Multiplicity of carotene patterns derives from competition between phytoene desaturase diversification and biological environments

Phytoene desaturases catalyse from two to six desaturation reactions on phytoene, generating a large diversity of molecules that can then be cyclised and produce, depending on the organism, many different carotenoids. We constructed a phylogenetic tree of a subset of phytoene desaturases from the CrtI family for which functional data was available. We expressed in a bacterial system eight codon optimized CrtI enzymes from different clades. Analysis of the phytoene desaturation reactions on crude extracts showed that three CrtI enzymes can catalyse up to six desaturations, forming tetradehydrolycopene. Kinetic data generated using a subset of five purified enzymes demonstrate the existence of characteristic patterns of desaturated molecules associated with various CrtI clades. The kinetic data was also analysed using a classical Michaelis–Menten kinetic model, showing that variations in the reaction rates and binding constants could explain the various carotene patterns observed. Competition between lycopene cyclase and the phytoene desaturases modified the distribution between carotene intermediates when expressed in yeast in the context of the full β-carotene production pathway. Our results demonstrate that the desaturation patterns of carotene molecules in various biological environments cannot be fully inferred from phytoene desaturases classification but is governed both by evolutionary-linked variations in the desaturation rates and competition between desaturation and cyclisation steps.

Test-System for Bacteria Sensing Based on Peroxidase-Like Activity of Inkjet-Printed Magnetite Nanoparticles

Rapid detection of bacterial contamination is an essential task in numerous medical and technical processes and one of the most rapidly developing areas of nano-based analytics. Here, we present a simple-to-use and special-equipment-free test-system for bacteria detection based on magnetite nanoparticle arrays. The system is based on peroxide oxidation of chromogenic substrate catalyzed by magnetite nanoparticles, and the process undergoes computer-aided visual analysis. The nanoparticles used had a pristine surface free of adsorbed molecules and demonstrated high catalytic activities up to 6585 U/mg. The catalytic process showed the Michaelis–Menten kinetic with Km valued 1.22 mmol/L and Vmax of 4.39 µmol/s. The nanoparticles synthesized were used for the creation of inkjet printing inks and the design of sensor arrays by soft lithography. The printed sensors require no special equipment for data reading and showed a linear response for the detection of model bacteria in the range of 104–108 colony-forming units (CFU) per milliliter with the detection limit of 3.2 × 103 CFU/mL.

Modulation of plant Acetyl CoA Synthetase activity by acetylation

ABSTRACTAcetyl-CoA synthetase (ACS) is one of several enzymes that generate the key metabolic intermediate, acetyl-CoA. In microbial cells ACS generates the precursor for fatty acid and polyketide biosynthesis, and is regulated by the post-translational acetylation of a key lysine residue that inhibits catalytic activity. In contrast, ACS in plant cells is part of a two-enzyme system that maintains acetate homeostasis. Despite these different metabolic roles, this study demonstrates that the plant ACS can also be regulated by the acetylation of a specific lysine residue. The lysine residue that is targeted for this post translational modification reaction is positioned in a homologous region of the microbial and plant ACS sequences, occurring in the middle of a conserved motif. The inhibitory effect of the acetylation of residue Lys-622 of the Arabidopsis ACS was demonstrated by site-directed mutagenesis of this residue, including its genetic substitution with the non-canonical N-ε-acetyl-lysine residue. These modifications lowered the catalytic efficiency of the enzyme by a factor of more than 500-fold, and Michaelis-Menten kinetic analysis of the mutant enzyme indicates that this acetylation affects the first half-reaction of the ACS catalyzed reaction, namely the formation of the acetyl adenylate enzyme intermediate.

Development of New Biokinetic-Dosimetric Models for the Simulation of Iodine Blockade in the Case of Radioiodine Exposure in Man

In the case of nuclear incidents, radioiodine may be liberated. After incorporation it accumulates in the thyroid and by internal irradiation enhances the risk of cancer occurrence. By administering a large dose of non-radioactive iodine the uptake of radioiodine into the gland can be inhibited (“iodine blockade”). Biokinetic models using first order kinetics are not suited to simulate iodine blockade, as the uptake into the gland is mediated by a saturable active transport. Therefore, we integrated an uptake mechanism described by a Michaelis-Menten kinetic into a simple ICRP biokinetic model. We moreover added a total uptake blocking mechanism representing the Wolff-Chaikoff effect becoming active when the gland is saturated with iodine. The validity of the model was ascertained by comparison with IMBA software. The competition of radioiodine and stable iodine at the membrane carrier site was modeled according to the rate law for monomolecular reactions for competing substrates. Our simulations show that competition for the uptake at the membrane carrier site accounts for about 60% and the saturation of the gland with iodine for over 35% of the total protective efficacy that exceeds 95%. Following acute radioiodine exposure, it is preferable to administer a single large dose of stable iodine. In the case of continuous radioiodine exposure, a single dose of stable iodine is less effective than after an acute exposure and splitting the total available dose and shortening the dosage intervals enhance efficacy. Model-based simulations may be a useful tool to develop antidote dosage schemes for uncommon emergencies.

Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450

NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.

Microwave Irradiation-Assisted Chitosan Hydrolysis Using Cellulase Enzyme

The influence of microwave irradiation on the chitosan hydrolysis catalyzed by cellulase enzyme was studied. The hydrolyzed chitosan was characterized by measuring its viscosity and reducing sugar. Further, it was also characterized by Fourier-Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD), and Scanning Electron Microscope (SEM). The classical Michaelis-Menten kinetic parameters were measured by analyzing the amount of reducing sugars. The results were compared with the hydrolysis by using conventional shaker incubator. The hydrolysis reaction time needed to obtain similar reducing sugar yield was significantly lower for microwave irradiation than shaker incubator. On the other hand, the reduction rate of the relative viscosity was significantly higher for the hydrolysis of chitosan using shaker incubator. A significant difference in chemical structure was observed between hydrolysis using microwave irradiation and shaker incubator. Overall, the result showed that the hydrolysis behavior of chitosan using microwave irradiation is significantly different with using shaker incubator. Copyright © 2018 BCREC Group. All rights reservedReceived: 19th March 2018; Revised: 19th June 2018; Accepted: 25th June 2018How to Cite: Rokhati, N., Pramudono, B., Istirokhatun, T., Susanto, H. (2018). Microwave Irradiation-Assisted Chitosan Hydrolysis Using Cellulase Enzyme. Bulletin of Chemical Reaction Engineering & Catalysis, 13 (3): 466-474 (doi:10.9767/bcrec.13.3.2378.466-474)Permalink/DOI: https://doi.org/10.9767/bcrec.13.3.2378.466-474

Enzyme kinetics from circular dichroism of insulin reveals mechanistic insights into the regulation of insulin-degrading enzyme

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that selectively degrades biologically important substrates associated with type 2 diabetes and Alzheimer’s disease (AD). As such, IDE is an attractive target for therapeutic innovations. A major requirement is an understanding of how other molecules present in cells regulate the activity of the enzyme toward insulin, IDE’s most important physiologically relevant substrate. Previous kinetic studies of the IDE-dependent degradation of insulin in the presence of potential regulators have used iodinated insulin, a chemical modification that has been shown to alter the biological and biochemical properties of insulin. Here, we present a novel kinetic assay that takes advantage of the loss of helical circular dichroic signals of insulin with IDE-dependent degradation. As proof of concept, the resulting Michaelis–Menten kinetic constants accurately predict the known regulation of IDE by adenosine triphosphate (ATP). Intriguingly, we found that when Mg2+ is present with ATP, the regulation is abolished. The implication of this result for the development of preventative and therapeutic strategies for AD is discussed. We anticipate that the new assay presented here will lead to the identification of other small molecules that regulate the activity of IDE toward insulin.

Novel DyP from the basidiomycete Pleurotus sapidus: substrate screening and kinetics

A novel Dye-decolorizing peroxidase from the basidiomycete Pleurotus sapidus was screened for dyedecolorizing peroxidase activity with 2,2‘-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid), Remazol Brilliant Blue R and Guaiacol. Additionally, the catalytic efficiency on degrading β-carotene into volatile products, and the catalyst storage stability with three different additives were also studied. The apparent inhibition constant (KS) was 51.7 μM. Optimal reaction rates (Vmax) and affinity constants (Km) towards the reducing substrates were obtained using Michaelis-Menten kinetic theory. The trend in the calculated Km’s was found to be 7.0 mM > 0.524 mM > 0.051 mM for Guaiacol, 2,2‘-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and Remazol Brilliant Blue R. The storage stability of the catalyst was evaluated with 7.0% w/v PEG400, 7.0% w/v PEG1450 and 0.1% w/v Tween®80 at 5°C over a period of 45 days. The study revealed the longest activity conservation with PEG1450, where rDyP had lost 30% of initial activity. The enzyme solution presented similar pH and temperature dependence to known fungal dye-decolorizing peroxidases with most prolific enzymatic activities registered at pH 4.0 and temperatures below 30°C. An interesting property of the catalyst was oxidation observed in the absence of hydrogen peroxide.

Optimization and Kinetic Modelling of The Enzymatic Hydrolysis of Oil Palm Petioles

Oil palm petiole is the solid waste of the crude palm oil industry. It contains about 35% cellulose, 18% hemicellulose and 22-25% lignin. During hydrolysis lingo celllulosic, cellulose and hemicellulose are gradually degraded into fermentable sugars, such as glucose and xylose. Enzymatic hydrolysis of oil palm petioleby xylanase could be an effective biotechnological process, since it can be performed at ambient temperature and pressure. Further glucose and xylose can be used as raw material for the production of a wide variety of chemicals such as xylitol and bioethanol. The aim of this study wasto examine the optimum conditions needed for the enzymatic hydrolysis of oil palm petioles, particularly temperature and pH. A surface Response Method Methodologies (RSM) by central composite design (CCD) was employed to obtain the optimum xylose concentration. The dynamics of enzymatic hydrolysis process was modelled using the Michaelis Menten kinetic model with kinetic parameters obtained from experimental data. The results of this study lead to an enhanced process of the enzymatic hydrolysis of oil palm petiole, whichwas shown to follow the Michaelis Menten kinetic model and the kinetic parameters including Km and Vm were obtained, they were 6.433 g/L andVm= 0.042 g/L/min. The optimum hydrolysis condition wereobserved to be at temperature 50oC and pH 4.8. Keywords: enzymatic hydrolysis; glucose; kinetic modelling; oil palm petioles; xylose

Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant (Km) was 1.7 mM with a kcat of 75 s−1. Two crystal structures are presented, the apoPvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310–320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Finally, these results contribute to knowledge of mechanistic details of the function of arginine kinase.