Relative Rates Of Hydrolysis Research Articles

Synthesis of porous gas materials for a semiconductor carbon(ii) oxide sensor and their investigation

In this article, the process and conditions for the synthesis of porous silica materials based on alkoxysilanes and metal oxides are studied. the main stages are discussed. A change in the pH value of a solution affects the relative rate of hydrolysis and condensation reactions of the solution, which makes it possible to obtain various structures from weakly branched silicate polymers to ordered, densely packed particles. The H2O/TEOS molar ratio (R) also has a significant effect on the structure of the synthesized silica materials. By changing R, silica materials can be obtained in the form of solid gels and colloidal particles. The composition and properties of the developed gas sensitive materials were studied by SEM, XRD and thermal analysis.

Quantitative peptide release kinetics to describe the effect of pH on pepsin preference

The preference of pepsin to hydrolyse certain peptide bonds is typically determined by counting peptides after hydrolysis, without considering concentrations and kinetics. In this study, peptide release was quantified to describe proteolysis by pepsin. The aim was to investigate whether pH affects individual hydrolysis rates of peptide bonds. α-Lactalbumin was hydrolysed by porcine pepsin systematically at pH 1–5 and peptides were identified and quantified with UPLC-PDA-MS at eight time points. Apparent pH-based differences in specificity were caused by differences in total hydrolysis rate but the relative hydrolysis rates of cleavage sites were generally independent of pH. Previous statements of pepsin preference for amino acids in the P3-P3′ positions withstand when considering the hydrolysis rates of cleavage sites and was pH independent. Despite the a-specificity of pepsin, many bonds were not or slowly hydrolysed, some cleavage sites became more accessible during hydrolysis (demasking) and some became less accessible (masking).

Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses

Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.

Weak Temperature Dependence of the Relative Rates of Chlorination and Hydrolysis of N2O5 in NaCl–Water Solutions

We have measured the temperature dependence of the ClNO2 product yield in competition with hydrolysis following N2O5 uptake to aqueous NaCl solutions. For NaCl-D2O solutions spanning 0.0054-0.21 M, the ClNO2 product yield decreases on average by only 4 ± 3% from 5 to 25 °C. Less reproducible measurements at 0.54-2.4 M NaCl also fall within this range. The ratio of the rate constants for chlorination and hydrolysis of N2O5 in D2O is determined on average to be 1150 ± 90 at 25 °C up to 0.21 M NaCl, favoring chlorination. This ratio is observed to decrease significantly at the two highest concentrations. An Arrhenius analysis reveals that the activation energy for hydrolysis is just 3.0 ± 1.5 kJ/mol larger than for chlorination up to 0.21 M, indicating that Cl- and D2O attack on N2O5 has similar energetic barriers despite the differences in charge and complexity of these reactants. In combination with the measured preexponential ratio favoring chlorination of 300-200+400, we conclude that the strong preference of N2O5 to undergo chlorination over hydrolysis is driven by dynamic and entropic, rather than enthalpic, factors. Molecular dynamics simulations elucidate the distinct solvation between strongly hydrated Cl- and the hydrophobically solvated N2O5. Combining this molecular picture with the Arrhenius analysis implicates the role of water in mediating interactions between such distinctly solvated species and suggests a role for diffusion limitations on the chlorination reaction.

Zearalenone-14-Glucoside Is Hydrolyzed to Zearalenone by β-Glucosidase in Extracellular Matrix to Exert Intracellular Toxicity in KGN Cells.

As one of the most important conjugated mycotoxins, zearalenone-14-glucoside (Z14G) has received widespread attention from researchers. Although the metabolism of Z14G in animals has been extensively studied, the intracellular toxicity and metabolic process of Z14G are not fully elucidated. In this study, the cytotoxicity of Z14G to human ovarian granulosa cells (KGN) and the metabolism of Z14G in KGN cells were determined. Furthermore, the experiments of co-administration of β-glucosidase and pre-administered β-glucosidase inhibitor (Conduritol B epoxide, CBE) were used to clarify the mechanism of Z14G toxicity release. Finally, the human colon adenocarcinoma cell (Caco-2) metabolism model was used to verify the toxicity release mechanism of Z14G. The results showed that the IC50 of Z14G for KGN cells was 420 μM, and the relative hydrolysis rate of Z14G on ZEN was 35% (25% extracellular and 10% intracellular in KGN cells). The results indicated that Z14G cannot enter cells, and Z14G is only hydrolyzed extracellularly to its prototype zearalenone (ZEN) by β-glucosidase which can exert toxic effects in cells. In conclusion, this study demonstrated the cytotoxicity of Z14G and clarified the toxicity release mechanism of Z14G. Different from previous findings, our results showed that Z14G cannot enter cells but exerts cytotoxicity through deglycosylation. This study promotes the formulation of a risk assessment and legislation limit for ZEN and its metabolites.

Examining the Role of Atomic Scale Heterogeneity on the Thermal Conductivity of Transparent, Thermally Insulating, Mesoporous Silica–Titania Thin Films

This paper examines the effect of compositional heterogeneity on the thermal conductivity of transparent, mesoporous silica–titania composites that contain either 10 or 20 mol % titania. The relative hydrolysis rates of the silica and titania precursors were modified to control their compositional heterogeneity, while the ratio of polymer to inorganic precursors (silica + titania) was varied to control the porosity of the films. All films were optically transparent at thicknesses up to 1 μm with transmittance above 90% and haze below 5% at visible wavelengths. It was found that the heterogeneity of the titania species in the 10 mol % titania samples could be easily tailored from highly dispersed titania to a composition with heterogeneous silica-rich and titania-rich domains. By contrast, samples with 20 mol % titania always showed a heterogeneous titania distribution. The results show that mesoporous films with more homogeneously distributed titania had a lower thermal conductivity at all porosities, likely due to increases in propagon and diffuson scattering as a result of the increased number density of titania heteroatom scattering centers. These results increase our understanding of heat carrier propagation in amorphous materials and add to the design rules for creating amorphous, optically clear, low thermal conductivity materials.

Use of Silica Based Materials as Modulators of the Lipase Catalyzed Hydrolysis of Fats under Simulated Duodenal Conditions.

The effect of silica materials and their functionalization in the lipase catalyzed fat hydrolysis has been scarcely studied. Fifteen silica materials were prepared and their effect on the fat hydrolysis was measured, under simulated duodenal conditions, using the pH-stat method. The materials are composed of the combination of three supports (Stöber massive silica nanoparticles, Stöber mesoporous nanoparticles and UVM-7) and four surface functionalizations (methyl, trimethyl, propyl and octyl). In addition, the non-functionalized materials were tested. The functional groups were selected to offer a hydrophobic character to the material improving the interaction with the fat globules and the lipase. The materials are able to modulate the lipase activity and their effect depending on the support topology and the organic covering, being able to increase or reduce the fat hydrolysis. Depending of the material, relative fat hydrolysis rates of 75 to 140% in comparison with absence of the material were obtained. The results were analyzed by Partial Least Square Regression and suggest that the alkyl modified mesopores are able to improve the fat hydrolysis, by contrast the non-porous nanoparticles and the textural pores tend to induce inhibition. The effects are more pronounced for materials containing long alkyl chains and/or in absence of taurodeoxycholate.

On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation.

Chemical group‐transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial‐scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α‐d‐glucose 1‐phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β‐glucosyl‐enzyme intermediate (E‐Glc), but additionally from a noncovalent complex of E‐Glc and substrate which unlike E‐Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate‐bound E‐Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate‐bound E‐Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E‐Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group‐transfer reactions by hydrolytic enzymes.

Unexpected Anomeric Acceptor Preference Observed Using dDNP NMR for Transglycosylation Studies of β-Galactosidases

The transglycosylation abilities of β-galactosidases were investigated using hyperpolarized [U-13C,U-2H]glucose as an acceptor and o-nitrophenyl β-galactopyranoside as a donor. Several products were readily observable, and at least in the case when O3 acted as an acceptor, the enzymes showed a clear selectivity toward the β-anomer of glucose. Additionally, it was possible to determine the relative hydrolysis rates of the formed transglycosylation products, providing information on the selectivity as well. Using this method, the transglycosylation abilities of the enzymes could be studied at a very high temporal resolution as well as with high sensitivity, and due to the relative ease of the setup, this method could be more generally applied to investigate glycosidases.

Extensively Drug-Resistant Pseudomonas aeruginosa ST309 Harboring Tandem Guiana Extended Spectrum β-Lactamase Enzymes: A Newly Emerging Threat in the United States.

BackgroundTreatment of serious infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa remains a challenge, despite the introduction of novel therapeutics. In this study, we report 2 extensively drug-resistant clinical isolates of sequence type (ST) 309 P aeruginosa resistant to all β-lactams, including the novel combinations ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam.MethodsIsolates were sequenced using both short-read (Illumina) and long-read technology to identify resistance determinants, polymorphisms (compared with P aeruginosa PAO1), and reconstruct a phylogenetic tree. A pair of β-lactamases, Guiana extended spectrum β-lactamase (GES)-19 and GES-26, were cloned and expressed in a laboratory strain of Escherichia coli to examine their relative impact on resistance. Using cell lysates from E coli expressing the GES genes individually and in tandem, we determined relative rates of hydrolysis for nitrocefin and ceftazidime.ResultsTwo ST309 P aeruginosa clinical isolates were found to harbor the extended spectrum β-lactamases GES-19 and GES-26 clustered in tandem on a chromosomal class 1 integron. The presence of both enzymes in E coli was associated with significantly elevated minimum inhibitory concentrations to aztreonam, cefepime, meropenem, ceftazidime/avibactam, and ceftolozane/tazobactam, compared with those expressed individually. The combination of ceftazidime/avibactam plus aztreonam was active in vitro and used to achieve cure in one patient. Phylogenetic analysis revealed ST309 P aeruginosa are closely related to MDR strains from Mexico also carrying tandem GES.ConclusionsThe presence of tandem GES-19 and GES-26 is associated with resistance to all β-lactams, including ceftolozane/tazobactam. Phylogenetic analysis suggests that ST309 P aeruginosa may be an emerging threat in the United States.

Molecular insight into chymotrypsin inhibitor 2 resisting proteolytic degradation

Chymotrypsin inhibitor 2 (CI2) is a special serine protease inhibitor which can resist hydrolysis for several days with a rapid equilibrium between the Michaelis complex and acyl-enzyme intermediate. The energies and conformational changes for subtilisin-catalyzed proteolysis of CI2 were examined in this paper for the first time by employing pseudo bond ab initio QM/MM MD simulations. In the acylation reaction, a low-barrier hydrogen bond between His64 and Asp32 in the transition state together with the lack of covalent backbone constraints makes the peptide bonds of CI2 break more easily than in other serine protease inhibitors. After acyl-enzyme formation, molecular dynamics simulations showed that the access of hydrolytic water to the active site requires partial dissociation of the leaving group. However, retention of the leaving group mainly by the intra- and inter-molecular H-bonding networks hinders the access of water and retards the deacylation reaction. Instead of the dissociation constant of inhibitors, we suggest employing the free energy at the acyl-enzyme state to predict the relative hydrolysis rates of CI2 mutants, which are testified by the experimental relative hydrolysis rates.

A plasmid borne, functionally novel glycoside hydrolase family 30 subfamily 8 endoxylanase from solventogenic Clostridium.

Glycoside hydrolase family 30 subfamily 8 (GH30-8) β-1,4-endoxylanases are known for their appendage-dependent function requiring recognition of an α-1,2-linked glucuronic acid (GlcA) common to glucuronoxylans for hydrolysis. Structural studies have indicated that the GlcA moiety of glucuronoxylans is coordinated through six hydrogen bonds and a salt bridge. These GlcA-dependent endoxylanases do not have significant activity on xylans that do not bear GlcA substitutions such as unsubstituted linear xylooligosaccharides or cereal bran arabinoxylans. In the present study, we present the structural and biochemical characteristics of xylanase 30A from Clostridium acetobutylicum (CaXyn30A) which was originally selected for study due to predicted structural differences within the GlcA coordination loops. Amino acid sequence comparisons indicated that this Gram-positive-derived GH30-8 more closely resembles Gram-negative derived forms of these endoxylanases: a hypothesis borne out in the developed crystallographic structure model of the CaXyn30A catalytic domain (CaXyn30A-CD). CaXyn30A-CD hydrolyzes xylans to linear and substituted oligoxylosides showing the greatest rate with the highly arabinofuranose (Araf)-substituted cereal arabinoxylans. CaXyn30A-CD hydrolyzes xylooligosaccharides larger than xylotriose and shows an increased relative rate of hydrolysis for xylooligosaccharides containing α-1,2-linked arabinofuranose substitutions. Biochemical analysis confirms that CaXyn30A benefits from five xylose-binding subsites which extend from the −3 subsite to the +2 subsite of the binding cleft. These studies indicate that CaXyn30A is a GlcA-independent endoxylanase that may have evolved for the preferential recognition of α-1,2-Araf substitutions on xylan chains.

Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR.

Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.

Towards predicting protein hydrolysis by bovine trypsin

Abstract The extent of protein enzymatic hydrolysis is considered to be mostly determined by protease specificity and the number of cleavage sites (CS) on the substrate. However, this theoretical maximum is typically not reached. The limited hydrolysis of certain CS may be due to the differences in enzyme preference resulting from neighbouring amino acids (AA) of the CS (secondary specificity). This study aims to find the link between enzyme secondary specificity and the relative hydrolysis rate constants (selectivity) of individual CS in a protein, to better predict the maximum experimental degree of hydrolysis. Bovine tryptic hydrolysis of α-lactalbumin and β-casein showed that ≥50% of the CS were inefficiently hydrolysed. The selectivity depended on the number of charged AA at P2 and P2′ positions of a CS. Trypsin efficiently cleaves CS with neutral AA at these two positions. The selectivity towards 12 out of 18 (67%) CS in β-lactoglobulin was correctly predicted. The predicted maximum degree of hydrolysis is within ∼13% error of the experimental value, which is ∼5 times better than the prediction based only on enzyme specificity. This work helps to estimate the extent of hydrolysis and the peptide formation of bovine tryptic hydrolysis of other substrates.

Partial Purification and Characterization of Cellulase Produced by <i>Bacillus sphaericus</i> CE-3

Cellulase is an enzyme produced by fungi, bacteria, protozoa and termite, that hydrolyze cellulose. They are known for their diverse applications in industry and medicine. The aim of this study is to purify and investigate cellulolytic properties of cellulase enzyme produced by Bacillus sphaericus CE-3 isolated from refuse dump in Nnamdi Azikiwe University, Awka, Nigeria. Enzyme was produced by submerged fermentation at 30°C for 30 h. The enzyme was purified to homogeneity by dialysis in 4M sucrose solution, ion-exchange chromatography on Q-Sepharose FF and by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The relative molecular mass of the enzyme was estimated using SDS-Polyacrylamide gel electrophoresis. Effects of temperature, pH and metals on enzyme activity and stability and the relative rate of hydrolysis of various substrates were also studied. The Purification fold for the enzyme was 7.8, with 66.4 μ/mg specific activity protein and overall yield of 35.8. The relative molecular mass range of the enzyme was estimated between 22.3 kDa - 26.3 kDa. The enzyme was optimally active at pH 9.0 and 40°C, stable at pH 9.0 and unusually retained over 90% activity between 50°C - 100°C after 30 min incubation. It was strongly activated by Mn2+ but inhibited by Ba2+, Co2+, Hg2+, Pb2+, Cu2+, Sr2+, Fe2+, Ca2+ and Zn2+. The cellulase displayed high catalytic activity with untreated sawdust, followed by carboxymethyl cellulose, while sodium hydroxide treated sawdust was the least hydrolyzed. Since the enzyme is thermo-stable, alkalophilic and could utilize natural wastes like sawdust as substrate, it is obvious that it would be of great use in textile, starch processing and pulp and paper industries.

Phenyl carbamate end-capped oligoesters: a model for hydrolytic stability of ester-based urethanes

A series of hydroxyl-terminated oligoesters were synthesized to determine the structural effects on the relative rates of hydrolysis in both acidic and alkaline media. Model compounds consisting of one diol and one dibasic acid were used because the simplicity of the chemical structure allowed for the analysis of individual effects. Linear, cycloaliphatic, and aromatic dibasic acids were used. The diacids were reacted with a series of linear and bulky diols. All hydroxyl-terminated oligoesters were end-capped with phenyl isocyanate. Three different stages were distinguished in the case of aliphatic oligoesters, while more bulky structures (cycloaliphatic and aromatics) only presented one or two stages. Hydrolysis rates showed a complex behavior where steric and anchimeric effects play a mutual role on initial hydrolysis rates.

Arrays of Hollow Silica Half‐Nanospheres Via the Breath Figure Approach

Breath figures (BFs) are patterns of liquid droplets that usually form upon condensation on a cold surface. Earlier work has shown that BFs can be used to produce continuous films of porous honeycomb‐structured patterns on various types of materials, paving the path to a number of important applications such as the manufacturing of highly ordered nano‐ and micron‐sized templates, micro lenses, and superhydrophobic coatings. It is worth noting, however, that few new findings have been reported in this area in recent years, limiting pursuits of novel architectures and key applications. In this report, an alternative method is described by which arrays of hollow silica half‐nanospheres can be produced via BF templates. In the present method, a chemical vapor deposition (CVD) protocol performed while the BF is formed on a glass substrate yields a nanostructured pattern of silica half‐spheres, which size (100–700 nm) and density across the glass surface vary with substrate modification and with the relative rates of water condensation and hydrolysis from silica precursors (a process carried out at room temperature). This method of forming arrays of hollow half‐nanospheres via the BF approach may be applicable to various other oxides and a broad range of substrates including large‐area flexible plastics.

Synthesis and hydrolysis of gas-phase lanthanide and actinide oxide nitrate complexes: a correspondence to trivalent metal ion redox potentials and ionization energies.

Several lanthanide and actinide tetranitrate ions, M(III)(NO3)4(-), were produced by electrospray ionization and subjected to collision induced dissociation in quadrupole ion trap mass spectrometers. The nature of the MO(NO3)3(-) products that result from NO2 elimination was evaluated by measuring the relative hydrolysis rates under thermalized conditions. Based on the experimental results it is inferred that the hydrolysis rates relate to the intrinsic stability of the M(IV) oxidation states, which correlate with both the solution IV/III reduction potentials and the fourth ionization energies. Density functional theory computations of the energetics of hydrolysis and atoms-in-molecules bonding analysis of representative oxide and hydroxide nitrates substantiate the interpretations. The results allow differentiation between those MO(NO3)3(-) that comprise an O(2-) ligand with oxidation to M(IV) and those that comprise a radical O(-) ligand with retention of the M(III) oxidation state. In the particular cases of MO(NO3)3(-) for M = Pr, Nd and Tb it is proposed that the oxidation states are intermediate between M(III) and M(IV).

Determination of the Influence of Substrate Concentration on Enzyme Selectivity Using Whey Protein Isolate and Bacillus licheniformis Protease

Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each cleavage site in the protein. It was determined from the identification and quantification of the peptides present in the hydrolysates. Solutions of 0.1-10% (w/v) whey protein isolate (WPI) were hydrolyzed by Bacillus licheniformis protease at constant enzyme-to-substrate ratio. The cleavage sites were divided into five groups, from very high (>10%) to very low selectivity (<0.1%). The selectivity toward cleavage sites after Glu 62 and 134 was 2 times higher at 10% (w/v) WPI than at the lower protein concentrations. This finding shows that both the rate of hydrolysis and the enzyme selectivity were influenced by the substrate concentration.

Synthesis and properties of lipophilic derivatives of 5-fluorouracil

A series of N-acyl derivatives of 5-fluorouracil (5-FU) bearing residues of palmitic, p-myristoylaminobenzoic, p-oleoylaminobenzoic, and 1-adamantanecarbonic acids was synthesized. Relative rates of hydrolysis of derivatives mentioned under physiological conditions, at pH 7.2 and 37 degrees C, have shown that stability of these compounds increases with reducing of spatial accessibility of amide group at N1 in 5-FU. These substances incorporate easily into lipid bilayer; their liposomal preparations showed substantial cytostatic activity on HBL (human breast lymphoma) cells and are of interest as potential antitumor preparations. Also, a fluorescent analog, 1-[8-(3-perylenyl)octanoyl]-5-fluorouracil, destined for studies of the 5-FU derivatives behavior in cells and tissues was prepared.