Subsequent Hydrolysis Step Research Articles

Recently evolved combination of unique sulfatase and amidase genes enables bacterial degradation of the wastewater micropollutant acesulfame worldwide.

Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and an acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA amidase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and likely evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics.

Development of a GC–MS strategy for the determination of cross-linked proteins in 20th century paint tubes

In this paper we present the technical study of protein-drying oil mixtures in the paint formulation of original manufacturers paint tubes from Fabbrica di colori per Belle Arti Maimeri dating back to the 1950s. The study is part of a wider research project aimed at understanding the chemistry of protein-drying oil mixtures, and how the oil curing in the presence of proteins may affects the protein analysis with mass spectrometric based techniques.Tempera grassa, according to traditional Late Medieval and Renaissance painting techniques, entails the use, as paint binder, of a mixture of a drying oil with a water miscible medium, commonly egg. The paints analysed in this study were, according to technical documentation available at the manufacturer, based on a mixture of linseed oil, ovalbumin and mastic resin. All the components of the paints were detected but proteins proved very challenging. They could not be detected by pyrolysis coupled to gas chromatography-mass spectrometry, nor by using classical procedures for gas chromatography – mass spectrometry analysis of amino acids after hydrolysis. A new analytical procedure based on two subsequent hydrolysis steps assisted by microwaves was implemented and proved to be successful. The first step with trifluoroacetic acid was aimed at a partial hydrolysis of proteins into peptides, for their efficient extraction. The second with hydrochloric acid in the vapour phase was used for a complete hydrolysis of peptides into amino acids for the GC–MS analysis. Linking experimental results on the tempera grassa paints to data obtained on the curing of oil/protein mixtures in paint mock-ups, the hypothesis that copolymerisation of proteins and the oil has taken place is proposed, and a discussion on how this may affect protein detection is carried out.

Glucan Conversion and Membrane Recovery of Biomimetic Cellulosomes During Lignocellulosic Biomass Hydrolysis.

Enzyme immobilization has been identified as one way to recycle enzymes and reduce processing costs during enzymatic hydrolysis of lignocellulosic materials. However, most immobilization methods have not been attractive to lignocellulosic processing plants. In this study, cellulase enzymes were attached to a copolymer of glycidyl methacrylate (GMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) to make polymer-enzyme conjugates (PECs) and facilitate recovery using a 50-kDa molecular weight cutoff membrane. Glucan conversion during biomass hydrolysis was investigated using new PECs and PECs recovered after an initial hydrolysis stage. Enzyme immobilization on PECs did not reduce effectiveness during the initial hydrolysis. Temperature and pH showed similar effects on free enzymes and PECs. PECs facilitated higher conversion rates than free enzymes at high biomass loadings. Recovered PECs were used to achieve approximately 100% glucan conversion in a subsequent hydrolysis step when supplemented with 40% of the free enzyme used in the first stage. The combination of PECs and membrane recovery has the potential to reduce hydrolysis cost during cellulosic bioprocessing.

Polymer Extraction from Lignocellulosic Biomass (Water Hyacinth)

Bioconversion of renewable lignocellulosic biomass to biofuel and value-added products is globally gaining significant importance. Lignocellulosic wastes are the most promising feedstock considering its great availability and low cost. The biomass conversion process involves mainly two steps: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars and fermentation of the sugars to ethanol and other bioproducts. However, sugars necessary for fermentation are trapped inside the recalcitrant structure of the lignocellulose. Hence, pretreatment of lignocellulosic wastes is always necessary to alter and/or remove the surrounding matrix of lignin and hemicelluloses in order to improve the hydrolysis of cellulose. These pretreatments cause physical and/or chemical changes in the plant biomass in order to achieve this result. Each pretreatment has a specific effect on the cellulose, hemicellulose, and lignin fraction. Thus, the pretreatment methods and conditions should be chosen according to the process configuration selected for the subsequent hydrolysis steps. In general, pretreatment methods can be classified into four categories, including physical, physicochemical, chemical, and biological pretreatment. Bioresource utilization of biopolymeric materials has now gained recent attention. Cellulose was extracted from water hyacinth by acid, alkali treatment & extracted cellulose was grafted with curcumin, pesticide, grape juice, magnetorheological fluid, and the grafted composite material was evaluated for release of respective grafted materials. In the present study, a polymer extracted from water hyacinth was evaluated for various applications. The present study would suggest the possible utilization of water hyacinth composite as the biomaterial for diverse applications.

Improving the extraction efficiency of sedimentary carbohydrates by sequential hydrolysis

Carbohydrates serve as structural materials, energy storage compounds, and energy carriers in organisms and provide insights into the dominant sources and the degradation state of organic matter (OM). Several studies have analyzed dissolved and/or particulate carbohydrates in the environment, often with the aim of studying particular carbohydrate groups of interest. These studies have often employed different acids and hydrolytic conditions, making comparisons across studies a challenge. Here, we introduce a sequential acid hydrolysis protocol for the comprehensive extraction, quantification and compositional investigation of carbohydrates in sediment samples. This protocol has four acid hydrolysis steps that sequentially increase in extraction strength, where each subsequent hydrolysis step is performed on the unextracted remains previously hydrolyzed. The four hydrolysis steps consist of 1 M HCl for 2 h at (1) room temperature, (2) 50 °C, and (3) 105 °C, followed by incubation for 8 h using (4) 6 M HCl and 105 °C. Based on tests using diverse carbohydrate standard compounds, the protocol recovers most di- and oligosaccharides and soluble polysaccharides during the initial hydrolysis steps, whereas the recovery of most insoluble polysaccharides is highest during the final extraction step. Applying this protocol to different sediment types shows that recoveries of neutral sugars, amino sugars and sugar alcohols are on average ~60% higher than with a reference one-step extraction method using only hydrolysis with hot 6 M HCl. This sequential extraction protocol thus provides an important new tool for the quantitative and compositional analysis of carbohydrates in aquatic sediments.

ChemInform Abstract: Triphenylphosphinecarboxamide: An Effective Reagent for the Reduction of Azides and Its Application to Nucleic Acid Detection.

AbstractIn contrast to conventional methods in this procedure no subsequent hydrolysis step is necessary.

ChemInform Abstract: A Novel Synthesis of Antiviral Nucleoside Phosphoramidate and Thiophosphoramidate Prodrugs via Nucleoside H‐Phosphonamidates.

AbstractA series of novel H‐phosphonamidates (III) is synthesized by reaction of stavudine (I) with PCl3 followed by coupling with amino acid esters (II) and final subsequent hydrolysis step.

18F]Fluoropyruvate: radiosynthesis and initial biological evaluation

The radiosynthesis of [(18)F]fluoropyruvate was investigated using numerous precursors were synthesized from ethyl 2,2-diethoxy-3-hydroxypropanoate (5) containing different leaving groups: mesylate, tosylate, triflate, and nonaflate. These precursors were evaluated for [(18)F]fluoride incorporation with triflate being superior. The subsequent hydrolysis step was investigated, and an acidic hydrolysis was optimized. After establishing suitable purification and formulation methods, the [(18)F]fluoropyruvate could be isolated in ca. 50% d.c. yield. The [(18)F]fluoropyruvate was evaluated in vitro for its uptake into tumor cells using adenocarcinomic human alveolar basal epithelial cells (A549) and unfortunately showed an uptake of approximately 0.1% of the applied dose per 100,000 cells after 30 min. Initial pharmacokinetic properties were assessed in vivo using nude mice showed a high degree of bone uptake from defluorination, which will limit its potential as an imaging agent for metabolic processes.

Синтез ионных жидкостей на основе катионов 1,3-диалкилимидазолия и тетраалкиламмония для проведения предобработки лигноцеллюлозного сырья с целью увеличения его гидролизуемости

In this paper, based on the theoretical analysis of the properties of ionic liquids dependence on their structure a number of compounds of this class with the optimal structure was synthesized for continuing the process of pretreatment of lignocelluloses raw material. The purpose of preprocessing is to remove lignin and hemicelluloses, as well as to reduce degree of crystallinity and increase the porosity of the cellulose, making it more accessible to the enzymes in the subsequent hydrolysis step in a process of production of bioethanol. As the optimal structures ionic liquids were proposed containing acetate and iodide anions.

Stabilization of the ADP/Metaphosphate Intermediate during ATP Hydrolysis in Pre-power Stroke Myosin: QUANTITATIVE ANATOMY OF AN ENZYME

It has been proposed recently that ATP hydrolysis in ATPase enzymes proceeds via an initial intermediate in which the dissociated γ-phosphate of ATP is bound in the protein as a metaphosphate (PγO3(-)). A combined quantum/classical analysis of this dissociated nucleotide state inside myosin provides a quantitative understanding of how the enzyme stabilizes this unusual metaphosphate. Indeed, in vacuum, the energy of the ADP(3-) · PγO3(-) · Mg(2+) complex is much higher than that of the undissociated ATP(4-). The protein brings it to a surprisingly low value. Energy decomposition reveals how much each interaction in the protein stabilizes the metaphosphate state; backbone peptides of the P-loop contribute 50% of the stabilization energy, and the side chain of Lys-185(+) contributes 25%. This can be explained by the fact that these groups make strong favorable interactions with the α- and β-phosphates, thus favoring the charge distribution of the metaphosphate state over that of the ATP state. Further stabilization (16%) is achieved by a hydrogen bond between the backbone C=O of Ser-237 (on loop Switch-1) and a water molecule perfectly positioned to attack the PγO3(-) in the subsequent hydrolysis step. The planar and singly negative PγO3(-) is a much better target for the subsequent nucleophilic attack by a negatively charged OH(-) than the tetrahedral and doubly negative PγO4(2-) group of ATP. Therefore, we argue that the present mechanism of metaphosphate stabilization is common to the large family of nucleotide-hydrolyzing enzymes. Methodologically, this work presents a computational approach that allows us to obtain a truly quantitative conception of enzymatic strategy.

Lewis Base-Promoted Hydrosilylation of Cyclic Malonates: Synthesis of β-Substituted Aldehydes and γ-Substituted Amines

The Lewis base-promoted hydrosilylation of cyclic malonates provides a convenient synthesis of beta-substituted aldehydes. No over-reduction to the primary alcohol is observed as the aldehyde functionality is protected until a subsequent hydrolysis step. The utility of the method is demonstrated in a number of examples and further in the synthesis of gamma-substituted propylamines in a one-pot hydrosilylation/reductive amination process.

Hydrolysis of Fluorosilanes: A Theoretical Study

Hydrolysis and condensation of simple trifluorosilanes, HSiF3 and MeSiF3, was studied by quantum mechanical methods. Hydrolysis of fluorosilanes is highly endothermic. The Gibbs free energy of the first reaction step in the gas phase is 31.4 kJ/mol, which corresponds to an equilibrium constant of 10(-6). Hydrolysis of the subsequent fluorine atoms in trifluorosilanes is thermodynamically more unfavorable than the first step of substitution. No significant difference in thermodynamics of hydrolysis was found between HSiF3 and MeSiF3. The activation energy for hydrolysis by a water dimer is significantly lower than that for hydrolysis by a water monomer. The former reaction is also less unfavorable thermodynamically, due to a high binding energy of the HF-H2O complex formed as a product of hydrolysis. Self-consistent reaction field (SCRF) calculations show that hydrolysis of trifluorosilanes in aqueous medium has lower activation energy than in the gas phase. It is also thermodynamically less unfavorable, due to better solvation of the products. Homofunctional condensation of HSiF2OH is thermodynamically favored. The equilibrium mixture for hydrolysis/condensation of RSiF3 in water is predicted to contain ca. 2.3% disiloxane (HF2Si)2O, if 100-fold excess of water relative to silane is assumed. Further hydrolysis of (HF2Si)2O is negligible. The thermodynamics of fluorosilane hydrolysis contrasts with that of chlorosilanes, where both hydrolysis and condensation are strongly favorable. Moreover, in the case of trichlorosilanes each subsequent hydrolysis step is more facile, leading to the product of full hydrolysis, RSi(OH)3.

Electrostatic influence of active-site waters on the nucleophilic aromatic substitution catalyzed by 4-chlorobenzoyl-CoA dehalogenase

The 4-chlorobenzoyl-CoA dehalogenase catalyzes the hydrolytic dechlorination of 4-chlorobenzoyl-CoA via a two-step mechanism, namely nucleophilic aromatic substitution and ester hydrolysis. The mutation of an active-site Histidine residue has been shown to reduce the catalytic activity in both the substitution and subsequent hydrolysis steps. In this communication, we report a quantum mechanical/molecular mechanical simulation of the potential of mean force for the substitution step, which confirms the increased barrier height in the H90Q mutant and provides evidence on the electrostatic influence of two active-site waters on the rate-limiting barrier.

A Simple Approach to Synthesize and Bond Organosiloxane Films on Injection-Molded Polyolefins

A simple method was devised to fabricate and anchor organosiloxane coatings along low-surface-energy plastics without resorting to chemical pre-treatment of the polymer surface. Polypropylene tubes were purposely distended by exposure to toluene-based solutions. An established solvent of many organic polymers and compounds, toluene not only perfused into the plastic via gradual intercalation of accessible polymer chains, but it also facilitated the entry of less compatible reagents as co-perfusates. The reagents, typically aminoalkyl si lanes, were activated either concomitant to perfitsion of polypro-pylene or during a subsequent hydrolysis step. Freed silanol groups, interacting along and beneath the Surface of the plastic, reacted to form a network held together by siloxane bonds. The type of network formed and presented herein was interpreted as an amorphous aminopropylsiloxane-silanol hydrate film, with overall thickness partially submerged and caught in the matrix of the plastic. This concept to intertwine a growing organosiloxane network and a compliant matrix precluded the conventional methods normally used to improve bonding on hydrophobic plastics. A comparison of ninhydrin color yields suggested that films were more effectively and conveniently synthesized when aminopropyltri-methoxysilane was applied in water-saturated toluene. It follows that by integrating low-temperature hydrothermal processing into this strategy, inorganic-organic hybrids or inorganic oxide films may be formed oil pre-molded plastics and feature traits such as covalent-like fastness and gas impermeability.

Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue.

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.

Reaction Kinetics of Protease with Substrate Phage: KINETIC MODEL DEVELOPED USING STROMELYSIN

Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.

Structure of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 A resolution: an enzyme catalyst generated via adaptive mutation.

Here we describe the three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3. This enzyme catalyzes the hydrolysis of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA. The molecular structure of the enzyme/4-hydroxybenzoyl-CoA complex was solved by the techniques of multiple isomorphous replacement, solvent flattening, and molecular averaging. Least-squares refinement of the protein model reduced the crystallographic R factor to 18.8% for all measured X-ray data from 30 to 1.8 A resolution. The crystallographic investigation of this dehalogenase revealed that the enzyme is a trimer. Each subunit of the trimer folds into two distinct motifs. The larger, N-terminal domain is characterized by 10 strands of beta-pleated sheet that form two distinct layers which lie nearly perpendicular to one another. These layers of beta-sheet are flanked on either side by alpha-helices. The C-terminal domain extends away from the body of the molecule and is composed of three amphiphilic alpha-helices. This smaller domain is primarily involved in trimerization. The two domains of the subunit are linked together by a cation, most likely a calcium ion. The 4-hydroxybenzoyl-CoA molecule adopts a curved conformation within the active site such that the 4-hydroxybenzoyl and the adenosine moieties are buried while the pantothenate and pyrophosphate groups of the coenzyme are more solvent exposed. From the three-dimensional structure it is clear that Asp 145 provides the side-chain carboxylate group that adds to form the Meisenheimer intermediate and His 90 serves as the general base in the subsequent hydrolysis step. Many of the structural principles derived from this investigation may be directly applicable to other related enzymes such as crotonase.

Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].