Glucose Isomerase Research Articles

A Novel Glucose Isomerase from Caldicellulosiruptor bescii with Great Potentials in the Production of High-Fructose Corn Syrup.

Glucose isomerase (GI) that catalyzes the conversion of D-glucose to D-fructose is one of the most important industrial enzymes for the production of high-fructose corn syrup (HFCS). In this study, a novel GI (CbGI) was cloned from Caldicellulosiruptor bescii and expressed in Escherichia coli. The purified recombinant CbGI (rCbGI) showed neutral and thermophilic properties. It had optimal activities at pH 7.0 and 80°C and retained stability at 85°C. In comparison with other reported GIs, rCbGI exhibited higher substrate affinity (Km = 42.61 mM) and greater conversion efficiency (up to 57.3% with 3M D-glucose as the substrate). The high catalytic efficiency and affinity of this CbGI is much valuable for the cost-effective production of HFCS.

Continuous 2G ethanol production from xylose in a fixed-bed reactor by native Saccharomyces cerevisiae strain through simultaneous isomerization and fermentation

The yeast Saccharomyces cerevisiae is the most used microorganism for ethanol production, however, on its wild form it cannot assimilate xylose. The previous ex-vivo isomerization of xylose to xylulose catalyzed by the enzyme xylose isomerase (XI) is an alternative to overcome this limitation. The present work evaluated continuous 2G ethanol production through xylose simultaneous isomerization and fermentation (SIF) using xylose isomerase co-immobilized with yeast. From the initial studies carried out with five different industrial S. cerevisiae strains, Itaiquara® baker´s yeast was selected due to its good performance in terms of ethanol yield (0.34 g/g) and productivity (2.1 g/L/h) having xylose as a carbon source. Continuous xylose SIF in a fixed-bed reactor was run for 7 days with high values of ethanol yield (0.37 g/g) and productivity (1.9 g/L/h). The operation for longer periods could be possible upon implementing strategies for pH and contamination control, showing great progress to achieve a feasible industrial 2G ethanol production process.

Permeabilized TreS-Expressing Bacillus subtilis Cells Decorated with Glucose Isomerase and a Shell of ZIF-8 as a Reusable Biocatalyst for the Coproduction of Trehalose and Fructose.

Metal-organic frameworks (MOFs) are a class of porous materials with versatile properties. In this study, ZIF-8 was employed to establish a two-enzyme system by encapsulating permeabilized Bacillus subtilis cells coated with glucose isomerase. B. subtilis was constructed by introducing the shuttle plasmid PMA5 associated with the overexpression of trehalose synthase. Using this two-enzyme system, trehalose was produced by trehalose synthase and the byproduct glucose was converted to fructose with the help of glucose isomerase. The decrease in glucose production not only relieved the inhibition of the entire reaction chain but also increased the final yield of trehalose. The highest trehalose production rate reached 67.7% and remained above 50% after 20 batches. In addition, the toxicity of the ZIF-8 coating for B. subtilis was investigated by fluorescence microscopy and was found to be negligible. By simulating an extreme environment, the ZIF-8 coating was demonstrated to have a protective effect on the cells and enzymes. This study provides a theoretical basis for the application of MOFs in the immobilization of microorganisms and enzymes.

Optimization of simultaneous saccharification and isomerization of dextrin to high fructose syrup using a mixture of immobilized amyloglucosidase and glucose isomerase

High fructose syrup is a sweetener widely used as a substitute for sucrose in the food and beverage industry with its current industrial production being carried out in three sequential processes using three different enzymes that operate under different pH and temperature conditions. In this study, a single step synergistic saccharification and isomerization process has been proposed, using the commercial amyloglucosidase from Aspergillus niger immobilized by the CLEA technique (co-aggregated with magnetic nanoparticles and polyethyleneimine), and the commercial immobilized glucose isomerase (IGI) from Streptomyces murinus, Sweetzyme® IT Extra. Immobilization stabilized both enzymes, widening the operation window, thus, by applying a factorial design with a central composite rotatable design it was possible to define an optimal condition of pH and temperature of the process, as well as the best ratio between the two enzymes (pH 5.7, 50 °C and UGI/UAMG ratio of 1.56). Simultaneous saccharification and isomerization of 35 % (w/v) dextrin solution produced a Dextrose Equivalent yield over 95 %, with a glucose-to-fructose conversion around 48 % after 30 h of reaction. In addition, both biocatalysts could be reused for six consecutive cycles, maintaining glucose-to-fructose conversions without loss of activity and with easy recovery of the biocatalysts. Furthermore, because they are of different natures (magnetic CLEA of amyloglucosidase and non-magnetic IGI pellets), if one of these biocatalysts is inactivated, they can be easily separated and reloaded individually.

Development of Some Properties of a Thermophilic Recombinant Glucose Isomerase by Mutation

Site-directed mutagenesis was made to obtain a glucose isomerase (GI) working at high temperatures and low pH values and having high affinity to the substrate and more pH and termal stability in comparison with the native enzyme. For this purpose, GI gene from Geobacillus caldoxylosilyticus TK4 was cloned to pET-28a(+) vector and H99Q, V184T and D102N mutations were performed. Biochemical features of the recombinant and mutant enzymes were identified. These mutations enabled the mutant enzymes to increase the optimum temperature and KM and decrease the optimum pH and Vmax values of the reaction. Furthermore, the mutant proteins had more thermal and pH stability than that of the recombinant GI. The mutant enzymes showed the highest activities in the presence of Co2+, Cu2+ and Mn2+ and were more resistant to some metals than the recombinant GI. In conclusion, the site-directed mutations led to improve the performance of the recombinant enzyme, which means that the mutant enzyme (TK4GI mutation) may be practicable in high fructose corn syrup production process.

Proteomic identification of allergenic proteins in red oak (Quercus rubra) pollen

BackgroundRed oak pollen is an important cause of allergic respiratory disease and it is widely distributed in North America and central Europe. To date, however, red oak pollen allergens have not been identified. Here, we describe the allergenic protein profile from red oak pollen. MethodsTotal proteins were extracted from red oak pollen using a modified phenolic extraction method, and, subsequently, proteins were separated by two-dimensional gel electrophoresis (2DE) for both total protein stain (Coomassie Blue) and immunoblotting. A pool of 8 sera from red oak sensitive patients was used to analyze blotted proteins. Protein spots were analyzed by Mass Spectrometry. ResultsElectrophoretic pattern of total soluble proteins showed higher intensity bands in the regions of 26–40 and 47–52 kDa. Two dimensional immunoblots using pool sera from patients revealed four allergenic proteins spots with molecular masses in the range from 50 to 55 kDa. Mass spectrometry analysis identified 8 proteins including Enolase 1 and Enolase 1 chloroplastic, Xylose isomerase (X1 isoform), mitochondrial Aldehyde dehydrogenase, UTP-Glusose-1-phosphate uridylyltransferase, Betaxylosidase/alpha-l-arabinofuranosidase and alpha- and beta subunits of ATP synthase. ConclusionsThis study has identified for first time 8 IgE binding proteins from red oak pollen. These findings will pave the way towards the development of new diagnostic and therapeutic modalities for red oak allergy.

Application of mannitol producing Leuconostoc citreum TR116 to reduce sugar content of barley, oat and wheat malt-based worts

Achieving a high monosaccharide composition in malt wort is instrumental to achieve successful lactic acid bacteria fermentation of malt based beverages. The conversion of monosaccharides to alternative metabolites such as the sweet polyol, mannitol with heterofermentative strains presents a novel approach for sugar reduction and to compensate for the loss of sweetness. This work outlines the application of an adopted mashing regimen with the addition of exogenous enzymes to produce wort with high fructose content which can be applied to different malted grain types with consistently efficacious monosaccharide production for bacterial fermentation. The so produced worts are then fermented with Leuconostoc citreum TR116 a mannitol hyper-producer. Malted barley, oat and wheat were mashed to stimulate protein degradation and release of free amino acids along with the enzymatic conversion of starch to fermentable sugars. Amyloglucosidase and glucose isomerase treatment converted di- and oligo-saccharides to glucose and provided a moderate fructose concentration in malt worts which was consistent across the three cereals. Fructose was completely depleted during fermentation with Lc. Citreum TR116 and converted to mannitol with high efficiency (>90%) while overall sugar reduction was >25% in all malt worts. Differences in amino acid composition of malt worts did not significantly affect growth of Lc. Citreum TR116 but did affect the formation of the aroma compounds diacetyl and isoamyl alcohol. Organic acid production and acidification of wort was similar across cereal substrates and acetic acid formation was linked to yield of mannitol. The results suggest that differences in amino acid and fructose content of malt worts considerably change metabolite formation during fermentation with Lc. Citreum TR116, a mannitol hyper-producer. This work gives new insight into the development of consumer acceptable malt based beverages which will provide further options for the health conscious and diabetic consumer, an important step in the age of sugar overconsumption.

Application of enzyme cocktails from Indonesian isolates to corncob (Zea mays) waste saccharification

High contents of cellulose and hemicellulose of corncob waste makes it suitable as a raw material for sugar saccharification that can be used as sugar source for bioethanol fermentation. However, the presence of lignin inhibits the complete sugar conversion, necessitating pretreatment of the corncob material. Therefore, our study aimed to introduce a new pretreatment method for corncob to minimize lignin contents, hydrolyze it using an enzyme cocktail, and ferment it to bioethanol. Firstly, we studied pretreatment of corncob powder to minimize lignin interference using various reagents like 2% H2SO4, 2% NaOH, 2% H2SO4 – 2% NaOH, and 15% NH4OH, which revealed that 15% NH4OH was the most efficient reagent. Then, several enzyme cocktails consisting of cellulase, xylanase, and xylose isomerase, isolated from microbes collected in Indonesia, in ratios of 1:1:1, 2:1:1, 1:2:1, and 1:1:2 were subjected to the pretreated corncob material. We found that hydrolysis with the enzyme cocktails produced significant amounts of reducing sugars, and 1:1:1 enzyme cocktail produced the highest amounts with amazing capability to ferment to bioethanol.

Crystal structure of a novel xylose isomerase from Streptomyces sp. F-1 revealed the presence of unique features that differ from conventional classes

BackgroundEnzymatic isomerization is a promising strategy to solve the problem of xylose fermentation and, consequently, to leverage the production of advanced biofuels and biochemicals. In a previous work, our research group discovered a new strain of Streptomyces with great biotechnological potential due to its ability to produce a broad arsenal of enzymes related to lignocellulose degradation. MethodsWe applied a multidisciplinary approach involving enzyme kinetics, biophysical methods, small angle X-ray scattering and X-ray crystallography to investigate two novel xylose isomerases, XylA1F1 and XylA2F1, from this strain. ResultsWe showed that while XylA1F1 prefers to act at lower temperatures and relatively lower pH, XylA2F1 is extremely stable at higher temperatures and presents a higher turnover number. Structural analysis revealed that XylA1F1 exhibits unique properties in the active site not observed in classical XylAs from classes I and II nor in its ortholog XylA2F1. It encompasses the natural substitutions, M86A and T93K, that create an extra room for substrate accommodation and narrow the active-site entrance, respectively. Such modifications may contribute to the functional differentiation of these enzymes. ConclusionsWe have characterized two novel xylose isomerases that display distinct functional behavior and harbor unprecedented amino-acid substitutions in the catalytic interface. General significanceOur findings contribute to a better understanding of the functional and structural aspects of xylose isomerases, which might be instrumental for the valorization of the hemicellulosic fraction of vegetal biomass.

Stable sample delivery in viscous media via a capillary for serial crystallography

Serial crystallography (SX) is an innovative technology in structural biology that enables the visualization of the molecular dynamics of macromolecules at room temperature. SX experiments always require a considerable amount of effort to deliver a crystal sample to the X-ray interaction point continuously and reliably. Here, a sample-delivery method using a capillary and a delivery medium is introduced. The crystals embedded in the delivery medium can pass through the capillary tube, which is aligned with the X-ray beam, at very low flow rates without requiring elaborate delivery techniques, drastically reducing sample consumption. In serial millisecond crystallography using a viscous medium via a capillary, crystals of lysozyme embedded in agarose, which produce an unstable injection stream at atmospheric pressure, and crystals of glucose isomerase embedded in gelatin, which is known to be problematic for open-extruder operation, were stably delivered at a flow rate of 100 nl min−1. The room-temperature crystal structures of lysozyme and glucose isomerase were successfully determined at 1.85 and 1.70 Å resolutions, respectively. This simple but highly efficient sample-delivery method can allow researchers to deliver crystals precisely to an X-ray beam in SX experiments.

Fructose syrup production from Onggok with isomerization process by Mg/Al hydrotalcite catalyst and glucose isomerase enzyme

Onggok, solid waste of tapioca still contains high carbohydrate. This component can be converted to liquid sugar (glucose and fructose). Hydrolysis process convert carbohydrate become glucose syrup while isomerization process would change those glucose become fructose syrup. This study aims to produce fructose from Onggok with isomerization by Mg/Al hydrotalcite catalyst and compare the productivity 500ml Onggok substrate (10.32 percent DS) was hydrolyzed become glucose in liquefaction and saccharification process. Liquefaction took placed in 95 degree C, pH 6.5, for 1 hour, by adding 0.067 percent (v/v) α-amylase while the saccharification was start by adding 0.067 percent (v/v) glucoamylase at 60 degree C, pH 5, for 1 hour. Isomerization with Mg/Al hydrotalcite took placed in 50 ml hydrolysate sugar, 1 percent (b/v) catalyst, and 100 degree C while process with glucose isomerase was at 100 ml substrate, 60 degree C, pH 8.2, and same catalyst ratio (1 percent). Isomerization process for each catalyst would be hold in 3, 5, and 7 hours. The best result of isomerization productivity was 39.29 g/L. h for process with Mg/Al hydrotalcite catalyst, while isomerization yield was 6.18 percent for process with glucose isomerization enzyme.

Recombinant xylose-fermenting yeast construction for the co-production of ethanol and cis,cis-muconic acid from lignocellulosic biomass

New exogenous cis,cis-muconic acid biosynthetic pathway genes were expressed in Saccharomyces cerevisiae. The xylose isomerase gene from Bacteroides valgutus and pentose phosphate pathway genes from S. cerevisiae were overexpressed in the yeast strain. The strain was further modified by the overexpression of gene Aro1 (with a stop codon of AroE) and a feedback-resistant Aro4opt mutant gene from S. cerevisiae. Under oxygen-limited conditions, it produced 65 mg/L cis,cis-muconic acid from xylose. Co-fermentation of 88 g/L glucose and 50 g/L xylose generated 54 g/L ethanol and 248 mg/L cis,cis-muconic acid. Under aerobic conditions, muconic acid titer reached 424 mg/L. With the supplement of 1 g/L catechol, 1286 mg/L muconic acid was produced. Fermentation of an oil palm empty fruit bunch hydrolysate resulted in 31.3 g/L ethanol and 53.4 mg/L muconic acid. This is the first report on the production of muconic acid from lignocellulosic biomass hydrolysate using a recombinant xylose-fermenting yeast.

Enhancing the sweetening power of lactose by enzymatic modification in the reformulation of dairy products

Lactose solutions of up to 50% (w/v) were incubated with lactases and glucose isomerases for subsequent implementation in dairy product samples to enhance sweetness. A degree of hydrolysis of >90% and of isomerisation of 50% were attainable. The sensory sweetening power of lactose in solutions of up to 50% (w/v) can be enhanced 2–3 times. Based on sensory experiments, application of this bi‐enzymatic system in yoghurt and pudding samples allowed for a 10–20% (w/w) reduction in the total sugar content, whilst retaining equal sweetness. The growth of yoghurt starter cultures was not affected, yet furosine formation more than doubled in high heated, enzyme‐modified milk.

Comparison of Isomerase and Weimberg Pathway for γ-PGA Production From Xylose by Engineered Bacillus subtilis.

The production of poly-γ-glutamic acid (γ-PGA), a biopolymer consisting of D- and L-glutamic acid monomers, currently relies on L-glutamate, or citrate as carbon substrates. Here we aimed at using plant biomass-derived substrates such as xylose. γ-PGA producing microorganisms including Bacillus subtilis natively metabolize xylose via the isomerase pathway. The Weimberg pathway, a xylose utilization pathway first described for Caulobacter crescentus, offers a carbon-efficient alternative converting xylose to 2-oxoglutarate without carbon loss. We engineered a recombinant B. subtilis strain that was able to grow on xylose with a growth rate of 0.43 h−1 using a recombinant Weimberg pathway. Although ion-pair reversed-phase LC/MS/MS metabolome analysis revealed lower concentrations of γ-PGA precursors such as 2-oxoglutarate, the γ-PGA titer was increased 6-fold compared to the native xylose isomerase strain. Further metabolome analysis indicates a metabolic bottleneck in the phosphoenolpyruvate-pyruvate-oxaloacetate node causing bi-phasic (diauxic) growth of the recombinant Weimberg strain. Flux balance analysis (FBA) of the γ-PGA producing B. subtilis indicated that a maximal theoretical γ-PGA yield is achieved on D-xylose/ D-glucose mixtures. The results of the B. subtilis strain harboring the Weimberg pathway on such D-xylose/ D-glucose mixtures demonstrate indeed resource efficient, high yield γ-PGA production from biomass-derived substrates.

Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

BackgroundEfficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts.ResultsWe constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal.ConclusionThis study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.

Shortening injection matrix for serial crystallography

Serial crystallography allows crystal structures to be determined at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5–2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments.

Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21.

Cell lysate of Escherichia coli strain BL21 showed significant D-glucose isomerase activity. The rate of glucose conversion was increased up to 40% when cells were induced with 1% D-xylose. E. coli BL21 xylose isomerase (ECXI-BL21) was purified to homogeneity, up to 1.9-fold with overall 10.88% enzyme yield by heat shock, salting out and electro-elution. The molecular mass of ECXI-BL21 was estimated as 43.9kDa on SDS-PAGE. pHopt. and Topt. of the enzyme were calculated as 7.0 and 50°C, respectively. Activation energy (E a) of ECXI-BL21 was 45kJ/mol. Enzyme was stable from 30 to 55°C and at pH range 6.0-8.0. ECXI-BL21(holo) was activated by 10mM magnesium (35%), 0.5mM cobalt (20%) and manganese (25%), and 0.5/10mM Mn2+/Mg2+ (50%) and Co2+/Mg2+ (30%) as compared to ECXI-BL21(apo). Catalytic affinity (K m) of ECXI-BL21 for D-glucose was calculated as 0.82mM, while maximum velocity (V max) of the reaction D-glucose(aldo) ⇌ D-fructose(keto) was 108μmol/mg/min. D-fructose formed was identified on silica gel plate. This thermophilic enzyme, T m = 75°C, has great potential for high fructose syrup production used in food and soft drink industries.

High Gravity Fermentation of Sugarcane Bagasse Hydrolysate by Saccharomyces pastorianus to Produce Economically Distillable Ethanol Concentrations: Necessity of Medium Components Examined

A major economic obstacle in lignocellulosic ethanol production is the low sugar concentrations in the hydrolysate and subsequent fermentation to economically distillable ethanol concentrations. We have previously demonstrated a two-stage fermentation process that recycles xylose with xylose isomerase to increase ethanol productivity, where the low sugar concentrations in the hydrolysate limit the final ethanol concentrations. In this study, three approaches are combined to increase ethanol concentrations. First, the medium-additive requirements were investigated to reduce ethanol dilution. Second, methods to increase the sugar concentrations in the sugarcane bagasse hydrolysate were undertaken. Third, the two-stage fermentation process was recharacterized with high gravity hydrolysate. It was determined that phosphate and magnesium sulfate are essential to the ethanol fermentation. Additionally, the Escherichia coli extract and xylose isomerase additions were shown to significantly increase ethanol productivity. Finally, the fermentation on hydrolysate had only slightly lower productivity than the reagent-grade sugar fermentation; however, both fermentations had similar final ethanol concentrations. The present work demonstrates the capability to produce ethanol from high gravity sugarcane bagasse hydrolysate using Saccharomyces pastorianus with low yeast inoculum in minimal medium. Moreover, ethanol productivities were on par with pilot-scale commercial starch-based facilities, even when the yeast biomass production stage was included.

Mining Xylose Isomerase Producing Microbes

An aerobic, rod shaped, mesophilic, gram positive xylitol producing bacterial isolates were made from different enriched substrates. Xylose isomerase is an enzyme that catalyzes the interconversion of D-xylose and D-xylulose. The microbes producing xylulose from xylose via xylose isomerase enzyme were screened for the xylose isomerase activity based on 2, 3, 5-Triphenyl tetrazolium dye retention capacity. Among the fourteen isolates screened, only six isolates showed the positive results by retaining the dye of which XLY3 was the best. Phylogenetic and sequence analysis of 16SrRNA gene showed 98 % homology to Bacillus endophyticus

Safety evaluation of the food enzyme xylose isomerase from the genetically modified Streptomyces rubiginosus strain DP-Pzn37.

The food enzyme is a d‐xylose aldose‐ketose‐isomerase (EC 5.3.1.5) produced with the genetically modified Streptomyces rubiginosus strain DP‐Pzn37 by Danisco US Inc. Although the production strain contains antibiotic resistance genes, the food enzyme was shown to be free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in an immobilised form for the isomerisation of glucose for the production of high fructose syrups. Residual amounts of total organic solids (TOS) are eliminated by the use of an immobilised food enzyme and further removed by the purification steps applied during the production of high fructose syrups using the immobilised enzyme; consequently, dietary exposure was not calculated. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 85.2 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the immobilisation process and the removal of total organic solids during the production of high fructose syrups, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.