Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6 displays activity on oat fiber xylan for prebiotic production.

Carbohydrate active enzymes are valuable tools in cereal processing to valorize underutilized side streams. By solubilizing hemicellulose and modifying the fiber structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibers: arabinoxylo-oligosaccharides. The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61°C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan. HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, whereas the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat ß-glucan. In contrast to the commercially available homolog CtXyn5A, HhXyn5A gave a more specific HPAEC-PAD oligosaccharide product profile when using wheat arabinoxylan and alkali extracted oat bran fibers as the substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modeling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX and X5 concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to the lack of important ligand-interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.

Engineering Saccharomyces cerevisiae for production of the capsaicinoid nonivamide.

BACKGROUND: Capsaicinoids are produced by plants in the Capsicum genus and are the main reason for the pungency of chili pepper fruits. They are strong agonists of TRPV1 (the transient receptor potential cation channel subfamily V member 1) and used as active ingredients in pharmaceuticals for the treatment of pain. The use of bioengineered microorganisms in a fermentation process may be an efficient route for their preparation, as well as for the discovery of (bio-)synthetic capsaicinoids with improved or novel bioactivities. RESULTS: Saccharomyces cerevisiae was engineered to over-express a selection of amide-forming N-acyltransferase and CoA-ligase enzyme cascades using a combinatorial gene assembly method, and was screened for nonivamide production from supplemented vanillylamine and nonanoic acid. Data from this work demonstrate that Tyramine N-hydroxycinnamoyl transferase from Capsicum annuum (CaAT) was most efficient for nonivamide formation in yeast, outcompeting the other candidates including AT3 (Pun1) from Capsicum spp. The CoA-ligase partner with highest activity from the ones evaluated here were from Petunia hybrida (PhCL) and Spingomonas sp. Ibu-2 (IpfF). A yeast strain expressing CaAT and IpfF produced 10.6 mg L-1 nonivamide in a controlled bioreactor setup, demonstrating nonivamide biosynthesis by S. cerevisiae for the first time. CONCLUSIONS: Baker's yeast was engineered for production of nonivamide as a model capsaicinoid, by expressing N-acyltransferases and CoA-ligases of plant and bacterial origin. The constructed yeast platform holds potential for in vivo biocatalytic formation of capsaicinoids and could be a useful tool for the discovery of novel drugs.

Shared decision-making in hemodialysis vascular access practice.

Shared decision-making (SDM) is a process of collaborative deliberation in the dyadic patient-physician interaction whereby physicians inform the patients about the pros and cons of all available treatment options and reach an agreement with the patients on their preferred treatment plan. In hemodialysis vascular access practice, SDM advocates a deliberative approach based on the existence of reasonable alternatives-that is, arteriovenous fistula, arteriovenous graft, and central venous catheter-so that patients are able to form and share preferences about access options. In spite of its ethical imperative, SDM is not broadly applied in hemodialysis vascular access planning. Physicians and surgeons commonly deliver prescriptive fistula-centered recommendations concerning the approach to vascular access care. This paternalistic approach has been shaped by directions from long-held clinical practice guidelines and is reinforced by financial payment models linked with the prevalence of arteriovenous fistula in patients on hemodialysis. Awareness is growing that what may have initially seemed a medically and surgically appropriate approach might not always be focused on each individual's goals of care. Clinician's recommendations for vascular access often do not sufficiently consider the uncertainty surrounding the potential benefits of the decision or the cumulative impact of the decision on patient's quality of life. In the evolving health care landscape, it is time for the practice of hemodialysis vascular access to shift from a hierarchical doctor-patient approach to patient-centered care. In this article we review the current state of vascular access practice, present arguments why SDM is necessary in vascular access planning, review barriers and potential solutions to SDM implementation, and discuss future research contingent on an effective system of physician-patient participative decision-making in hemodialysis vascular access practice.

Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)-catalyzed transglycosylation.

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of a long-carbohydrate-chain alkyl glycoside and cyclodextrin glucanotransferase (CGTase)-catalyzed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl ß- d-maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase-catalyzed coupling/disproportionation reactions with α-cyclodextrin and dodecyl ß- d-maltoside diuronic acid (DDM-2COOH) or octyl ß- d-glucuronic acid (OG-COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non-ionic substrates (dodecyl ß- d-maltoside and octyl ß- d-glucoside), Toruzyme exhibited high catalytic activity at pH 5-6, but for the acidic substrates (DDM-2COOH and OG-COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM-2COOH and OG-COOH, which exist at lower pH (pKa about 3).

Chemoenzymatic synthesis of the pH responsive surfactant octyl ß-D-glucopyranoside uronic acid.

Methodology was developed to expand the range of benign alkyl glycoside surfactants to include also anionic types. This was demonstrated possible through conversion of the glycoside to its carboxyl derivative. Specifically, octyl ß-D-glucopyranoside (OG) was oxidised to the corresponding uronic acid (octyl ß-D-glucopyranoside uronic acid, OG-COOH) using the catalyst system T. versicolor laccase/2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and oxygen from air as oxidant. The effects of oxygen supply methodology, concentrations of laccase, TEMPO and OG as well as reaction temperature were evaluated. At 10 mM substrate concentration, the substrate was almost quantitatively converted into product, and even at a substrate concentration of 60 mM, 85% conversion was reached within 24 h. The surfactant properties of OG-COOH were markedly dependent on pH. Foaming was only observed at low pH, while no foam was formed at pH values above 5.0. Thus, OG-COOH can be an attractive low-foaming surfactant, for example for cleaning applications and emulsification, in a wide pH range (pH 1.5-10.0).

Effect of cholesterol on the molecular structure and transitions in a clinical-grade lung surfactant extract.

The lipid-protein film covering the interface of the lung alveolar in mammals is vital for proper lung function and its deficiency is related to a range of diseases. Here we present a molecular-level characterization of a clinical-grade porcine lung surfactant extract using a multitechnique approach consisting of [Formula: see text]-[Formula: see text] solid-state nuclear magnetic spectroscopy, small- and wide-angle X-ray scattering, and mass spectrometry. The detailed characterization presented for reconstituted membranes of a lung extract demonstrates that the molecular structure of lung surfactant strongly depends on the concentration of cholesterol. If cholesterol makes up about 11% of the total dry weight of lung surfactant, the surfactant extract adopts a single liquid-ordered lamellar phase, [Formula: see text], at physiological temperatures. This [Formula: see text] phase gradually changes into a liquid-disordered lamellar phase, [Formula: see text], when the temperature is increased by a few degrees. In the absence of cholesterol the system segregates into one lamellar gel phase and one [Formula: see text] phase. Remarkably, it was possible to measure a large set of order parameter magnitudes [Formula: see text] from the liquid-disordered and -ordered lamellar phases and assign them to specific C-H bonds of the phospholipids in the biological extract with no use of isotopic labeling. These findings with molecular details on lung surfactant mixtures together with the presented NMR methodology may guide further development of pulmonary surfactant pharmaceuticals that better mimic the physiological self-assembly compositions for treatment of pathological states such as respiratory distress syndrome.

Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes.

Mycobacteriophages possess different sets of lytic enzymes for disruption of the complex cell envelope of the mycobacteria host cells and release of the viral progeny. Lysin B (LysB) enzymes are mycolylarabinogalactan esterases that cleave the ester bond between the arabinogalactan and mycolic acids in the mycolylarabinogalactan-peptidoglycan (mAGP) complex in the cell envelope of mycobacteria. In the present study, four LysB enzymes were produced recombinantly and characterized with respect to their enzymatic and antibacterial activities. Examination of the kinetic parameters for the hydrolysis of para-nitrophenyl ester substrates, shows LysB-His6 enzymes to be active against a range of substrates (C4-C16), with a catalytic preference towards p-nitrophenyl laurate (C12). With p-nitrophenyl butyrate as substrate, LysB-His6 enzymes showed highest activity at 37 °C. LysB-His6 enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca2+ and Mn2+ enhanced the enzymatic activity of LysB-His6 enzymes, while transition metal ions like Zn2+ and Cu2+ inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His6 enzymes against mAGP complex was confirmed by LC-MS. LysB-His6 enzymes showed marginal antibacterial activity when tested alone against Mycobacterium smegmatis, however a synergetic activity was noticed when combined with outer membrane permealizers. These results confirm that LysB enzymes are lipolytic enzymes with potential application as antimycobacterials.

Self-Assembly in Ganglioside‒Phospholipid Systems: The Co-Existence of Vesicles, Micelles, and Discs.

Ganglioside lipids have been associated with several physiological processes, including cell signaling. They have also been associated with amyloid aggregation in Parkinson's and Alzheimer's disease. In biological systems, gangliosides are present in a mix with other lipid species, and the structure and properties of these mixtures strongly depend on the proportions of the different components. Here, we study self-assembly in model mixtures composed of ganglioside GM1 and a zwitterionic phospholipid, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). We characterize the structure and molecular dynamics using a range of complementary techniques, including cryo-TEM, polarization transfer solid state NMR, diffusion NMR, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and calorimetry. The main findings are: (1) The lipid acyl chains are more rigid in mixtures containing both lipid species compared to systems that only contain one of the lipids. (2) The system containing DOPC with 10 mol % GM1 contains both vesicles and micelles. (3) At higher GM1 concentrations, the sample is more heterogenous and also contains small disc-like or rod-like structures. Such a co-existence of structures can have a strong impact on the overall properties of the lipid system, including transport, solubilization, and partitioning, which can be crucial to the understanding of the role of gangliosides in biological systems.

Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

Development of in situ product removal strategies in biocatalysis applying scaled-down unit operations.

An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi-automatically characterize ω-transaminases in a scaled-down packed-bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two-step liquid-liquid extraction (LLE) ISPR concept was tested using scaled-down unit operations combined in a plug-and-play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 gMPPA · L-1 , a purity up to 70% gMPPA · gtot-1 and a recovery in the range of 80% mol · mol-1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600-609. © 2016 Wiley Periodicals, Inc.

Acceleration of α-synuclein aggregation by exosomes.

Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes, we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins Flotillin-1 and Alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as a low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.

Considerations of prescription opioid abuse and misuse among older adults in West Virginia--An Under-Recognized Population at Risk.

Opioid abuse, misuse and overdose is now a public health epidemic receiving political, medical, and media attention at all levels. Despite the fact that many people know someone suffering from addiction, there is very little research focusing on this issue in older adults. Chronic pain, a highly prevalent affliction for the aging population, has been accompanied by a significant increase in opioid use. This, along with some unique aspects of older adults (increased susceptibility to illness, higher likelihood of altered presentation of illness, and impaired recovery), means that great care needs to be taken when considering opioids for treatment. Prudent prescribing is possible, but universal precautions should be taken to reduce the risk of opioid abuse, misuse, and addiction. This review provides education, summarizes current literature, and gives guidance in universal precautions for prescribing opioids.

A GH57 4-α-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production.

4-α-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of α-glucans by cleaving and reforming α-1,4 glycosidic bonds in α-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long α-glucan chains from dodecyl-ß-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 °C and an activity half-life of 150 min at 90 °C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.

Xylanases and high-degree wet milling improve soluble dietary fibre content in liquid oat base.

The growth of plant-based food and drink substitutes has led to increased interest in oat-based milk substitute as a dairy milk alternative. Conventional liquid oat base (LOB) production results in a fibre-rich insoluble by-product and loss of valuable macronutrients. This study investigates the use of xylanase enzymes to release insoluble arabinoxylan (AX) fibre and employs different degrees of milling in the LOB manufacturing process, with the aim to reduce insoluble waste and simultaneously increase soluble dietary fibre in oat-based milk substitutes. The combination of decreased mill gap space from 1 to 0.05 mm and addition of GH10 xylanase, resulted in a homogenous LOB product and solubilization of all available AX. Potential prebiotic arabinoxylooligosaccharides of DP3-7 from GH10 hydrolysis were identified using HPAEC-PAD and MS analysis. These findings demonstrate the value of utilizing xylanases and fine-milling in LOB manufacturing, offering a sustainable approach to maximize health benefits of oat-based beverages.

Immobilization techniques improve volumetric hydrogen productivity of Caldicellulosiruptor species in a modified continuous stirred tank reactor.

BACKGROUND: Co-cultures and cell immobilization have been used for retaining biomass in a bioreactor, with the aim to improve the volumetric hydrogen productivity (QH2). Caldicellulosiruptor kronotskyensis is a strong cellulolytic species that possesses tapirin proteins for attaching on lignocellulosic materials. C. owensensis has its reputation as a biofilm former. It was investigated whether continuous co-cultures of these two species with different types of carriers can improve the QH2. RESULTS: QH2 up to 30 ± 0.2 mmol L-1 h-1 was obtained during pure culture of C. kronotskyensis with combined acrylic fibres and chitosan. In addition, the yield of hydrogen was 2.95 ± 0.1 mol H2 mol-1 sugars at a dilution rate (D) of 0.3 h-1. However, the second-best QH2 26.4 ± 1.9 mmol L-1 h-1 and 25.4 ± 0.6 mmol L-1 h-1 were obtained with a co-culture of C. kronotskyensis and C. owensensis with acrylic fibres only and a pure culture of C. kronotskyensis with acrylic fibres, respectively. Interestingly, the population dynamics revealed that C. kronotskyensis was the dominant species in the biofilm fraction, whereas C. owensensis was the dominant species in the planktonic phase. The highest amount of c-di-GMP (260 ± 27.3 µM at a D of 0.2 h-1) were found with the co-culture of C. kronotskyensis and C. owensensis without a carrier. This could be due to Caldicellulosiruptor producing c-di-GMP as a second messenger for regulation of the biofilms under the high dilution rate (D) to prevent washout. CONCLUSIONS: The cell immobilization strategy using a combination of carriers exhibited a promising approach to enhance the QH2. The QH2 obtained during the continuous culture of C. kronotskyensis with combined acrylic fibres and chitosan gave the highest QH2 among the pure culture and mixed cultures of Caldicellulosiruptor in the current study. Moreover, it was the highest QH2 among all cultures of Caldicellulosiruptor species studied so far.

Team-Based Hospice Referrals: A Potential Quality Metric for Lung Cancer in the Immunotherapy Era.

BACKGROUND: Immune checkpoint inhibitors (ICIs) can lead to durable responses in patients with lung cancer but may delay transitions to hospice at the end of life (EOL). We aimed to test the association of continuity of care with EOL outcomes in the ICI era. METHODS: We collected retrospective data on all patients with lung cancer who started ICI treatment at a single comprehensive cancer center in the United States (1/1/14-5/1/18) and subsequently died. We defined a hospice referral as having continuity of care if placed by a provider from the patient's multidisciplinary cancer team (e.g., a medical oncologist, palliative care specialist, intensivist, and hospitalist). RESULTS: In this cohort of 143 patients, 58% had a team-based hospice referral which was associated with a lower risk of death in the hospital. The most common reason patients declined hospice at EOL was an unwillingness to discontinue cancer-directed therapy. As compared to a similar historical cohort of patients treated with chemotherapy alone (2008-2010), there was a similar rate of hospice referral (68% vs 74%) but higher rates of new systemic therapy initiated within 30 days of death (17% vs 6%, p .001) and last dose within 14 days of death (13% vs 5%, p .005). CONCLUSIONS: Future studies should test the continuity of care at EOL as a new quality metric for advanced NSCLC.

Clinician Perceptions of Barriers and Facilitators for Delivering Early Integrated Palliative Care via Telehealth.

Early integrated palliative care (EIPC) significantly improves clinical outcomes for patients with advanced cancer. Telehealth may be a useful tool to deliver EIPC sustainably and equitably. Palliative care clinicians completed a survey regarding their perceptions of the barriers, facilitators, and benefits of using telehealth video visits for delivering EIPC for patients with advanced lung cancer. Forty-eight clinicians across 22 cancer centers completed the survey between May and July 2022. Most (91.7%) agreed that telehealth increases access to EIPC and simplifies the process for patients to receive EIPC (79.2%). Clinicians noted that the elderly, those in rural areas, and those with less-resourced backgrounds have greater difficulty using telehealth. Perceived barriers were largely patient-based factors, including technological literacy, internet and device availability, and patient preferences. Clinicians agreed that several organizational factors facilitated telehealth EIPC delivery, including technological infrastructure (85.4%), training (83.3%), and support from study coordinators (81.3%). Other barriers included systems-based factors, such as insurance reimbursement and out-of-state coverage restrictions. Patient-, organization-, and systems-based factors are all important to providing and improving access to telehealth EIPC services. Further research is needed to investigate the efficacy of telehealth EIPC and how policies and interventions may improve access to and dissemination of this care modality.

Towards engineered yeast as production platform for capsaicinoids.

Capsaicinoids are bioactive alkaloids produced by the chili pepper fruit and are known to be the most potent agonists of the human pain receptor TRPV1 (Transient Receptor Potential Cation Channel Subfamily V Member 1). They are currently produced by extraction from chili pepper fruit or by chemical synthesis. Transfer of the biosynthetic route to a microbial host could enable more efficient capsaicinoid production by fermentation and may also enable the use of synthetic biology to create a diversity of new compounds with potentially improved properties. This review summarises the current state of the art on the biosynthesis of capsaicinoid precursors in baker's yeast, Saccharomyces cerevisiae, and discusses bioengineering strategies for achieving total synthesis from sugar.

Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy.

There is a great need for efficient analysis of the composition of vegetable oils and fats, since it affects the physical and technical properties. However, due to the complex nature of these kind of samples, it is often difficult and costly. In the present study, we developed a Non-Aqueous Reversed-Phase HPLC method that can be used to separate and quantify different free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides and triacylglycerides, including regioisomers such as SOS/SSO and 1,2- and 1,3-diolein. Two 25 cm Nucleodur C18 Isis columns in series, sub-ambient column temperature and a mobile phase gradient composed of acetonitrile, acetic acid, isopropanol and heptane were used for the separation. The lipids were detected and quantified using a charged aerosol detector and it was found that the peak shape highly affected the detector response as well as the response uniformity, even when inverse gradient compensation was employed. Thus, calibration and determination of response factors were necessary for reliable quantification. A correlation between response factors and peak width at half peak height was found and used for quantification of non-calibrated components. A quantification approach was suggested including an appropriate selection of calibrated components, depending on sample composition and the accuracy required. It was shown in a complex oil sample that the reduced calibration approach, using only 6 instead of 33 calibrated components, resulted in virtually the same composition, but yielded a more accurate result compared to using relative area that neglects response factors. The method validation showed good reproducibility and accuracy, making it an excellent tool for extensive analysis of complex lipid mixtures.

Characterization of simultaneous uptake of xylose and glucose in Caldicellulosiruptor kronotskyensis for optimal hydrogen production.

BACKGROUND: Caldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C. kronotskyensis in the presence of mixtures of glucose-xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study, we aimed to investigate further whether C. kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). RESULTS: Interestingly, growth of C. kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus, C. kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C. kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (µmax = 0.57 h-1) as on glucose (µmax = 0.28 h-1). A study of the sugar uptake was made with different glucose-xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be KI,glu = 0.01 cmol L-1 and KI,xyl = 0.001 cmol L-1, hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatics analysis could not exclude that C. kronotskyensis possesses more than one transporter for glucose. As a next step it was investigated whether glucose uptake by C. kronotskyensis improved in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose; nevertheless, the growth rate on each sugar remained similar. CONCLUSIONS: C. kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C. kronotskyensis has adapted more strongly to pentose rather than hexose, thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.