (EPS-1) and 0.27 (EPS-2). EPS-1 and EPS-2 demonstrated high degradation temperatures of 319 °C and 314 °C, respectively, and non-cytotoxicity to HEK-293 cells at 2 and 3 mg/mL, respectively. In addition, both showed antioxidant activities. EPSs from strain WSUCF1 may expand the applications of microorganisms isolated from extreme environments and provide a valuable resource for exploitation in biomedical fields such as drug delivery carriers.

Biofilm formation of Geobacillus thermodenitrificans, Geobacillus thermoglucosidans and Anoxybacillus flavithermus in milk on stainless steel were monitored at 55 degrees C, 60 degrees C, and 65 degrees C for various incubation times. Although species of Geobacillus showed a rapid response and produced biofilm within 4 h on stainless steel, a delay (lag time) was observed for Anoxybacillus. A hyperbolic equation and a hyperbolic equation with lag could be used to describe the biofilm formation of Geobacillus and Anoxybacillus, respectively. The highest biofilm formation amount was obtained at 60 degrees C for both Geobacillus and Anoxybacillus. However, the biofilm formation rates indicated that the lowest rates of formation were obtained at 60 degrees C for Geobacillus. Moreover, biofilm formation rates of G. thermodenitrificans (1.2-1.6 Log(10)CFU/mL.h) were higher than G. thermoglucosidans (0.4-0.7 Log(10)CFU/mL.h). Although A. flavithermus had the highest formation rate values (2.7-3.6 Log(10)CFU/mL.h), this was attained after the lag period (4 or 5 h). This study revealed that modeling could be used to describe the biofilm formation of thermophilic bacilli in milk.

In this study lipolytic biocatalysts GD-95RM, GDEst-95 and GDEst-lip were immobilized by encapsulation in calcium alginate beads. All three immobilized biocatalysts demonstrated significantly increased thermal stability at 60–70 °C temperatures and the activity of GD-95RM lipase increased by 50% at 70–80 °C following the immobilization. Moreover, encapsulated GDEst-95 esterase retained higher than 50% lipolytic activity after 3 months of incubation with butanol (25%) and ethanol (50%); GDEst-lip enzyme possessed 50% activity after 2 months of treatment with ethanol (25%) and methanol (25%); and GD-95RM lipase displayed higher that 50% activity after two-week incubation with methanol (50%). All three immobilized enzymes displayed long-term storage capability (>50% activity) at least until 3 months at 4 °C. It was also detected that immobilized GD-95RM and GDEst-lip can perform flow hydrolysis of both avocado oil and p-NP dodecanoate in prototype packed-bed column reactor. The analysis of continuous transesterification of avocado or sunflower oil with ethanol or methanol as substrates confirmed that encapsulated GD-95RM and GDEst-lip enzymes is a useful approach to produce fatty acid alkyl esters.

Though the biofilms of thermophilic, endospore-forming bacilli have received increasing interest in recent years, their biofilms are still unclear. In the current study, a comparative characterization of biofilm-forming patterns of totally 105 isolates and reference strains belonging to Anoxybacillus (85) and Geobacillus (20) genera were investigated. Various species belonging to these two genera have been screened for their biofilm production responses in milk, and their biofilm production patterns were also compared with their lactose and casein utilization. Thereby, this is the first report which displays strong biofilm producing behaviours of some thermophilic species in milk such as G. thermoglucosidans, A. caldiproteolyticus, A. suryakundensis, A. salavatliensis, and A. kamchatkensis subsp. asaccharedens except for well-known members like G. steraothermophilis and A. flavithermus. It was also assigned that elevated incubation temperature (65°C) had a stimulative effect on both biofilms forming capacities of Geobacillus and Anoxybacillus. Nevertheless, no direct correlation was found between biofilm formation capacity and lactose or casein catabolism characteristics. Finally, crystal violet binding assay, allowing the determination of the biofilm production amounts of microorganisms relatively in a short time, has been optimized. In this context, a methodological contribution has been provided to the literature in order to determine the possible biofilm production responses of thermophilic biofilms in the dairy industry.

The current study focuses on optimizing, characterizing, and purifying a thermostable amylase from thermophilic bacteria Geobacillus icigianus (BITSNS038). The amylase was produced optimally at 18–24 h of incubation in the presence of starch and tryptone at 7.5 and 3.0 g/L, respectively (pH 7.0, 70°C, and 150 rpm). Km, Vmax values for starch were 2.17 mg/mL and 4.16 U/mL respectively. The enzyme showed excellent thermal stability at 70°C, retaining 62.5% residual activity after 8 h and a reduced deactivation rate constant (kd) of 0.001. When tested for its efficacy in starch hydrolysis, it showed 34.5% hydrolysis of corn starch slurry and antibiofilm activity, but no antimicrobial activity was noticed. The ultrafiltration led to partial purification of amylase showed 2.67-fold purification with a molecular weight in the range of 45 and 66 kDa. This is probably the first-ever report on a thermostable amylase from G. icigianus and its extensive potential uses.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.

Selenium is a nutrient element essential for the growth and reproduction of plants and animals. Generally, selenium nanoparticle has a high level of absorption in regular supplementation comparative to selenium. In this study, selenium nanoparticles have been synthesized using a cell-free extract of Geobacillus as a reducing and stabilizing agent. This is for the first time; Geobacillus has been used for the synthesis of selenium nanoparticles. The cell-free extract of bacteria has active enzymes that are responsible for the reduction of sodium selenite to prepare selenium nanoparticles. The cell-free extract increased the reduction process comparative to the bacterial reduction. The biosynthesized nanoparticles were characterized by UV-visible spectrophotometry, transmission electron microscopy along with selected area electron diffraction, Fourier transform infrared spectroscopy, and energy-dispersive X-ray spectroscopy. The spherical nanoparticles have diameters in the range of 30 to 60 nm. The antioxidant activity was evaluated by various antioxidant assays. This study revealed that biofunctionalized selenium nanoparticle has more antioxidant activity than sodium selenite and cell-free extract. Abbreviations: ABTS: 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid); DPPH: 1,1-diphenyl-2-picrylhydrazyl; EDTA: ethylenediaminetetraacetic acid; EDAX: energy dispersive X-ray analysis; FRAP: ferric reducing antioxidant power; FTIR: Fourier transform infrared spectroscopy; PBS: phosphate buffered saline; PMSF: phenylmethylsulfonyl fluoride; SAED: selected area electron diffraction; SeNP: selenium nanoparticles; TEM: transmission electron microscopy; TPTZ- 2,4,6-Tri(2-pyridyl)-s-triazine; YAG: yttrium aluminium garnet

A lignocellulolytic and thermophilic bacterium Geobacillus sp. WSUCF1 was investigated for its production of biopolymers (exopolysaccharides, EPSs) using agricultural waste corn stover. The maximum...

A mannose-6-phosphate isomerase (MPI) from Geobacillus thermodenitrificans was expressed and successfully encapsulated into the Saccharomyces cerevisiae spores. Our results demonstrated that compared to the free enzyme, the MPI triple mutant encapsulated in osw2Δ spores exhibited much preferred enzymatic properties, such as enhanced catalytic activity, excellent reusability, thermostability, and tolerance to various harsh conditions. In combination with an l-arabinose isomerase (AI) also from G. thermodenitrificans, this technique of spore encapsulation was applied for producing a high-value rare sugar l-ribose from biomass-derived l-arabinose. Using a 10 mL reaction system, 350 mg of l-ribose was produced from 1 g of l-arabinose with a conversion yield of 35% by repeatedly reacting with 200 mg of AI-encapsulated spores and 300 mg of MPI-encapsulated spores. This study provides a very useful and concise approach for the synthesis of rare sugars and other useful compounds.

Background Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. Results The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of pNP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate pNP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60 °C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). Conclusions This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

Protein thermostability is important to evolution, diseases and industrial applications. Proteins use diverse molecular strategies toachieve stability at high temperature, yet reducing the entropy of unfolding seems required. We investigated five small α-proteins and five β-proteins with known, distinct structures and thermostability (Tm ) using multi-seed molecular dynamics simulations at 300, 350, and 400 K. The proteins displayed diverse changes in hydrogen bonding, solvent exposure and secondary structure with no simple relationship to Tm . Our dynamics were in good agreement with experimental B-factors at 300 K and insensitive to force field choice. Despite the very distinct structures, the native-state (300 + 350 K) free energy landscapes were significantly broader for the two most thermostable proteins and smallest for the three least stable proteins in both the α- and β-group and with both force fields studied independently (tailed t-test, 95% confidence level). Our results suggest that entropic ensembles stabilize proteins at high temperature due to reduced entropy of unfolding, viz. ΔG = ΔH - TΔS. Supporting this mechanism, the most thermostable proteins were also the least kinetically stable, consistent with broader free energy landscapes, typified by villin headpiece and confirmed by specific comparison to a mesophilic ortholog of Thermus thermophilus apo-pyrophosphate phosphohydrolase. We propose that molecular strategies of protein thermostabilization, although diverse, tend to converge towards highest possible entropy in the native state consistent with the functional requirements. We speculate that this tendency may explain why many proteins are not optimally structured and why molten globule states resemble native proteins so much. This article is protected by copyright. All rights reserved.

In this study three lipases GD-28, GD-95 and GD-66 (all 43 kDa in size), isolated from Geobacillus spp. were subjected to directed evolution experiments to yield a new synthetic lipolytic enzyme. This new lipase, obtained by DNA shuffling and epPCR, was named GDlip43 (also 43 kDa in size). It demonstrated increased thermoactivity, thermostability, an ability to hydrolyze short and long acyl chain p-NP esters and was activated by different organic solvents. Different activity of GDlip43 raised the hypothesis of new candidate amino acids which could be important for the activity of Geobacillus lipases. Based on the sequence alignment of parental and GDlip43 lipase, three candidate amino acids were selected. The importance of these amino acids, localized at positions 153, 154 and 247 (all of which are distant from the catalytic center of Geobacillus lipases) was investigated using site-directed mutagenesis. Directed evolution experiments also yielded another new lipase - GDlip30 (30 kDa in size). This low molecular mass derivative of GDlip43 had clearly detectable lipolytic activity (40 U/mg) and is the smallest currently known active Geobacillus lipase variant.

The search for novel DNA Polymerases I, with higher fidelity and better polymerization rate, is essential to improve the Polymerase Chain Reaction method. A thermo-halophilic bacterium has been isolated from an undersea hot spring, dubbed Pria Laot Sabang (PLS) A strain. The 0.9 kb DNA Polymerase I gene fragments from the isolate were amplified, sequenced, and identified. The fragments were part of the polymerization domain of the enzyme. Homological analysis of the gene sequence showed that the PLS A strain was closely related to Bacillus caldolyticusstrain XM. However, Swissprot structural analysis reveals that PLS A strain had high homology to Geobacillus stearathermophilus. Full sequence analysis is still needed to identify the species and evaluate the intact enzyme structure.

Pullulanase type I (PulA) is a debranching enzyme that specifically cleaves α-1,6-glycosidic linkages in pullulan. Pullulan has not only diverse applications in food industry but also has immune-stimulatory effects on B and T cells, and found to enhance the production of various anti-inflammatory cytokines in human. Moreover, pullulan has been suggested as a possible anti-cancer drug delivery agent without adjuvant due to its unique structure. The process of pullulan degradation is unresolved due to imprecise pullulanase structural characteristics. Therefore, the present study aimed to understand the structural and functional characteristics of pullulanase enzyme from Geobacillus thermopakistaniensis MAS1 strain using various computational approaches. The physio-chemical topographies and secondary structure of GT_PulA were explored using ProPram, InterPro and SMART. Various tools like I-TASSER, ModRefiner, RAMPAGE, PROCHECK and MOE 2009.10 were used to construct and verify the 3D structural model. The structural elucidation confirmed the significant domains, i.e., CBM48, CBM2, and TIM barrel having catalytically active residues, and conserved region YNGWDP. CBM2 domain along with TIM barrel has a capacity to bind different ligands and proved favorable for multiple substrate catalyses. These structural properties can have a potential effect on enhancing enzymatic activity of GT_PulA enzyme.

The study of the mechanism of endogenous microbial flooding in the reservoir, this paper analyzed the effect of microbial and its product on the stability of paraffin wax using Geobacillus stearoth...

The enzyme Urocanate Hydratase (UH) participates in the catabolic pathway of L-histidine. Trypanosoma cruzi Urocanate Hydratase (TcUH) is identified as a therapeutic molecular target in the WHO/TDR Targets Database. We report the 3D structure determination and number of features of TcUH, and compared it to other few available bacterial UH structures. Each monomer presents two domains and one NAD+ molecule. Superpositions revealed differences in the relative orientation of domains within monomers, such that TcUH monomer A resembles Urocanate Hydratase from Geobacillus kaustophilus (GkUH) (open conformation), while monomer C resembles Urocanate Hydratase from Pseudomonas putida (PpUH) and Urocanate Hydratase from Bacillus subtilis (BsUH) (closed conformations). We use the structure of TcUH to make considerations about 3 non-deleterious and 2 deleterious mutations found in human UHs: non-deleterious mutations could be accommodated without large displacements or interaction interruptions, whereas deleterious mutations in one case might disrupt an α-helix (as previously suggested) and in the other case, besides disrupting the enzyme interaction with the substrate, might interfere with interdomain movement.

Summary The potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l−1). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribC Gtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpN Gtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l−1 after 12‐h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high‐temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature., Taking advantage of Geobacillus thermoglucosidasius with the characteristics of fast growth and utilization of diverse inexpensive substrates, here we demonstrated that this strain could be used as a promising alternative host for biosynthesis of riboflavin. Afterward, a sequential of engineering strategy was applied to improve riboflavin titers in the G. thermoglucosidasius producer to 1034.5 mg l−1 at 12‐h flask fermentation in a simple mineral salt medium. As proof‐of‐concept, our work presented the feasibility of riboflavin production by engineering the thermophilic G. thermoglucosidasius. More importantly, the present work paves the way to further systematic metabolic engineer this thermophilic host for riboflavin production.

Significance X-ray crystallography often fails to determine the positions of hydrogen atoms, which play crucial roles in enzymatic reactions. Despite many X-ray crystallographic studies, the reaction mechanism of copper-containing nitrite reductases (CuNIRs), which reduce nitrite using two protons, has been controversial. The high-resolution neutron structure of a CuNIR reveals the protonation states of catalytic residues and key water molecules, thus providing insights into the catalytic mechanism. The catalytic Cu is shown to be coordinated by a hydroxide ion and not water. Furthermore, the hydrogen-deuterium exchange ratio suggests that intramolecular electron transfer is involved in a hydrogen-bond jump. These observations are consistent with previous computational chemistry; therefore, our study forms a bridge between the structural biology and quantum chemistry of CuNIRs., Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

The microbial degradation of lignocellulose in natural ecosystems presents numerous biotechnological opportunities, including biofuel production from agricultural waste and feedstock biomass. To explore the degradation potential of specific thermophiles, we have identified and characterized extremophilic microorganisms isolated from hot springs environments that are capable of biodegrading lignin and cellulose substrates under thermoalkaline conditions, using a combination of culturing, genomics, and metabolomics techniques. Organisms that can use lignin and cellulose as a sole carbon source at 60 to 75°C were isolated from sediment slurry of thermoalkaline hot springs (71 to 81°C and pH 8 to 9) of Yellowstone National Park. Full-length 16S rRNA gene sequencing indicated that these isolates were closely related to Geobacillus thermoleovorans. Interestingly, most of these isolates demonstrated biofilm formation on lignin, a phenotype that is correlated with increased bioconversion. Assessment of metabolite level changes in two Geobacillus isolates from two representative springs were undertaken to characterize the metabolic responses associated with growth on glucose versus lignin carbon source as a function of pH and temperature. Overall, results from this study support that thermoalkaline springs harbor G. thermoleovorans microorganisms with lignocellulosic biomass degradation capabilities and potential downstream biotechnological applications. IMPORTANCE Since lignocellulosic biomass represents a major agro-industrial waste and renewable resource, its potential to replace nonrenewable petroleum-based products for energy production is considerable. Microbial ligninolytic and cellulolytic enzymes are of high interest in biorefineries for the valorization of lignocellulosic biomass, as they can withstand the extreme conditions (e.g., high temperature and high pH) required for processing. Of great interest is the ligninolytic potential of specific Geobacillus thermoleovorans isolates to function at a broad range of pH and temperatures, since lignin is the bottleneck in the bioprocessing of lignocellulose. In this study, results obtained from G. thermoleovorans isolates originating from YNP springs are significant because very few microorganisms from alkaline thermal environments have been discovered to have lignin- and cellulose-biodegrading capabilities, and this work opens new avenues for the biotechnological valorization of lignocellulosic biomass at an industrial scale.

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine.

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

Bean dregs can be prepared into organic fertilizer by microbial fermentation. Geobacillus toebii GT-02, which has promoting effect on bean dregs fermentation, was isolated from horse dung and it grows within a range of 40–75 °C and pH 6.50–9.50. The effectiveness of GT-02 addition on composition transformations and the microbial community in bean dregs thermophilic fermentation at 70 °C for 5 days was investigated (T1). Fermentation of bean dregs without GT-02 served as control (CK). The results showed that T1 (the germination index (GI) = 95.06%) and CK (GI = 86.42%) reached maturity (defined by GI ≥ 85%) on day 3 and day 5, respectively. In addition, the total nitrogen loss of T1 (18.46%) on day 3 was lower than that in CK (24.12%). After thermophilic fermentation, the total organic carbon and dry matter loss of T1 (53.51% and 54.16%) was higher than that in CK (41.72% and 42.82%). The mean microbial number in T1 was 4.94 × 107 CFUs/g dry matter, which was 5.37 times higher than that in CK. 16S rDNA sequencing identified Bacillus, Geobacillus and Thermobacillus as dominant in CK, while Bacillus, Ammoniibacillus and Geobacillus were dominant in T1. A canonical correspondence analysis showed that Geobacillus and Ammoniibacillus were positively correlated with the GI. Thus, thermophilic fermentation with GT-02 can promote the maturity of bean dregs, which indicated the potential application value of GT-02 in thermophilic fermentation.

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme. Resumo A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60 °C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.

The growing industrial demand for stable proteases has driven this study to purify and partially characterize a protease from a bacterial strain isolated from undersea fumaroles. Phylogenetic analysis of the 400bp conserved area of the 16S rRNA gene indicated that the isolate was related to Geobacillus thermoleovorans strain SGAir 0734. Purification by ammonium sulfate precipitation and anion exchange chromatography produced 118-fold purity. The optimum activity (646 U mL−1) of the pure enzyme was observed at 60 °C, pH 7 and with 5M sodium chloride addition. The enzyme retained 73% of its activity when in 50% n-hexane, while the activity was only 40% of the control when in 50% ethyl acetate. The enzyme was tested for its leather dehairing capability. It only caused a slight dehairing and produced soft leather. The enzyme may be applied in skin rejuvenation, wrinkle-smoothing, and high-quality suede production industries.

1,4-α-Glucan branching enzyme (GBE, EC. 2.4.1.18), which plays a key role in the synthesis of starch and glycogen, has been overexpressed in E. coli as an intracellular enzyme by many researchers. In this study, it was found that the GBEs from Geobacillus thermoglucosidans and Rhodothermus obamensis were secreted into the culture medium when they were expressed separately, in E. coli. This occurred despite the absence of any signal peptide. In fact, although bioinformatics tools predicted that both of these proteins would localize to the cytoplasm, a high level of expression and non-classical secretion was found to achieve without addition of the inducer isopropyl β-d-thiogalactopyranoside. Further experiments revealed that secretion was a two-step process that occurred via the periplasmic space. Results excluded the involvement of the Sec pathway or the TAT pathway. Instead, the findings indicated a relationship between cell membrane integrity and the secretion of the two GBEs, and suggested that their N-termini play an essential role in their expression and secretion.

Cyclomaltodextrinases show diverse hydrolyzing and/or transglycosylation activities against cyclodextrins, starch and pullulan. A gene annotated as cyclomaltodextrinase from Geobacillus thermopakistaniensis was cloned and overexpressed in Escherichia coli. The gene product, CDaseGt, was purified and biochemically characterized. The recombinant enzyme exhibited highest activity with α-cyclodextrin at 55 °C and pH 6.0. Specific hydrolytic activities towards α-, β- and γ-cyclodextrin were 1200, 735 and 360 μmol min−1 mg−1, respectively. To the best of our knowledge, the activity against α-cyclodextrin is the highest among the reported enzymes. Next to cyclodextrins, pullulan was the most preferred substrate with a specific activity of 105 μmol min−1 mg−1. CDaseGt was capable of hydrolysis of maltotriose and acarbose as well as transglycosylation of their hydrolytic products. At 65 °C, there was no significant loss in enzyme activity even after overnight incubation. Activity of CDaseGt was not metal ions dependent, however, the presence of Mn2+ significantly enhanced the α-CDase activity. EDTA had no significant effect on the CDaseGt activity, however, it enhanced the thermostability of the enzyme. CDaseGt existed in monomeric as well as dimeric form in solution. Dimeric form is more active compared to the monomeric one. Equilibrium between the two forms seems to be concentration dependent.

GULER, Halil Ibrahim/0000-0002-7261-6790 WOS: 000478684300072 PubMed: 31201678 The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542(T) was cloned into pET28a(+) expression vector and characterized and is being reported in this study for the first time in Geobacillus. The enzyme, designated as GthFAE, was purified by heat shock and ion-exchange column chromatography. In addition, a second clone containing a Histidine tag was expressed and purified by affinity column chromatography demonstrating future potential for scale-up. FAE gene contains an open reading frame (ORF) of 759-bp encoding a hypothetical 252 amino acid protein, a molecular mass of 28.11kDa and an isoelectric point of 5.53. From this study it was found that GthFAE had optimal activity at 50 degrees C and pH of 8.5. Furthermore, the enzyme has been found to retain 64% of its activity after two days incubation at 50 degrees C and exhibited a high level of functionality with p-nitrophenyl caprylate (C8). K-m, V-max, k(cat) and k(cat)/K-m values for p-nitrophenyl caprylate were determined as 0.035mM, 11,735 mu mol/min/mg protein, 5491 (1/s) and 156,885s(-1)mM(-1) respectively. The combination of higher activity and stability compared to previously reported FAEs makes GthFAE a potential candidate for use in the paper manufacturing industry. Karadeniz Technical University Research Foundation [BAP01-8704] We would like to thank Dr. Asif Rahman (Space Biosciences Division, NASA) for critical reading and language editing of this manuscript. This work was supported by the Karadeniz Technical University Research Foundation [Grant No. BAP01-8704].

Although nitrite in powdered milk formula (PIF) is a recognized health risk for infants, the presence of nitrite in PIF has only been investigated as a chemical contaminant during the inspection of end-products. The risk posed by microbial sources of nitrite during the PIF manufacturing process has not been considered. This is the first study to report the taxonomy and physiological characteristics of nitrite-producing bacteria isolated from PIF processing environments. All isolates identified as nitrite-producers (133 out of 501 strains collected over four years) from work-in-process and end-products of PIF were spore-forming bacilli. Nitrite-producing metabolism under PIF processing conditions was found in not only thermophilic isolates (3 Bacillus, 60 Geobacillus from 63 strains; 100%) but also in mesophilic isolates (65 Bacillus, 1 Anoxybacillus from 70 strains; 65.7%). Geobacillus was the only highly heat-resistant sporeformer and vigorous nitrite-producer exhibiting dramatic increases in nitrite over short periods of incubation (a maximum value within 3 h). High conversions of nitrate to nitrite (up to 88.8%) was also observed, highlighting bacteria as a key source of nitrite in PIF processing lines. Further research into the diversity of metabolic activity observed in this study can facilitate specialized management of nitrite-producers in PIF processing lines.

Anoxybacillus (A. flavithermus, A. kamchatkensis subsp. asachharedens, A. caldiproteolyticus and A. tepidamans) and Geobacillus (two strains of G. thermodenitrificans, G. thermoglucosidans ...

Ecological and efficient alternatives to industrial processes have sparked interest for using microorganisms and enzymes as biocatalysts. One of the difficulties is finding candidates capable of resisting the harsh conditions in which industrial processes usually take place. Extremophiles, microorganisms naturally found in "extreme" ecological niches, produce robust enzymes for bioprocesses and product development. Thermophiles like Geobacillus, Alyciclobacillus, Anoxybacillus, Pyrococcus and Thermoccocus are some of the extremophiles containing enzymes showing special promise for biocatalysis. Glutamate dehydrogenase used in food processes, laccases and xylanases in pulp and paper processes, nitrilases and transaminases for pharmaceutical drug synthesis and lipases present in detergents, are examples of the increasing use of enzymes for biocatalytic synthesis from thermophilic microorganisms. Some of these enzymes from thermophiles have been expressed as recombinant enzymes and are already in the market. Here we will review recent discoveries of thermophilic enzymes and their current and potential applications in industry.

The aim of this study was to investigate the effect of atmospheric pressure cold plasma on the microbial substrate utilization and biomass yield in a thermophilic strain. Geobacillus sp. strain WSUCF1, a thermophile capable of producing cellulolytic enzymes with higher activity was used for this investigation. Treatment with cold plasma for 4 min increased the rates of glucose utilization by 74% and biomass yield by 60% when compared with the control. WSUCF1 treated with plasma also displayed enhanced biofilm formation. This study for the first time, reports the use of cold plasma for enhancing the substrate utilization and biofilm formation in a thermophile.

In this study, a thermophilic facultative anaerobic strain Geobacillus thermoglucosidasius W-2 was found to degrade nitroalkane under both aerobic and anaerobic conditions. Bioinformatical analysis revealed three putative nitroalkane-oxidizing enzymes (Gt-NOEs) genes from the W-2 genome. The three identified proteins Gt2929, Gt1378, and Gt1208 displayed optimal activities at high temperatures (70, 70, and 80 °C, respectively). Among these, Gt2929 exhibited excellent degradation capability, pH stability, and metal ion tolerance for nitronates under aerobic condition. Interestingly, under anaerobic condition, only Gt1378 still maintained high activity for 2-nitropropane and nitroethane, indicating that the W-2 strain utilized various pathways to degrade nitronates under aerobic and anaerobic conditions, respectively. Taken together, the first revelation of thermophilic nitroalkane-degrading mechanism under both aerobic and anaerobic conditions provides guidance and platform for biotechnological and industrial applications.

Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43 kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14 A resolution. Structure of the lipase showed an alpha-beta fold with 19 α-helices and 10 β-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6 A) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with Tm of 76 °C. 13C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O2) activation, catalyzes formation of a tyrosine–valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O2, but prefers manganese in the presence of O2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation. Electronic supplementary material The online version of this article (10.1007/s00775-019-01639-4) contains supplementary material, which is available to authorized users.

Xylanases have great attention to act as a potential role in agro-industrial processes. In this study, production, characterization, and fruit juice application of novel xylanase from thermophilic Geobacillus vulcani GS90 (GvXyl) were performed. GvXyl was purified via acetone precipitation and gel-filtration chromatography. The results showed that GvXyl had 1,671.4 U/mg of specific activity and optimally worked at pH 8 and 55°C. It was also active in a wide pH (3-9) and temperature (30-90oC) ranges. GvXyl was highly stable at 90oC and relatively stable at pH 3-9. The kinetic parameters of GvXyl were obtained as Km , Vmax , and kcat ; 10.2 mg/ml, 4,104 µmol min-1 mg-1 , and 3,542.6 s-1 , respectively. GvXyl had higher action than commercial xylanase in fruit juice enrichment. These results revealed that GvXyl might possess a potential influence in fruit juice processing because of its high specific activity and great thermal stability. PRACTICAL APPLICATIONS: Polysaccharides include starch, pectin, and hemicellulose create problems by lowering fruit juice quality in beverages. To overcome this problem, various clarification processes might be applied to natural fruit juices. Even though chemicals are widely used for this purpose, recently enzymes including xylanases are preferred for obtaining high-quality products. In this study, we reported the production and biochemical characterization of novel thermostable xylanase from thermophilic G. vulcani GS90 (GvXyl). Also, apple and orange juice enrichment were performed with the novel xylanase to increase the quality in terms of yield, clarity, and reducing sugar substance. The improved quality features of apple and orange juices with GvXyl was then compared to commercially available β-1,4-xylanase. The results revealed that GvXyl might possess a potential influence in fruit juice processing because of its high specific activity and great thermal stability.

A thermostable T1 lipase from Geobacillus zalihae exhibits broad substrate specificity and good potential application in fats and oils. However, structural insight into the enzyme against substrates is poorly understood at the molecular level. Herein, the study aimed to examine interactions between a mutant T1 lipase (Mut-T1 lipase) and selected fatty acids (caprylic, myristic, stearic, oleic, linoleic and linolenic acids) by performing molecular docking and molecular dynamics (MD) simulation. The structure of Mut-T1 lipase obtained by homology modeling was reliable for molecular docking and MD simulation. Molecular docking revealed that Mut-T1 lipase showed low binding affinity for caprylic acid (−4.97 kcal/mol) compared to the other fatty acids (−5.65 to −6.88 kcal/mol). However, the conformation of Mut-T1 lipase-caprylic acid complex was comparably stable during the simulation, in terms of less root-mean square fluctuation. Besides, solvent accessible surface area value of Mut-T1 lipase-fatty acid complexes decreased with increasing chain length of fatty acid. van der Waals interactions were requisite in maintaining complex stability during the binding process. This work provides structural insight into interactions between the lipase and the fatty acids, which will facilitate design and applications of new mutants of T1 lipase in modifying fats and oils.

Luo Han Guo fruit extract (Siraitia grosvenorii), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources (Paenibacillus macerans, Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes., This research was funded by OptiBiotix Health Plc (York, U.K.), the Spanish Ministry of Science, Innovation and Universities (Project AGL2017-84614-C2-1-R), and FEDER Spanish Ministry of Science and Innovation (Project RTI2018-101273-J-I00).

International audience; Thiamine diphosphate-dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon-carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high-throughput solid-phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α-ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction.