Low Enzyme Concentrations Research Articles

Prevalence, incidence, and clinical features of cardiac involvement in patients with pulmonary sarcoidosis.

The epidemiology and characteristics of cardiac involvement in patients with pulmonary sarcoidosis remain unclear. We aimed to determine the prevalence, incidence, and clinical features of cardiac sarcoidosis in patients with pulmonary sarcoidosis. The characteristics of patients with biopsy-proven pulmonary sarcoidosis were retrospectively evaluated. Cardiac sarcoidosis was diagnosed via evaluations, including 18F-fluorodeoxyglucose positron emission tomography at the time of diagnosis of pulmonary sarcoidosis and during follow-up. Characteristics of patients with and without cardiac complications were compared. In total, 438 patients with pulmonary sarcoidosis were included, of which 40 (9.1%) were diagnosed with cardiac sarcoidosis at the time of diagnosis of pulmonary sarcoidosis. During the follow-up period, 14 patients (4%) developed cardiac complications (0.0075/person-years). Electrocardiographic abnormalities were the most common findings leading to the diagnosis of cardiac sarcoidosis (85%). Compared to patients without cardiac involvement, those with cardiac sarcoidosis had lower serum angiotensin converting enzyme concentration [19.9 (15.5 - 25.1) vs. 17.4 (12.6 - 23.8) U/L)], higher rates of kidney complications (3 vs. 13%), fewer ocular complications (78 vs. 17%), and lower lymphocyte levels [35.8 (18.6 - 53) vs. 25.1 (14.2 - 38.2)%] and CD4/CD8 ratios [4.8 (3.1 - 7.5)% vs. 3.9 (1.8 - 6)%] in bronchoalveolar lavage fluid analysis. At the time of diagnosis of pulmonary sarcoidosis, cardiac complications occurred in approximately 10% of the patients and developed in 0.0075/person-year during follow-up. Low serum angiotensin converting enzyme concentration, lymphocyte level, and CD4/CD8 ratio in the bronchoalveolar lavage fluid may be unique features of patients with cardiac sarcoidosis.

Cipaglucosidase alfa plus miglustat: linking mechanism of action to clinical outcomes in late-onset Pompe disease.

Enzyme replacement therapy (ERT) is the only approved disease-modifying treatment modality for Pompe disease, a rare, inherited metabolic disorder caused by a deficiency in the acid α-glucosidase (GAA) enzyme that catabolizes lysosomal glycogen. First-generation recombinant human GAA (rhGAA) ERT (alglucosidase alfa) can slow the progressive muscle degeneration characteristic of the disease. Still, most patients experience diminished efficacy over time, possibly because of poor uptake into target tissues. Next-generation ERTs aim to address this problem by increasing bis-phosphorylated high mannose (bis-M6P) N-glycans on rhGAA as these moieties have sufficiently high receptor binding affinity at the resultant low interstitial enzyme concentrations after dosing to drive uptake by the cation-independent mannose 6-phosphate receptor on target cells. However, some approaches introduce bis-M6P onto rhGAA via non-natural linkages that cannot be hydrolyzed by natural human enzymes and thus inhibit the endolysosomal glycan trimming necessary for complete enzyme activation after cell uptake. Furthermore, all rhGAA ERTs face potential inactivation during intravenous delivery (and subsequent non-productive clearance) as GAA is an acid hydrolase that is rapidly denatured in the near-neutral pH of the blood. One new therapy, cipaglucosidase alfa plus miglustat, is hypothesized to address these challenges by combining an enzyme enriched with naturally occurring bis-M6P N-glycans with a small-molecule stabilizer. Here, we investigate this hypothesis by analyzing published and new data related to the mechanism of action of the enzyme and stabilizer molecule. Based on an extensive collection of in vitro, preclinical, and clinical data, we conclude that cipaglucosidase alfa plus miglustat successfully addresses each of these challenges to offer meaningful advantages in terms of pharmacokinetic exposure, target-cell uptake, endolysosomal processing, and clinical benefit.

Microbial transglutaminase in cashew‐based vegan cheese: an innovative approach in achieving ideal texture and meltability

SummaryThe aim of this study was to investigate the effects of microbial transglutaminase (MTGase) on the textural properties and meltability of vegan cheese formulated using cashew paste, tapioca starch, coconut oil, and pea protein. The parameters were evaluated at various MTGase concentrations (0.1, 0.25, 0.5, 0.75, and 1%) and different incubation times (30, 60, and 90 min). A MTGase concentration of 0.25% resulted in higher meltability Index of 3.02 ± 0.14 and acceptable hardness of 3479.44 ± 14.49 g at 30 min of incubation time. However, prolonged incubation times were found to adversely affect the texture and meltability of the cheese. A similar adverse effect on texture was observed at an MTGase concentration of 0.1% at 60 and 90 min of incubation. The results revealed that lower MTGase concentrations with longer incubation times produced similar effects as higher MTGase concentrations with shorter incubation times. However, at a lower enzyme concentration of 0.1%, meltability was significantly higher (3.24 ± 0.33, P < 0.05) at 60 min of incubation compared to 90 min. The findings suggest that vegan cheese with improved texture, meltability, and sensory properties can be developed by combining cashew, tapioca starch, coconut oil, and pea protein in a ratio of 50:25:20:10, with 1% each of carrageenan and agar, and 60% water, and incubating the mixture with 0.25% MTGase for 30 min at 40 °C.

Boosting Chemiexcitation of Phenoxy-1,2-dioxetanes through 7-Norbornyl and Homocubanyl Spirofusion.

The chemiluminescent light-emission pathway of phenoxy-1,2-dioxetane luminophores is increasingly attracting the scientific community's attention. Dioxetane probes that undergo rapid, flash-type chemiexcitation demonstrate higher detection sensitivity than those with a slower, glow-type chemiexcitation rate. This is primarily because the rapid flash-type produces a greater number of photons within a given time. Herein, we discovered that dioxetanes fused to 7-norbornyl and homocubanyl units present accelerated chemiexcitation rates supported by DFT computational simulations. Specifically, the 7-norbornyl and homocubanyl spirofused dioxetanes exhibited a chemiexcitation rate 14.2-fold and 230-fold faster than that of spiro-adamantyl dioxetane, respectively. A turn-ON dioxetane probe for the detection of the enzyme β-galactosidase, containing the 7-norbornyl spirofused unit, exhibited an S/N value of 415 at a low enzyme concentration. This probe demonstrated an increase in detection sensitivity toward β-galactosidase expressing bacteria E. coli with a limit-of-detection value that is 12.8-fold more sensitive than that obtained by the adamantyl counterpart. Interestingly, the computed activation free energies of the homocubanyl and 7-norbornyl units were correlated with their CCsC spiro-angle to corroborate the measured chemiexcitation rates.

Mechanistic Investigation of Enzyme Triggered Release from a Xyloglucan Matrix Tablet for Controlled Colonic Drug Delivery

The objective of this study was to investigate the mechanisms underlying drug release from a controlled colonic release (CCR) tablet formulation based on a xyloglucan polysaccharide matrix and identify the factors that control the rate of release for the purpose of fundamentally substantiating the concept and demonstrating its robustness for colonic drug delivery. Previous work demonstrated in vitro limited release of 5-aminosalicylic acid (5-ASA) and caffeine from these tablets in small intestinal environment and significant acceleration of release by xyloglucanase, an enzyme of the colonic microbiome. Targeted colonic drug delivery was verified in an animal study in vivo. In the present work, interaction of the xyloglucan matrix tablets with aqueous dissolution media containing xyloglucanase was found to lead to the spontaneous formation of a hydrated highly viscous gummy layer at the surface of the matrix which had a reduced drug content compared to the underlying regions and persisted with a nearly constant thickness that was inversely correlated to the enzyme concentration throughout the duration of the release process. Enzymatic hydrolysis of xyloglucan was determined to take place at the surface of the matrix leading to matrix erosion and a relation for the rate of enzymatic reaction as a function of bulk enzyme concentration and the concentration of dissolved xyloglucan in the gummy layer was derived. A mathematical model was developed encompassing aqueous medium ingress, matrix metamorphosis due to xyloglucan dissolution and matrix swelling, enzymatic hydrolysis of the polysaccharide and concomitant drug release due to matrix erosion and simultaneous drug diffusion. The model was fitted to data of reducing sugar equivalents in the medium reflecting matrix erosion and released drug amount. Enzymatic reaction parameters and reasonable values of medium ingress velocity, xyloglucan dissolution rate constant and drug diffusion coefficient were deduced that provided an adequate approximation of the data. Erosion was shown to be the overwhelmingly dominant drug release mechanism while the role of diffusion marginally increased at low enzyme concentration and high drug solubility. Changing enzyme concentration had a rather weak effect on matrix erosion and drug release rate as demonstrated by model simulations supported by experimental data, while xyloglucan dissolution was slow and had a stronger effect on the rate of the process. Therefore, reproducible colonic drug delivery not critically influenced by inter- and intra-individual variation of microbial enzyme activity may be projected.

BIOTECHNOLOGICAL PROCESSING OF LOW-GRADE MEAT WITH ENZYME PREPARATIONS

Biotechnological methods for processing raw materials in the meat industry are associated with the creation of advanced technologies. Domestic and world experience indicate the advisability of using enzymes in the meat industry to accelerate the maturation processes and increase the quality of semi-finished products. In order to intensify technological processes, increase the nutritional and biological value of finished products, enzyme preparations are used in the process of processing low-grade meat raw materials. Currently, herbal enzyme preparations phytosin, papain, bromelain, etc. are widely used. Processing meat with enzymes improves the consistency of meat, softens the structure of rough and strong muscle fibers and connective tissue; helps to increase the degree of digestibility of the product; improves taste, smell and color and accelerates the ripening process of meat [1].Enzymes are participants in both traditional and new technological processes used in food production. The muscles of farm animals are characterized by low concentrations of intracellular enzymes. Some anatomical parts of the carcass are characterized by a high content of connective tissue. This determines the toughness of such meat and its slow maturation. Beef under normal conditions matures after 10-14 days of aging at a temperature of 2-4°C. After treatment with enzymes, the meat matures in 1-2 days. The main purpose of aging beef is to break down the muscle fiber structure and enhance the natural flavor of the meat [2].Processing low-grade meat with plant enzymes will make it possible to use parts of the carcass that are full in composition, but have naturally increased rigidity: meat from the hind legs, shoulder blades, and brisket. Processing meat with a plant enzyme preparation leads to significant destructive changes that provide an effective softening effect, and is consistent with the obtained assessments of physicochemical and functional technological properties [3].The use of enzyme preparations has a positive effect on the tenderness, juiciness, nutritional value of raw materials, the formation of the required level of water-binding and water-holding capacity, and improves organoleptic characteristics due to the targeted effect of plant enzymes and enzymatic preparations on the components of muscle tissue [4].

Optimization of production conditions, isolation, purification, and characterization of tannase from filamentous fungi.

Tannase-producing filamentous fungi residing alongside tannin-rich ambient in the Northwest Himalayas were isolated at laboratory conditions and further identified by 18S ribosomal RNA gene sequencing. Five most potent tannase producing strains (EI ≥ 2.0), designated Aspergillus fumigatus AN1, Fusarium redolens AN2, Penicillium crustosum AN3, Penicillium restrictum AN4, and Penicillium commune AN5, were characterized. The strain Penicillium crustosum AN3 exhibited a maximum zone dia (25.66mm ± 0.38). During solid-state fermentation, a maximal amount of tannase was attained withPenicillium crustosum AN3 usingpine needles (substrate) by adopting response surface methodology for culture parameter optimization. Gel filtration chromatography yielded 46.48% of the partially purified enzyme with 3.94-fold of tannase purification. We found two subunits in enzyme-117.76 KDa and 88.51 KDa, respectively, in the SDS-PAGE. Furthermore, the characterization of partially purified tannase revealed a maximum enzyme activity of 8.36 U/mL at 30°C using a substrate concentration (methyl gallate) of 10mM. To broaden the knowledge of crude enzyme application, dye degradation studies were subjected to extracellular crude tannase fromPenicillium crustosum AN3 where the maximumdegradation achievedat a low enzyme concentration (5ppm).

Recombinant production of a highly efficient photolyase from Thermus thermophilus.

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋𝑚𝑎𝑥 ) of 2.0g L-1 was reached after 5h of cultivation, corresponding to 𝑃X =0.4g L-1 h. The µ𝑚𝑎𝑥 corresponded to 1.0 h-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25mg L-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15µg mL-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase kM of 9.5nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.

Desiccation cracking remediation through enzyme induced calcite precipitation in fine-grained soils under wetting drying cycles

The effects of desiccation cracking in clay soils on geotechnical constructions are substantial. This study investigates the viability of utilizing Enzyme-induced calcite precipitation (EICP), a bio inspired approach, as a potential solution for addressing desiccation cracking in fine-grain soils. For the EICP technique, crude soybean extract is employed for the purpose of urea hydrolysis. Multiple fluid samples, including a control sample, a cementation solution containing 1 M urea, 0.675 M CaCl2, and 4 g/L milk, along with various concentrations of enzyme solutions (3–80 g/L), were tested for the study. To evaluate the surface cracking patterns, the method involved constant monitoring and photo recording using a high-resolution camera aided by image processing software. The results showed that fine-grain soils improved from increased calcite precipitation and decreased desiccation cracking intensity when the EICP method was used. Cementation and enzyme solution with low concentrations (3 g/L and 10 g/L) had similar effects on crack remediation, suggesting a modest influence. In contrast to the sample treated with water, the crack network remained unaltered in this case. CaCO3 precipitation within the void area kept the crack network in place even as the void thickness decreased at increasing enzyme concentrations (30 g/L, 50 g/L, and 80 g/L). Wetting and drying cycles were found to decrease the crack ratio, crack width, and crack length in the EICP-treated sample, particularly under higher concentrations of urease enzyme. Lower enzyme concentrations of 3 g/L and 10 g/L have minimal impact on crack remediation but effectively inhibit new crack formation. Furthermore, higher enzyme concentrations result in calcium carbonate precipitates, forming a soil crust and increasing surface roughness. The study aims to enhance understanding of the EICP methodology and to provide novel perspectives on potential uses for soil enhancement.

An Efficient and Reproducible Method for the Isolation and Culture of Primary Cardiomyocytes from Adult Zebrafish.

Zebrafish is a popular animal model in regeneration studies due to their ability to regenerate the heart. Primary cardiomyocytes could be an alternative tool for studying the intrinsic mechanisms of cardiovascular disease in vitro. Thus, our objective is to develop an efficient protocol to isolate primary cardiomyocytes from zebrafish hearts. Low concentration of digestive enzyme (0.5 mg/mL collagenase type II) was utilized in our protocol to obtain single-cell suspension. The ventricles were fragmented, mechanically pipetted, and constantly shaken to ensure adequate contact between the tissues and the enzyme. Preplating the cell suspension onto culture plates for 2 h helped remove cardiac fibroblasts. The purity of isolated cells was validated by flow cytometry analysis of transgenic zebrafish with cardiomyocyte-specific expression of enhanced green fluorescent protein (EGFP) or endothelial cell-specific expression of mCherry. Quantitative real-time PCR analysis revealed a high level of the purity, with cardiac fibroblasts, endothelial cells, and epicardial cell markers scarcely detected in the purified cells. Altogether, this study established a reproducible protocol for isolating primary cardiomyocytes with high purity and activity from adult zebrafish hearts that can be cultured in vitro for up to 4 weeks. This protocol provides a valuable tool for studying the intrinsic mechanisms of cardiovascular disease in vitro using primary cardiomyocytes.

A γ-Glutamyl Transpeptidase (GGT)-Triggered Charge Reversal Drug-Delivery System for Cervical Cancer Treatment: In Vitro and In Vivo Investigation.

Neutral/negatively charged nanoparticles are beneficial to reduce plasma protein adsorption and prolong their blood circulation time, while positively charged nanoparticles easily transverse the blood vessel endothelium into a tumor and easily penetrate the depth of the tumor via transcytosis. Γ-Glutamyl transpeptidase (GGT) is overexpressed on the external surface of endothelial cells of tumor blood vessels and metabolically active tumor cells. Nanocarriers modified by molecules containing γ-glutamyl moieties (such as glutathione, G-SH) can maintain a neutral/negative charge in the blood, as well as can be easily hydrolyzed by the GGT enzymes to expose the cationic surface at the tumor site, thus achieving good tumor accumulation via charge reversal. In this study, DSPE-PEG2000-GSH (DPG) was synthesized and used as a stabilizer to generate paclitaxel (PTX) nanosuspensions for the treatment of Hela cervical cancer (GGT-positive). The obtained drug-delivery system (PTX-DPG nanoparticles) was 164.6 ± 3.1 nm in diameter with a zeta potential of -9.85 ± 1.03 mV and a high drug-loaded content of 41.45 ± 0.7%. PTX-DPG NPs maintained their negative surface charge in a low concentration of GGT enzyme (0.05 U/mL), whereas they showed a significant charge-reversal property in the high-concentration solution of GGT enzyme (10 U/mL). After intravenous administration, PTX-DPG NPs mainly accumulated more in the tumor than in the liver, achieved good tumor-targetability, and significantly improved anti-tumor efficacy (68.48% vs. 24.07%, tumor inhibition rate, p < 0.05 in contrast to free PTX). This kind of GGT-triggered charge-reversal nanoparticle is promising to be a novel anti-tumor agent for the effective treatment of such GGT-positive cancers as cervical cancer.

Engineering an Oxygen‐Binding Protein for Photocatalytic CO2Reductions in Water

AbstractWhile native CO2‐reducing enzymes display remarkable catalytic efficiency and product selectivity, few artificial biocatalysts have been engineered to allow understanding how the native enzymes work. To address this issue, we report cobalt porphyrin substituted myoglobin (CoMb) as a homogeneous catalyst for photo‐driven CO2to CO conversion in water. The activity and product selectivity were optimized by varying pH and concentrations of the enzyme and the photosensitizer. Up to 2000 TON(CO) was attained at low enzyme concentrations with low product selectivity (15 %), while a product selectivity of 74 % was reached by increasing the enzyme loading but with a compromised TON(CO). The efficiency of CO generation and overall TON(CO) were further improved by introducing positively charged residues (Lys or Arg) near the active stie of CoMb, which demonstrates the value of tuning the enzyme secondary coordination sphere to enhance the CO2‐reducing performance of a protein‐based photocatalytic system.

Engineering an Oxygen-Binding Protein for Photocatalytic CO2 Reductions in Water.

While native CO2 -reducing enzymes display remarkable catalytic efficiency and product selectivity, few artificial biocatalysts have been engineered to allow understanding how the native enzymes work. To address this issue, we report cobalt porphyrin substituted myoglobin (CoMb) as a homogeneous catalyst for photo-driven CO2 to CO conversion in water. The activity and product selectivity were optimized by varying pH and concentrations of the enzyme and the photosensitizer. Up to 2000 TON(CO) was attained at low enzyme concentrations with low product selectivity (15 %), while a product selectivity of 74 % was reached by increasing the enzyme loading but with a compromised TON(CO). The efficiency of CO generation and overall TON(CO) were further improved by introducing positively charged residues (Lys or Arg) near the active stie of CoMb, which demonstrates the value of tuning the enzyme secondary coordination sphere to enhance the CO2 -reducing performance of a protein-based photocatalytic system.

Intensified CO2 capture in wall-coated microreactors with immobilized carbonic anhydrase: Experimental and modeling

An intensified enzymatic CO2 capture process in a wall-coated enzyme-immobilized microreactor (WCEI_MR) was investigated experimentally and numerically under diverse flow conditions, at different human carbonic anhydrase II (hCA II) enzyme and buffer concentrations, and using different buffers. The motivation for using this type of microreactor for the CO2 capture process is to enhance the absorption rate by improving the flow and mass transfer through Taylor gas–liquid flow to efficiently accommodate the reduced time scales of CO2 absorption associated with the CA enzyme. hCA II enzyme was covalently immobilized on the internal surface of experimental microreactor (priori amine-functionalized via polydopamine & polyethyleneimine co-deposition) through glutaraldehyde. An unsteady-state two-scale model involving volume-averaged continuity and momentum balance equations, mass transport/reaction in bulk liquid & gas, and diffusion/enzymatic reaction equations in immobilized-enzyme layer was developed to simulate the wall-coated enzyme-loaded microreactor. Gas-liquid two phase Taylor flow in wall-coated microreactor generates an intensified gas–liquid/liquid–solid mass transfer which, coupled with the large surface area and small axial dispersion and backmixing, enhances the utilization of the enormous hCA II enzyme turnover number and ensures an enhanced enzyme-mediated CO2 absorption process at low pressure drop. WCEI_MR favors low enzyme concentrations, buffers with higher pKa2 constant that generates higher driving force for CO2 hydration or higher buffer concentration that improves enzymatic activity and the intermolecular proton-transfer step that stimulate the absorption process, all of which mitigate the impact of resistance to mass transfer. These findings suggest that WCEI_MR has a high potential for CO2 absorption and, when connected in flexible small-scale modular parallel systems, could represent a promising approach for large-scale green CO2 capture.

Mathematical expressions describing enzyme velocity and inhibition at high enzyme concentration.

Enzyme behaviour is characterised in the laboratory using diluted solutions of enzyme. However, in vivo processes usually occur at [S T ]≈[E T ]≈K m . Furthermore, thestudy of enzyme action involves characterisation of inhibitors and their mechanisms. However, to date, there have been no reports proposing mathematical expressions that can be used to describe enzyme activity at high enzyme concentration apart from the simplest single substrate, irreversible case. Using a continued fraction approach, equations can be easily derived for the most common cases in monosubstrate reactions, such as irreversible or reversible reactions and effector (inhibitor or activator) kinetic interactions. These expressions are an extension of the classical Michaelis-Menten equations. A first analysis using these expressions permits to deduce some differences at high versus low enzyme concentration, such as the greater effectiveness of allosteric inhibitors compared to catalytic ones. Also, they can be used to understand catalyst saturation in a reaction. Although they can be linearised, these equations also show differences that need to be taken into account. For example, the different meaning of line intersection points in Dixon plots. All in all, these expressions may be useful tools for modelling in vivo and biotechnological processes.

Experimental investigation on simultaneous production of bioethanol and biodiesel from macro-algae

The present work compares the biofuel production efficiency of Padina tetrastromatica and Sargassum swartzii macro-algae biomass by fermentation with Saccharomyces cerevisiae and Zymomonas Mobilis. The optimal pretreatment factors were identified by studying the effect of temperature, pH, and enzyme concentration on the conversion of macroalgae biomass to fermentable reducing sugars. The Low concentration of enzyme did not help conversion of biomass to reducing sugars even after 15 h of treatment with enzyme, but when the enzyme concentration was increased, the desired conversion was completed within 4 h of enzyme treatment. Both Padina tetrastromatica and Sargassum swartzii had an ethanol yield of above 80% irrespective of the difference in the species used for the fermentation. The highest possible ethanol yield (95.12%) was achieved for Padina tetrastromatica biomass when it was fermented with Zymomonas Mobilis. From the above results, it is clear that both Padina tetrastromatica and Sargassum swartzii have the potential to be used as reliable feedstock for biofuel production. The spent biomass after the fermentation process was used for the lipid extraction process and the extracted lipid was used for biodiesel production. The percentage of biodiesel yield obtained by means of the Soxhlet extraction process from Padina tetrastromatica and Sargassum swartzii macro-algae with the conventional NaOH catalyst was found to be 84% and 89% respectively. MgO.La2O3 was synthesized by means of the co-precipitation method. With the introduction of MgO.La2O3 nano-catalyst biodiesel yield was improved up to 88% and 92% for Padina tetrastromatica and Sargassum swartzii macro-algae, respectively.

HUG Domain Is Responsible for Active Dimer Stabilization in an NrdJd Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.

Enzymatic co-crosslinking of star-shaped poly(ethylene glycol) tyramine and hyaluronic acid tyramine conjugates provides elastic biocompatible and biodegradable hydrogels.

A combination of the viscoelastic properties of hyaluronic acid (HA) and the elastic properties of star shaped 8-arm poly(ethylene glycol) (8-arm PEG) was used to design in-situ forming hydrogels. Hydrogels were prepared by the enzymatic crosslinking of a partially tyramine modified 8-arm PEG and a tyramine conjugated HA using horseradish peroxidase in the presence of hydrogen peroxide. Hydrogels of the homopolymer conjugates and mixtures thereof were rapidly formed within seconds under physiological conditions at low polymer and enzyme concentrations. Elastic hydrogels with high gel content (≥95%) and high storage moduli (up to 22.4 kPa) were obtained. An in vitro study in the presence of hyaluronidase (100 U/mL) revealed that with increasing PEG content the degradation time of the hybrid hydrogels increased up to several weeks, whereas hydrogels composed of only hyaluronic acid degraded within 2 weeks. Human mesenchymal stem cells (hMSCs) incorporated in the hybrid hydrogels remained viable as shown by a PrestoBlue and a live-dead assay, confirming the biocompatibility of the constructs. The production of an extracellular matrix by re-differentiation of encapsulated human chondrocytes was followed over a period of 28 days. Gene expression indicated that these highly elastic hydrogels induced an enhanced production of collagen type II. At low PEG-TA/HA-TA ratios a higher expression of SOX 9 and ACAN was observed. These results indicate that by modulating the ratio of PEG/HA, injectable hydrogels can be prepared applicable as scaffolds for tissue regeneration applications.

Using parameter sampling to explore key properties of E. coli's anaerobic metabolism

The high uncertainty and incomplete knowledge of kinetic parameters is a major challenge for using kinetic metabolic models. Literature values are scarce and fitting procedures require large experimental data sets and complex computations. In this study we show that Monte-Carlo sampling of kinetic parameters allows the identification of system's properties such as stability and flux control patterns. Applying this computationally simple method to the anaerobic central metabolism of E. coli we determine that only few network parameters are directly correlated with stability. We show that low enzyme concentrations often correspond to positive eigenvalues, suggesting that enzyme-optimized pathways lack in flexibility. Analysis of the distribution of flux control coefficients reveals that the highest control on the network rates is exerted by reactions utilizing ATP. Finally, a comparison with experimental evidence confirms this method's potential to qualitatively analyze networks for which no information on parameters are available.

Beyond the Michaelis-Menten: Bayesian Inference for Enzyme Kinetic Analysis.

Although the Michaelis-Menten (MM) rate law has been widely used to estimate enzyme kinetic parameters, it works only under the condition of extremely low enzyme concentration. Furthermore, even when this condition is satisfied, parameter estimation is often imprecise due to the parameter identifiability issue. To overcome these limitations of the canonical approach to enzyme kinetics, we developed a Bayesian approach based on a modified form of the MM rate law, which is derived with the total quasi-steady state approximation. Here, we illustrate how to perform the Bayesian inference for the progress curve assay with our user-friendly computational R package. We also describe an optimal experimental design for the progress curve assay, with which enzyme kinetic parameters can be accurately and precisely estimated from minimal measurements of the progress curves.