Substrate Kinetics Research Articles

Alternative substrate kinetics of SARS-CoV-2 Nsp15 endonuclease reveals a specificity landscape dominated by RNA structure.

Coronavirus endoribonuclease Nsp15 contributes to the evasion of host innate immunity by suppressing levels of viral dsRNA. Nsp15 cleaves both ssRNA and dsRNA in vitro with a strong preference for unpaired or bulged U residues, and its activity is stimulated by divalent ions. Here, we systematically quantified effects of RNA sequence and structure context that define its specificity. The results show that sequence preference for U↓A/G, observed previously, contributes only ca. 2-fold to kcat/Km. In contrast, dsRNA structure flanking a bulged U residue increases kcat/Km by an order of magnitude compared to ssRNA while base pairing in dsRNA essentially blocks cleavage. Despite enormous differences in multiple turnover kinetics, the effect of RNA structure on the cleavage step is minimal. Surprisingly, although divalent ion activation of Nsp15 is widely considered to be important for its biological function, the effect on kcat/Km is only ∼2-fold and independent of RNA structure. These results reveal a specificity landscape dominated by RNA structure and provide a quantitative framework for identifying interactions that underlie specificity, determining mechanisms of inhibition and resistance and defining targets important for coronavirus biology.

Heparosan biosynthesis in recombinant Bacillus megaterium: Influence of N-acetylglucosamine supplementation and kinetic modeling.

Heparosan, an unsulfated polysaccharide, plays a pivotal role as a primary precursor in the biosynthesis of heparin-an influential anticoagulant with diverse therapeutic applications. To enhance heparosan production, the utilization of metabolic engineering in nonpathogenic microbial strains is emerging as a secure and promising strategy. In the investigation of heparosan production by recombinant Bacillus megaterium, a kinetic modeling approach was employed to explore the impact of initial substrate concentration and the supplementation of precursor sugars. The adapted logistic model was utilized to thoroughly analyze three vital parameters: the B. megaterium growth dynamics, sucrose utilization, and heparosan formation. It was noted that at an initial sucrose concentration of 30gL-1 (S1), it caused an inhibitory effect on both cell growth and substrate utilization. Intriguingly, the inclusion of N-acetylglucosamine (S2) resulted in a significant 1.6-fold enhancement in heparosan concentration. In addressing the complexities of the dual substrate system involving S1 and S2, a multi-substrate kinetic models, specifically the double Andrew's model was employed. This approach not only delved into the intricacies of dual substrate kinetics but also effectively described the relationships among the primary state variables. Consequently, these models not only provide a nuanced understanding of the system's behavior but also serve as a roadmap for optimizing the design and management of the heparosan production method.

Conditional Knockout of Glycosaminoglycan Synthesis in Bladder Epithelium Has No Effect on Urinary Voiding in Mice

Glycosaminoglycans (GAGs) on the luminal surface of the bladder epithelium have been suggested to play an important role in barrier function and in protection from infection. There is also a substantial literature that posits a breakdown in the GAG layer may underlie, or be associated with the development of painful bladder syndrome (PBS, aka interstitial cystitis) and indeed, GAG mimetics such as pentosan polysulphate provide symptomatic relief for some patients. However, opinions on the presence and role of GAGs vary, and incontrovertible evidence is lacking. We have taken a unique approach to answering this question by engineering a highly cell-specific genetic mouse model that lacks the obligatory initiating enzyme for GAG synthesis in urothelium. Uroplakin 2-Cre (UP2-Cre) mice were crossed with xylosyltransferase 2-floxed mice to knockout the xylt2 gene in urothelium. RT-PCR confirmed the tissue specificity of the knockout in urothelium but not in bladder smooth muscle. Further, lacZ reporter staining in cryosections demonstrated highly specific expression of the Cre construct in urothelium. Mammals express two xylosyltransferases, XylT1 and XylT2 in different proportions in different tissues. While functionally similar they appear to differ in their substrate specificity and kinetics. qPCR in dissected urothelium demonstrated that XylT2 was the dominant isoform, being expressed at approximately 80-fold higher level than XylT1. Void spot assays (VSA) on filter paper (4 hours) have proven to be a reliable indicator of voiding dysfunction in mice. We conducted VSAs on female Up2 cre/xylt2 fl/fl (n=12) and xylt2 fl/fl (n=11) littermate controls aged 8 – 16 weeks. There was no significant difference in void volumes, number of primary voids, volume/void or number of microvoids (<20 μl) between controls and knockouts, whereas transurethral instillation of protamine sulfate, which injures the apical cell layer, induced pain and a significantly greater number of microvoids, with reduced primary void volumes. We conclude preliminarily, that loss of GAGs in urothelium does not affect bladder function, suggesting that GAGs do not play a role in protecting the suburothelium and adjacent sensory afferents from noxious urine components. Future work will characterize and quantitate the distribution of GAGs within the urothelium to determine whether GAGs are deployed on the luminal surface and, whether they play a role in urothelial repair from injury. NIH grant P20DK097818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel experimental method to determine substrate uptake kinetics of gaseous substrates applied to the carbon monoxide-fermenting Clostridium autoethanogenum.

Syngas fermentation has gained momentum over the last decades. The cost-efficient design of industrial-scale bioprocesses is highly dependent on quantitative microbial growth data. Kinetic and stoichiometric models for syngas-converting microbes exist, but accurate experimental validation of the derived parameters is lacking. Here, we describe a novel experimental approach for measuring substrate uptake kinetics of gas-fermenting microbes using the model microorganism Clostridium autoethanogenum. One-hour disturbances of a steady-state chemostat bioreactor with increased CO partial pressures (up to 1.2 bar) allowed for measurement of biomass-specific CO uptake- and CO2 production rates ( , ) using off-gas analysis. At a pCO of 1.2 bar, a of -119 ± 1 mmol g-1 X h-1 was measured. This value is 1.8-3.5-fold higher than previously reported experimental and kinetic modeling results for syngas fermenters. Analysis of the catabolic flux distribution reveals a metabolic shift towards ethanol production at the expense of acetate at pCO 0.6 atm, likely to be mediated by acetate availability and cellular redox state. We characterized this metabolic shift as acetogenic overflow metabolism. These results provide key mechanistic understanding of the factors steering the product spectrum of CO fermentation in C. autoethanogenum and emphasize the importance of dedicated experimental validation of kinetic parameters.

Electrochemical Monitoring of Heterogeneous Peroxygenase Reactions Unravels LPMO Kinetics.

Biological conversion of plant biomass depends on peroxygenases and peroxidases acting on insoluble polysaccharides and lignin. Among these are cellulose- and hemicellulose-degrading lytic polysaccharide monooxygenases (LPMOs), which have revolutionized our concept of biomass degradation. Major obstacles limiting mechanistic and functional understanding of these unique peroxygenases are their complex and insoluble substrates and the hard-to-measure H2O2 consumption, resulting in the lack of suitable kinetic assays. We report a versatile and robust electrochemical method for real-time monitoring and kinetic characterization of LPMOs and other H2O2-dependent interfacial enzymes based on a rotating disc electrode for the sensitive and selective quantitation of H2O2 at biologically relevant concentrations. The H2O2 sensor works in suspensions of insoluble substrates as well as in homogeneous solutions. Our characterization of multiple LPMOs provides unprecedented insights into the substrate specificity, kinetics, and stability of these enzymes. High turnover and total turnover numbers demonstrate that LPMOs are fast and durable biocatalysts.

Study of composition of hydrogels and bioactive zinc (II) MOFs on plant tissue growth

In this work, we report the synthesis of metal-organic frameworks (MOFs) using zinc (II) and the amino acids L-phenylalanine (Phe), L-tryptophan (Trp), and L-histidine (His) as ligands. They were incorporated in hydrogel matrixes comprised of collagen, starch, green and red tomato cells to obtain composite hydrogels. The cell migration into the polymer matrices, the resistance to degradation and in vitro biocompatibility of hydrogels was studied. The cell migration assay showed a high confluence of plant cells, indicating a favorable environment for cell migration and proliferation in the presence of MOF-Zn(His) and MOF-Zn(Phe). During a degradation kinetics in vegetable substrate for 10 days, a rapid loss of water was observed in the first two days, indicating a remarkable water absorption capacity in the hydrogels. The results of metabolic activity in red and green tomato cells revealed a significant increase, exceeding 164±12 %, suggesting promising cell development in this type of composite hydrogels. These results support the potential of hydrogels synthesized with MOFs in agricultural and biotechnological applications, opening perspectives for future research and applications in plant tissue engineering.

Differences in venous clot structures between hemophilic mice treated with emicizumab versus factor VIII or factor VIIIFc.

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab prophylaxis led us to employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We therefore analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab than in the presence of rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab than as for rFVIII/rFVIIIFc. Scanning electron microscopy and immunofluorescent spinning-disk confocal microscopy of in vivo-generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFcand emicizumab-induced clots. Contrary to the regular fibrin mesh obtained with rFVIII/rFVIIIFc, fibrin fibers appeared to be fused into large patches upon emicizumab treatment. Moreover, fewer red blood cells were detected in regions in which these fibrin patches were present. The presence of highly dense fibrin structures associated with a diffuse fiber structure in emicizumab-induced clots was also observed when using super-resolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

Application of decomposition and integral manifolds to the singularly perturbed problem of kinetics of suicide substrate

Application of decomposition and integral manifolds to the singularly perturbed problem of kinetics of suicide substrate

Impact of P-Glycoprotein-Mediated Drug-Endogenous Substrate Interactions on Androgen and Blood-Brain Barrier Permeability

This report focuses on pharmacokinetic drug–endogenous substrate interactions (DEIs). We hypothesized that P-glycoprotein (P-gp)-mediated DEI might affect androgen kinetics, especially its blood–brain barrier (BBB) permeability. The intracellular accumulation of the endogenous substrates of P-gp, testosterone (TES) and androstenedione (ADO) was increased by several tested drugs in uptake studies using P-gp overexpressing cells, indicating that these drugs inhibit P-gp-mediated efflux of TES of ADO from the cells. In a transport study using rat BBB kit, we found that the BBB limited the penetration of TES and ADO into the central nervous system. In addition, tested drugs that cause DEI were found to increase BBB permeability of TES and ADO via P-gp inhibition. In short, this study provides new findings regarding the possibility that DEI may affect the kinetics of endogenous substrates of P-gp.

An amperometric biosensor and its steady state current in the case of substrate and product inhibition: Taylors series method and Adomian decomposition method

In this paper, an amperometric biosensor model with substrate and product inhibition kinetics is analysed. This model is a steady-state system of reaction-diffusion equations with non-linear terms related to non-Michaelis-Menten kinetics of an enzymatic reaction. We present the approximate analytical expression of the substrate and product concentrations using well-founded methods, namely the Taylors series method (TSM) and the Adomian decomposition method (ADM). These methods proved that they fit for all values of parameters in this model. The steady-state biosensor current, biosensor substrate sensitivity and resistance are also discussed. We also present the numerical solution of the described model using MATLAB programming, and it is noted that there is satisfactory agreement in comparing the analytical solution with numerical results for all possible values of parameters. The effects of the parameters, such as inhibition constants, diffusion parameters, bulk concentration and Michaelis-Menten constant on the sensitivity and the resistance of the biosensor are analysed.

Developing an in-house colorimetric method for detecting organophosphate and carbamate residue using cricket cholinesterase

BackgroundPesticides such as organophosphates and carbamates are used to control pests, leaving residues on fruits and vegetables. The assessment of residues in food materials is required for consumer and agricultural worker health protection. The purpose of this study was to develop an in-house colorimetric method for detecting organophosphate and carbamate residues using cricket cholinesterase that can be used as a low-cost, simple-to-use, and widely used test.ResultsThe method’s parameters, including substrate specificity, kinetics, and incubation time, were optimized. The results indicated that acetylthiocholine iodide was more active than butyrylthiocholine iodide. Using a buffer with a pH of 8.0 resulted in the greatest activity after 5 min. The same enzyme activity was observed at 25 °C and 30 °C, which was greater than at 35 °C and 40 °C. In this study, the Km and Vmax values for cricket cholinesterase were 3.75 mM and 35 µM/min, respectively. The specificity and purification factor of the produced ChE were 1.54 µM/min/mg and 1.43-fold, respectively. The limit of detection for detecting organophosphates and carbamates was in the range of 0.002–0.877 ppm. The validity of the method yielded the following results: relative sensitivity of 95%, relative specificity of 90%, relative accuracy of 93%, positive predictive value of 95%, and negative predictive value of 90%.ConclusionsThe developed method is simple and inexpensive, and it can analyze organophosphates and carbamates in a variety of vegetables at an appropriate detection level. Cholinesterase from cricket has the potential to be used for the development of a variety of methods for detecting residues, and the sensitivity could be enhanced using new techniques in the future.Graphical

Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5

In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarial Plasmodium falciparum transporter PfHT1, which is uniquely able to transport a wide range of different sugars. PfHT1 was captured in an intermediate ‘occluded’ state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity in PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resembles PfHT1. The coordination of D-fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D-fructose is consistent with biochemical analysis. Rather than a substrate-binding site that achieves strict specificity by having a high affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.

Structure and function characterization of the α-L-arabinofuranosidase from the white-rot fungus Trametes hirsuta

α-L-Arabinofuranosidases (Abfs) play a crucial role in the degradation of hemicelluloses, especially arabinoxylans (AX). Most of the available characterized Abfs are from bacteria, while fungi, as natural decomposers, contain Abfs with little attention given. An arabinofuranosidase (ThAbf1), belonging to the glycoside hydrolase 51 (GH51) family, from the genome of the white-rot fungus Trametes hirsuta, was recombinantly expressed, characterized, and functionally determined. The general biochemical properties showed that the optimal conditions for ThAbf1 were pH 6.0 and 50°C. In substrate kinetics assays, ThAbf1 preferred small fragment arabinoxylo-oligosaccharides (AXOS) and could surprisingly hydrolyze di-substituted 23,33-di-L-arabinofuranosyl-xylotriose (A2,3XX). It also synergized with commercial xylanase (XYL) and increased the saccharification efficiency of arabinoxylan. The crystal structure of ThAbf1 indicated the presence of an adjacent cavity next to the catalytic pocket which led to the ability of ThAbf1 to degrade di-substituted AXOS. The narrow binding pocket prevents ThAbf1 from binding larger substrates. These findings have strengthened our understanding of the catalytic mechanism of GH51 family Abfs and provided a theoretical foundation for the development of more efficient and versatile Abfs to accelerate the degradation and biotransformation of hemicellulose in biomass. KEY POINTS: • ThAbf1 from Trametes hirsuta degraded di-substituted arabinoxylo-oligosaccharide. • ThAbf1 performed detailed biochemical characterization and kinetics. • ThAbf1 structure has been obtained to illustrate the substrate specificity.

Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid–base catalysis and substrate-dependent metal ion activation

Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.

Comparing VFA Composition, Biomethane Potential, and Methane Production Kinetics of Different Substrates for Anaerobic Fermentation and Digestion

Solid waste is one of the largest sources of greenhouse gases (GHGs) today. The carbon footprint of landfills also has a large impact on global warming. Therefore, it is becoming more urgent to study the possibility of better environmentally friendly approaches for solid waste management and its safe disposal. The digestion of solid waste is a biological process that breaks down the organic content of the solid waste and thus stabilizes it. It also allows the recovery of valuable resources (such as biogas) and the utilization of stabilized waste in various industries. In this study, six substrates were studied to determine their biomethane potential (BMP) in anaerobic digestion. The substrates were fermented and digested anaerobically, and the biogas production was measured. The methane yield of food waste substrates had a higher methane yield between 354 and 347 mL/g-TCOD, and a biodegradability of 89–87%. Wastewater sludge substrates yielded between 324 and 288 mL/g-TCOD with a biodegradability of 81–73%. A kinetics analysis using first-order and Gompertz models was performed for biodegradation and methane production.

In vitro gas production kinetics are influenced by grain processing, flake density, starch retrogradation, and Aspergillus oryzae fermentation extract containing α-amylase activity.

Grain processing such as particle size, flake density, or starch retrogradation can influence ruminal degradability characteristics; however, it is unclear how exogenous α-amylase supplementation interacts with different processed grains. Four experiments were conducted to compare the effects of Aspergillus oryzae fermentation extract (Amaize; Alltech Biotechnology Inc., Nicholasville, KY) supplementation on in vitro gas production kinetics of grain substrates with different processing methods that are common in the feedlot industry. In experiment 1, corn processing (dry-rolled, high-moisture, steam-flaked) and Amaize supplementation (0 or 15 U α-amylase activity/100 mL) were evaluated in a 3 × 2 factorial arrangement of treatments. The rate of gas production for dry-rolled corn was higher (P < 0.001) with Amaize supplementation. In experiment 2, flake density (296, 322, 348, 373, and 399 g/L) and starch retrogradation (storage in heat-sealed foil bags for 3 d at 23 or 55°C) were evaluated in a 5 × 2 factorial arrangement of treatments. There was a flake density × starch retrogradation interaction (P < 0.01) for the rate of gas production because the decrease in the rate of gas production with starch retrogradation was greater at lighter flake densities compared with heavier flake densities. In experiment 3, Amaize supplementation was evaluated across flake densities of nonretrograded steam-flaked corn (stored at 23°C) used in experiment 2. There was a flake density × Amaize interaction (P < 0.01) for the rate of gas production where Amaize supplementation resulted in a lower rate of gas production at lighter flake densities (296, 322, and 348 g/L) but a higher rate of gas production at heavier flake densities (373 and 399 g/L). In experiment 4, Amaize supplementation was evaluated across flake densities of retrograded steam-flaked corn (stored at 55°C) used in experiment 2. Gas production was lower after 24 h with Amaize supplementation for retrograded flakes produced to a density of 322 and 399 g/L while Amaize supplementation did not influence gas production at 24 h at other flake densities. There was a flake density × Amaize interaction for the rate of gas production because Amaize supplementation resulted in a faster (P < 0.01) rate of gas production for all flake densities except retrograded flakes produced to a density of 296 g/L. Enzymatic starch availability was positively correlated with the rate of gas production. These data demonstrate that supplementation of 15 U/100 mL of Amaize resulted in greater rates of gas production for dry-rolled corn, corn steam-flaked to heavier densities, and retrograded steam-flaked corn.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-β-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.

In vitro methods to assess 11β-hydroxysteroid dehydrogenase type 1 activity.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive 11-keto-glucocorticoids to their active 11β-hydroxylated forms. It also catalyzes the oxoreduction of other endogenous and exogenous substrates. The ubiquitously expressed 11β-HSD1 shows high levels in liver and other metabolically active tissues such as brain and adipose tissue. Pharmacological inhibition of 11β-HSD1 was found to ameliorate adverse metabolic effects of elevated glucocorticoids in rodents and humans, improve wound healing and delay skin aging, and enhance memory and cognition in rodent Alzheimer's disease models. Thus, there is an interest to develop 11β-HSD1 inhibitors for therapeutic purposes. This chapter describes in vitro methods to assess 11β-HSD1 enzyme activity for different purposes, be it in disease models, for the assessment of the kinetics of novel substrates or for the screening and characterization of inhibitors. 11β-HSD1 protein expression and preparations of the different biological samples are discussed first, followed by a description of a well-established and easily adaptable 11β-HSD1 enzyme activity assay. Finally, different readout methods are shortly described. This chapter should provide the reader with a toolbox of methods to assess 11β-HSD1 activity with instructions in the form of a decision tree for the choice and implementation of an appropriate enzyme activity assay.

On the Use of the Adsorption Energy Distribution for the Analysis of Competing Substrate Kinetics

The use of renewable raw materials poses new challenges for the chemical and biotechnological industry. For the analysis of the reaction kinetics, elaborate procedures are necessary as the materials are more complex. Thus, a question arises whether interdisciplinary approaches may help to meet these challenges. In this study, the estimation of the adsorption energy distribution (AED) is used to characterize enzyme kinetics and, in particular, competing substrate kinetics for the example of xylose isomerase. Therefore, the kinetic parameters of two literature models are determined based on nonlinear regression and compared with parameter estimates derived from the estimation of the AED. It was shown that based on the AED, the number of competing substrates can be identified automatically, while this information must be provided a priori for the correct identification of the parameters of the literature models via direct nonlinear regression. Thus, the AED provides a promising tool for the characterization of competing substrate kinetics.

On the sliding wear and solid particle erosion behaviour of HVOF-sprayed CoNiCrAlY coatings and NiCrCoTi substrates in dependence of the oxidation dwell time at 900 °C

In high temperature sections up to 900 °C overlay MCrAlY coatings are frequently used to increase the life time of Ni based components in technical applications. Beside oxidation protection, coatings resistance to sliding wear and solid particle erosion is required during different service operation regimes. In the present work the focus is set on the wear behaviour of HVOF-sprayed CoNiCrAlY coatings deposited on a NiCrCoTi substrate (Nimonic 90) and of uncoated substrate, before and after oxidation for different dwell times up to 1200 h at 900 °C. The samples were investigated in respect to their behaviour under dry sliding wear conditions and solid particle erosion at ambient temperatures. Sliding wear was tested by means of pin-on-disc method. Solid particle erosion was conducted using an in-house developed air jet erosion testing rig. Moreover, microhardness indentations HV 0.1 were performed in order to determine changes of materials hardness depending on the heat exposure. Microstructural and phase analysis was performed by means of scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX) and by X-ray diffraction (XRD). Confocal laser scanning microscopy (CLSM) was used to measure thickness, hardness indentations and wear degree of the surfaces. Samples mass losses were considered as well. Coatings performance is pointed out in comparison with that of the uncoated NiCrCoTi substrate, set to same conditions. It could be shown, that the sliding wear and particle erosion behaviour of the investigated samples strongly depends on materials microstructure and chemical composition. The accelerated oxidation reaction kinetics of NiCrCoTi substrate lead to the formation of a thicker oxide scale, which exhibited protective effects of a glaze-layer during sliding wear. However, the oxide scales do not benefit the protection of solid particle erosion.