Hydrolysis Step Research Articles

Quantifying the reversibility of ATP hydrolysis in rabbit skeletal myosin subfragment 1 using a luciferase assay

BackgroundIt is known that the adenosine triphosphate (ATP) hydrolysis in a number of ATPases is reversible. Radioactive methods are standard in measuring ATPase reversibility. We used myosin as a well-studied model enzyme to develop a luciferase-based assay to quantify the reversibility of ATP hydrolysis. In myosin, ATP bound to the active site is hydrolyzed into adenosine diphosphate (ADP) and inorganic phosphate (Pi) and recombined back into ATP multiple times before products of hydrolysis are released. The reversibility of ATP hydrolysis by skeletal myosin was previously confirmed using radioactively labeled isotopes. Transient kinetics studies indicated that the ATP hydrolysis step is temperature-sensitive, with a dissociation equilibrium constant of 1.6 at 3 °C. Consequently, the association equilibrium constant at this temperature is 0.63. The goal of our work was two-fold, (a) to develop a luciferase-based assay to measure the equilibrium constant of enzymatic ATP hydrolysis, eliminating the need for radioactively labeled Pi, and (b) refine the value of the association equilibrium constant of the myosin ATPase hydrolysis step.MethodsIn this assay, a reaction mixture containing myosin and saturating levels of ADP and Pi was incubated to reach equilibrium, then the reaction was terminated, and the amount of ATP produced by myosin was quantified using the luciferase assay. The equilibrium constant of ATP hydrolysis was defined as the ratio of ATP to ADP bound to myosin.ResultsWe obtained a value of 0.78 ± 0.14 for the association equilibrium constant of ATP hydrolysis at 0 °C. 50 μM ADP bound to myosin is turned into 21.9 ± 3.0 μM ATP. Our result is in excellent agreement with the literature data, supporting the viability of the new methodology.DiscussionThis methodology allows a more accessible and safe option to measure ATP production and reversibility of ATPase enzymes. Standard laboratory equipment is utilized in the assay, and the number of steps in the developed assay is reduced significantly compared to the radioactive method.

Enhanced anaerobic digestion of freezing and thawing pretreated cow manure with increasing solid content: kinetics and microbial community dynamics

High solid anaerobic digestion has proved the mainstream technology for the treatment of organic wastes. However, the high molecular weight and complex lignocellulosic structure of cow manure (CM) make it indigestible and inefficient, leading to limit the hydrolysis step of anaerobic digestion at high solid content. To mitigate this bottleneck, an improved cost-effective freezing and thawing pretreatment technique was proposed in this study. The freezing and thawing pretreatment of raw CM without any dilution was done for 20 days. The maximum cumulative methane yield (487 mL CH4 g− 1VS) was achieved at a total solid (TS) of 5% followed by TS of 10% and 15%, which was 13%, 20% and 21% higher than obtained from untreated CM, respectively. The kinetic results revealed that the biodegradable materials could be utilized at increasing TS with decreasing hydrolysis rate. The pretreatment significantly enhanced the methylotrophic methanogenic pathway during high solid anaerobic digestion, which was contrary to the general concept that the process is usually dominated by acetoclastic and hydrogenotrophic methanogens. This study is very important to understand the effect of solid content but also important to understand the effect of freezing and thawing pretreatment on process parameters and microbial community dynamics in high solid anaerobic digestion.

Regarding Bioanalysis Lasting a Few Minutes: Automated Cooling-SPME and Fast-GC for Urinary 2-Phenyl-2-Propanol Monitoring.

An innovative SPME head space GC-MS method, in cooling mode, using a fully automated routine, was developed to detect 2-phenyl-2-propanol, a representative urinary metabolite of cumene. Following an acid hydrolysis and derivatization step with lowered quantities of reagents, acetic anhydride and pyridine, a 30 μm polydimethylsiloxane SPME fiber was used to sample derivatized 2-phenyl-2-propanol, such as benzenemethanol,α,α-dimethyl-acetate, from the headspace. Performances of the method, optimized through experimental design, provide an LOD of 0.034 mg/L and an LOQ 0.10 mg/L, with a short sampling time necessary per sample. The method, developed on standard solutions, will be applied to both occupationally exposed and non-exposed populations.

Remote Sulfonylation of Anilines with Sodium Sulfifinates Using Biomass-Derived Copper Catalyst

A biomass-based catalyst, CuxOy@CS-400, was employed as an excellent recyclable heterogeneous catalyst to realize the sulfonylation reaction of aniline derivatives with sodium sulfinates. Various substrates were compatible, giving the desired products moderate to good yields at room temperature. In addition, this heterogeneous copper catalyst was also easy to recover and was recyclable up to five times without considerably deteriorating in catalytic efficiency. Importantly, these sulfonylation products were readily converted to the corresponding 4-sulfonyl anilines via a hydrolysis step. The method offers a unique strategy for synthesizing arylsulfones and has the potential to create new possibilities for developing heterogeneous copper-catalyzed C-H functionalizations.

Endosomal actin branching, fission, and receptor recycling require FCHSD2 recruitment by MICAL-L1.

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process.

1O2 and Base Assisted Oxidative Conversion of β-Enaminoesters to α-Acyloxy-β-ketoesters under Visible Light Irradiation.

Singlet oxygen (1O2) and base assisted conversion of β-enaminoesters to α-acyloxy-β-ketoesters is demonstrated under visible light irradiation. The reaction involves formation of an imine intermediate via ene-type pathway initiated by 1O2 followed by base promoted dimerization and hydrolysis steps. The method is mild, environmentally friendly, requires air as the oxidant, and gives the products in moderate to high yields.

A multistage biogas slurry reflux and spray anaerobic digestion reactor for high solid anaerobic digestion: Performance and application evaluation

In this study, a new vertical plug-flow continuous dry anaerobic digestion reactor equipped with a spray percolation system was designed for rice straw and cow manure in dry AD. The findings indicated that the methane yield was 290.63 ± 19.16 mL/g VS when the optimal spray time (9 times/d) was used in batch AD. In addition, the results of continuous experiments under optimal spray time conditions showed that the volumetric methane production rate of the system was 1.55 L/(L·d) when the organic loading rate was 20 g VS/(L·d). In addition, hydrolysis and acidification steps were enhanced. This study demonstrated the potential of the new reactor for stable operation of high OLR dry AD. Digestate is already used as a substitute for synthetic fertilizers precisely because of the environmental benefits associated with its agronomic use.

Rational Engineering of a β-Glucosidase (H0HC94) from Glycosyl Hydrolase Family I (GH1) to Improve Catalytic Performance on Cellobiose.

The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. β-Glucosidases catalyze the final step in cellulose breakdown, producing glucose, and are often the rate-limiting step in biomass hydrolysis. The specific activity of most natural and engineered β-glucosidase is higher on the artificial substrate p-nitrophenyl β-d-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. We report an engineered β-glucosidase (Q319A H0HC94) with a 1.8-fold higher specific activity (366.3 ± 36 μmol/min/mg), a 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in catalytic efficiency (236.65 mM-1 s-1) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and protein structure network analysis indicate that the Q319A H0HC94 active site pocket is significantly remodeled compared to the WT, leading to changes in enzyme conformation, better accessibility of cellobiose inside the active site pocket, and higher enzymatic activity. This study shows the impact of rational engineering of a nonconserved residue to increase β-glucosidase substrate accessibility and catalytic efficiency by reducing crowding interaction between cellobiose and active site pocket residues near the gatekeeper region and increasing pocket volume and surface area. Thus, rational engineering of previously characterized enzymes could be an excellent strategy to improve cellulose hydrolysis.

Green directional conversion of food waste into glycerol in a two-step differentiation enzymolysis and facultative fermentation using Saccharomyces cerevisiae: Performance assessment and mechanism

Valorization of food waste into high value-added glycerol was designed and prepared via directional green transformation using multi-enzymolysis saccharification coupled facultative fermentation by Saccharomyces cerevisiae. The optimal conditions for the first step of multienzyme hydrolysis and the second step of facultative fermentation were 55 °C and pH 5.0 for 6 h, 30 °C and pH 7.0 for 70 h, respectively. The glycerol yield efficiency and kinetic rate constant were 73.6 % (w/w) and 0.681 h−1 for dry component of food waste, while the other 27.4 % was utilized as the growth energy substrate. The key points of transformation were the synergistic directed enzymolysis to produce 3 % glycerol and 95 % reducing sugars. Furthermore, the reducing sugars were converted into glycerol by switch the metabolic pathway from ethanol into glycerol through the quantitative expression of functional gpd1 and gpp2 genes activated GPD and GPP enzymatic activity and synchronous inhibition of adh gene for glycerol production with Na2SO3 regulation. The economic income was 135.52 USD/ton for converting 80 % water content of kitchen waste into glycerol. This finding provided an economic value-added approach to produce high-value glycerol for cost-effective recycling of food waste using commercial yeast.

Evaluation of a Benzodiazepine Immunoassay for Urine Drug Testing in Clinical Specimens.

Benzodiazepines are commonly prescribed medications frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated benzodiazepine metabolites, leading to false negatives. Clinical laboratories have addressed this deficiency by creating laboratory-developed tests (LDTs) that incorporate a beta-glucuronidase hydrolysis step to increase the clinical sensitivity of these assays. Performance characteristics of 2 FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a previously described benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 clinical urine specimens. The positive immunoassay cutoff was set at 200 ng/mL of nordiazepam and results were compared to an LC-MS/MS benzodiazepine LDT. Clinical sensitivity, specificity, precision, and immunoassay cross-reactivity were determined for all 3 immunoassays. The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all 3 assays. Immunoassay response of the Benzodiazepines II assay was greater across the range of concentrations tested (100-1000 ng/mL) relative to the other immunoassays and was the most sensitive immunoassay for the detection of lorazepam glucuronide. The Benzodiazepines II immunoassay demonstrated the greatest clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The incorporation of beta-glucuronidase was crucial, as the Benzodiazepines II and LDT immunoassays demonstrated superior clinical sensitivity when compared to the Benzodiazepines Plus immunoassay that does not incorporate a beta-glucuronidase hydrolysis step.

One‐Pot Cascade Reaction from Epoxide to Carboxylic Acids Using Bifunctional Fe‐ZSM‐5

AbstractThe quest for sustainable synthesis of carboxylic acids has led to the exploration of innovative routes and catalysts for the oxidative cleavage of carbon−carbon double bonds, prevalent in bio‐derived molecules. This process involves multiple steps including epoxidation, hydrolysis and vicinal diol cleavage, often catalysed by unsustainable homogeneous or noble‐metal catalysts. In this study, we present the utilization of Fe‐ZSM‐5 as a versatile bifunctional catalyst capable of catalysing both hydrolysis and vicinal diol cleavage steps, utilizing propylene oxide as model compound and H2O2 as oxidizing agent. The prepared Fe‐ZSM‐5 features the zeolite intrinsic Lewis and Brønsted acidity functions, essential for the hydrolysis step, as well as high fractions of monomeric FeOx species, crucial for the subsequent vicinal diol cleavage, leading to streamlined carboxylic acids formation in a one‐pot, two‐step process. Moreover, by combining Fe‐ZSM‐5 with TS‐1, renowned for its epoxidation activity, we demonstrate the complete cascade reaction in one pot, under mild conditions, starting from ethylene. Our study expands the utility of cost‐effective zeolite‐based catalysts enriched with abundant Fe metal for the one‐pot oxidative cleavage of C=C double bonds, offering a promising pathway towards sustainable carboxylic acid synthesis.

Enhanced cellobiose hydrolysis over fluorine-modulated carbon-based solid acid catalysts

In this study, we synthesized novel carbon-based solid acid catalysts using plasma engineering by integrating sulfonic acid groups as primary catalytic sites and supplemented these with fluorine-containing, chlorine-containing, and other oxygenated functional groups (OFGs). Notably, the catalyst with the fluorine-containing functional groups results superior glucose yield (58.2 %) than that containing only OFGs (37.6 %). Theoretical analysis of the reaction energetics revealed that the cleavage of cellobiose's 1,4-O linkage to form two glucose molecules potentially constitutes the rate-determining step (RDS) in cellobiose hydrolysis. The reaction energy for this RDS was found to increase in the sequence of G–SO3H–F, G–SO3H–Cl, and G-SO3H, consistent with the experimentally obtained catalytic activity trends. Through extensive characterization, including textural analysis, surface chemistry, and density functional theory calculations, we identified that the –F groups are the key to strengthening cellulose–catalyst interactions. This study is the first study to demonstrate the potential of fluorine-modulated carbon-based solid acid catalysts for upgrading cellulosic biomass to value-added compounds.

Structural Modification and Characteristics of Lappaconitine Alkaloid for the Discovery of Bioactive Components by Hypervalent Iodine Reagent.

Lappaconitine, a diterpene alkaloid isolated from Aconitum sinomontanum Nakai, exhibits a wide range of biological activities, making it a promising candidate for the development of novel derivatives with therapeutic potential. In our research, we executed a two-step transformation via oxidative cleavage of lappaconitine's vicinal diol using the hypervalent iodine reagent PhI(OAc)2, followed by strong alkaline hydrolysis. This approach yielded four new unanticipated compounds, whose structures were identified by spectroscopic methods and/or X-ray crystallography. Thus, we proposed plausible reaction mechanisms for their formations and particularly investigated the remarkable diastereoselectivity for the formation of single stereoisomer 8 observed during the alkaline hydrolysis step. Among them, compound 8 (code name: QG3030) demonstrated both enhanced osteogenic differentiation of human mesenchymal stem cells and significant osteogenic effect in an ovariectomized rat model with no acute oral toxicity.

Thermal pretreatment of swine waste to improve biodegradability in the anaerobic digestion process. Effect on the physicochemical characteristics of the substrate

In anaerobic digestion (AD) processes, hydrolysis is considered the limiting stage in the degradation of solid wastes. Such is the case of swine manure digestion, which due to the complex physical and chemical structure of the lignocellulosic material that composes it, an energy wastage has been observed in terms of methane production. Among the strategies used to improve the hydrolysis stage, it is possible to mention the thermal pretreatment of the substrate, which can significantly improve the biodegradability of the material used as raw material in AD. In this study, the effect of temperature (60–177 °C) and exposure time (30–60 min) on the physicochemical properties of the substrate such as pH, volatile organic acids (VOAs), total inorganic carbonates (TIC), volatile solids (SV), total solids (ST), soluble chemical oxygen demand (CODs) and biochemical methane potential (BMP) were determined. The temperature factor had a higher level of significance compared to the exposure time on the parameters of pH, VOAs, and TIC before and after the biogas generation process. However, the effect was different for CODs, where time was more significant than treatment temperature. As for the parameters of total and volatile solids content (ST and SV), the factors evaluated (temperature and exposure time) did not show significant effect. Also, the pretreatments showed an increase in biochemical methane potential, outperforming the untreated substrate by up to 70.4 % (121.74vs.71.44 mLCH4 gVS-1). The best combination of heating temperature and operating time was 120 °C and 45 min, which promoted the hydrolysis step that was reflected in an increase in CODs and improvement in methane production by 42 % over the untreated substrate

Benign and Selective Amination of Lignins towards Aromatic Biobased Building Blocks with Primary Amines.

Lignin is a widely available second-generation biopolymer and the main potential source of renewable aromatic building blocks. Lignin-based polyamines offer great potential in applications based on chemical and materials sciences. However, common aminations techniques for lignin usually involve toxic chemicals and generate hindered and low reactivity amines. In this study, we developed two new, simple, and benign 2-step methodologies for the elaboration of lignin-based polyamines from different technical lignins (kraft, soda and organosolv) with a selectivity towards reactive primary amines. These methods involve grafting amide groups onto lignin followed by a hydrolysis step. Non-toxic heterocyclic compounds N-acetyl-2-oxazolidinone and 2-methyl-2-oxazoline were used as amidation agents. Hydrolysis was performed in acetone-water mixtures. Reactions were studied on model compounds and optimized on lignins. Aminated lignins were fully characterized and primary amines were quantified using quantitative 19F NMR. Our methods generated aminated lignins with low apparent molar masses and high solubility in water and solvents. Nitrogen contents of the products ranged between 2.0 and 3.5 mmol/g with reactive primary amines counts up to 1.7 mmol/g. These soluble and reactive lignin-based polyamines offer great potential as a replacement for fossil-based polyamines in e.g., the synthesis of aromatic polymer materials or as potential chelating, antibacterial agents.

Comprehensive study on pilot nitrification-sludge fermentation coupled denitrification system with extended sludge retention time

The sludge fermentation-coupled denitrification process, utilized for sludge reduction and nitrogen removal from wastewater, is frequently hindered by its hydrolysis step’s efficacy. This study addresses this limitation by extending the sludge retention time (SRT) to 120 days. As a result, the nitrate removal efficiency (NRE) of the nitrification-sludge fermentation coupled denitrification (NSFD) pilot system increased from 67.1 ± 0.2 % to 96.7 ± 0.1 %, and the sludge reduction efficiency (SRE) rose from 40.2 ± 0.5 % to 62.2 ± 0.9 %. Longer SRT enhanced predation and energy dissipation, reducing intact cells from 99.2 % to 78.0 % and decreasing particle size from 135.2 ± 4.6 μm and 19.4 ± 2.1 μm to 64.5 ± 3.5 μm and 15.5 ± 1.6 μm, respectively. It also created different niches by altering the biofilm’s adsorption capacity, with interactions between these niches driving improved performance. In conclusion, extending SRT optimized the microbial structure and enhanced the performance of the NSFD system.

Benign and Selective Amination of Lignins towards Aromatic Biobased Building Blocks with Primary Amines

Lignin is a widely available second‐generation biopolymer and the main potential source of renewable aromatic building blocks. Lignin‐based polyamines offer great potential in applications based on chemical and materials sciences. However, common aminations techniques for lignin usually involve toxic chemicals and generate hindered and low reactivity amines. In this study, we developed two new, simple, and benign 2‐step methodologies for the elaboration of lignin‐based polyamines from different technical lignins (kraft, soda and organosolv) with a selectivity towards reactive primary amines. These methods involve grafting amide groups onto lignin followed by a hydrolysis step. Non‐toxic heterocyclic compounds N‐acetyl‐2‐oxazolidinone and 2‐methyl‐2‐oxazoline were used as amidation agents. Hydrolysis was performed in acetone‐water mixtures. Reactions were studied on model compounds and optimized on lignins. Aminated lignins were fully characterized and primary amines were quantified using quantitative 19F NMR. Our methods generated aminated lignins with low apparent molar masses and high solubility in water and solvents. Nitrogen contents of the products ranged between 2.0 and 3.5 mmol/g with reactive primary amines counts up to 1.7 mmol/g. These soluble and reactive lignin‐based polyamines offer great potential as a replacement for fossil‐based polyamines in e.g., the synthesis of aromatic polymer materials or as potential chelating, antibacterial agents.

Endosomal actin branching, fission and receptor recycling require FCHSD2 recruitment by MICAL-L1.

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homolog of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Since MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process.

Efficient recovery of antioxidants from olive leaves through green solvent extraction and enzymatic hydrolysis: Experimental evaluation and COSMO-RS analysis

The present study focuses on the valorization of olive leaves (Olea europaea L.) through the recovery of high-value antioxidants using green solvents (i.e., bio-based solvents (BioS) and natural eutectic solvents (NAES)) while evaluating the implementation of an enzymatic hydrolysis step for biomass pretreatment. Overall, 12 BioS including water, alcohols, ethers, esters, and terpenes, and 7 NAES composed of choline chloride, proline, and betaine combined with polyols, were studied as green solvents for the solid–liquid extraction of antioxidants from olive leaves. NAES generally performed better than BioS, with Proline:1,2-butanediol [1:4] being the most efficient, yielding 75 mg of total polyphenols/g of dried sample in the non-pretreated extract. As for the composition, 95 % of the non-pretreated extract was constituted by oleuropein, while, on average, 65 % of the pretreated extract was found to be a mixture of hydroxytyrosol and oleuropein. In all cases, the polyphenols extracted were determined by high-performance liquid chromatography. Finally, the experimental results were analyzed using COSMO-RS methodology, obtaining reliable solubility trends for target antioxidants in the proposed green solvents, while explaining the thermodynamic interactions of the systems in terms of molecular behavior. This work can serve as a guideline to develop flexible biorefinery schemes for the production of oleuropein and hydroxytyrosol based on different extraction approaches, with in silico tools providing further insights into the role of novel solvents in olive-derived polyphenols recovery processes.

2G-lactic acid from olive oil supply chain waste: olive leaves upcycling via Lactobacillus casei fermentation

The transition towards a sustainable model, particularly the circular economy, emphasizes the importance of redefining waste as a valuable resource, paving the way for innovative upcycling strategies. The olive oil industry, with its significant output of agricultural waste, offers a promising avenue for high-value biomass conversion into useful products through microbial processes. This study focuses on exploring new, high-value applications for olive leaves waste, utilizing a biotechnological approach with Lactobacillus casei for the production of second-generation lactic acid. Contrary to initial expectations, the inherent high polyphenol content and low fermentable glucose levels in olive leaves posed challenges for fermentation. Addressing this, an enzymatic hydrolysis step, following a preliminary extraction process, was implemented to increase glucose availability. Subsequent small-scale fermentation tests were conducted with and without nutrient supplements, identifying the medium that yielded the highest lactic acid production for scale-up. The scaled-up batch fermentation process achieved an enhanced conversion rate (83.58%) and specific productivity (0.26 g/L·h). This research confirms the feasibility of repurposing olive waste leaves for the production of lactic acid, contributing to the advancement of a greener economy through the valorization of agricultural waste.Key points• Olive leaves slurry as it did not allow L. casei to ferment.• High concentrations of polyphenols inhibit fermentation of L. casei.• Enzymatic hydrolysis combined to organosolv extraction is the best pretreatment for lactic acid production starting from leaves and olive pruning waste.Graphical