Kinetics of non-catalyzed hydrolysis of tannin in high temperature liquid water*

PubMed Central

Lu, Li-li; Lu, Xiu-yang; Ma, Nan

2008-01-01

High temperature liquid water (HTLW) has drawn increasing attention as an environmentally benign medium for organic chemical reactions, especially acid-/base-catalyzed reactions. Non-catalyzed hydrolyses of gallotannin and tara tannin in HTLW for the simultaneous preparation of gallic acid (GA) and pyrogallol (PY) are under investigation in our laboratory. In this study, the hydrolysis kinetics of gallotannin and tara tannin were determined. The reaction is indicated to be a typical consecutive first-order one in which GA has formed as a main intermediate and PY as the final product. Selective decomposition of tannin in HTLW was proved to be possible by adjusting reaction temperature and time. The present results provide an important basic data and reference for the green preparation of GA and PY. PMID:18500780

Rubber muscle actuation with pressurized CO2 from enzyme-catalyzed urea hydrolysis

NASA Astrophysics Data System (ADS)

Sutter, Thomas M.; Dickerson, Matthew B.; Creasy, Terry S.; Justice, Ryan S.

2013-09-01

A biologically inspired pneumatic pressure source was designed and sized to supply high pressure CO2(g) to power a rubber muscle actuator. The enzyme urease served to catalyze the hydrolysis of urea, producing CO2(g) that flowed into the actuator. The actuator’s power envelope was quantified by testing actuator response on a custom-built linear-motion rig. Reaction kinetics and available work density were determined by replacing the actuator with a double-action piston and measuring volumetric gas generation against a fixed pressure on the opposing piston. Under the conditions investigated, urease catalyzed the generation of up to 0.81 MPa (117 psi) of CO2(g) in the reactor headspace within 18 min, and the evolved gas produced a maximum work density of 0.65 J ml-1.

Understanding the hydrolysis mechanism of ethyl acetate catalyzed by an aqueous molybdocene: a computational chemistry investigation.

PubMed

Tílvez, Elkin; Cárdenas-Jirón, Gloria I; Menéndez, María I; López, Ramón

2015-02-16

A thoroughly mechanistic investigation on the [Cp2Mo(OH)(OH2)](+)-catalyzed hydrolysis of ethyl acetate has been performed using density functional theory methodology together with continuum and discrete-continuum solvation models. The use of explicit water molecules in the PCM-B3LYP/aug-cc-pVTZ (aug-cc-pVTZ-PP for Mo)//PCM-B3LYP/aug-cc-pVDZ (aug-cc-pVDZ-PP for Mo) computations is crucial to show that the intramolecular hydroxo ligand attack is the preferred mechanism in agreement with experimental suggestions. Besides, the most stable intermediate located along this mechanism is analogous to that experimentally reported for the norbornenyl acetate hydrolysis catalyzed by molybdocenes. The three most relevant steps are the formation and cleavage of the tetrahedral intermediate immediately formed after the hydroxo ligand attack and the acetic acid formation, with the second one being the rate-determining step with a Gibbs energy barrier of 36.7 kcal/mol. Among several functionals checked, B3LYP-D3 and M06 give the best agreement with experiment as the rate-determining Gibbs energy barrier obtained only differs 0.2 and 0.7 kcal/mol, respectively, from that derived from the experimental kinetic constant measured at 296.15 K. In both cases, the acetic acid elimination becomes now the rate-determining step of the overall process as it is 0.4 kcal/mol less stable than the tetrahedral intermediate cleavage. Apart from clarifying the identity of the cyclic intermediate and discarding the tetrahedral intermediate formation as the rate-determining step for the mechanism of the acetyl acetate hydrolysis catalyzed by molybdocenes, the small difference in the Gibbs energy barrier found between the acetic acid formation and the tetrahedral intermediate cleavage also uncovers that the rate-determining step could change when studying the reactivity of carboxylic esters other than ethyl acetate substrate specific toward molybdocenes or other transition metal complexes. Therefore

Theoretical studies of the transition state structures and free energy barriers for base-catalyzed hydrolysis of amides

PubMed Central

Xiong, Ying; Zhan, Chang-Guo

2010-01-01

The transition state structures and free energy barriers for the rate-determining step (i.e. the formation of a tetrahedral intermediate) of base-catalyzed hydrolysis of a series of amides in aqueous solution have been studied by performing first-principle electronic structure calculations using a hybrid supermolecule-polarizable continuum approach. The calculated results and a revisit of recently reported experimental proton inventory data reveal that the favorable transition state structure optimized for the tetrahedral intermediate formation of hydroxide ion-catalyzed hydrolysis of formamide may have three solvating water molecules remaining on the attacking hydroxide oxygen and two additional water molecules attached to the carbonyl oxygen of formamide. The calculated results have also demonstrated interesting substituent effects on the optimized transition state geometries, on the transition-state stabilization, and on the calculated free energy barriers for the base-catalyzed hydrolysis of amides. When some or all of the hydrogen atoms of formamide are replaced by methyl groups, the total number of water molecules hydrogen-bonding with the attacking hydroxide in the transition state decreases from three for formamide to two for N-methylacetamide, N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMA). The larger substituents of the amide hinder the solvent water molecules approaching the attacking hydroxide oxygen in the transition state and, therefore, destabilize the transition state structure and increase the free energy barrier. By using the optimized most favorable transition state structures, the calculated free energy barriers, i.e. 21.6 (or 21.7), 22.7, 23.1, and 26.0 kcal/mol for formamide, N-methylacetamide, DMF, and DMA, respectively, are in good agreement with the available experimental free energy barriers, i.e. 21.2, 21.5, 22.6, and 24.1 kcal/mol for formamide, N-methylacetamide, DMF, and DMA, respectively. PMID:17107116

Difference analysis of the enzymatic hydrolysis performance of acid-catalyzed steam-exploded corn stover before and after washing with water.

PubMed

Zhu, Junjun; Shi, Linli; Zhang, Lingling; Xu, Yong; Yong, Qiang; Ouyang, Jia; Yu, Shiyuan

2016-10-01

The difference in the enzymatic hydrolysis yield of acid-catalyzed steam-exploded corn stover (ASC) before and after washing with water reached approximately 15 % under the same conditions. The reasons for the difference in the yield between ASC and washed ASC (wASC) were determined through the analysis of the composition of ASC prehydrolyzate and sugar concentration of enzymatic hydrolyzate. Salts produced by neutralization (CaSO4, Na2SO4, K2SO4, and (NH4)2SO4), sugars (polysaccharides, oligosaccharides, and monosaccharides), sugar-degradation products (weak acids and furans), and lignin-degradation products (ethyl acetate extracts and nine main lignin-degradation products) were back-added to wASC. Results showed that these products, except furans, exerted negative effect on enzymatic hydrolysis. According to the characteristics of acid-catalyzed steam explosion pretreatment, the five sugar-degradation products' mixture and salts [Na2SO4, (NH4)2SO4] showed minimal negative inhibition effect on enzymatic hydrolysis. By contrast, furans demonstrated a promotion effect. Moreover, soluble sugars, such as 13 g/L xylose (decreased by 6.38 %), 5 g/L cellobiose (5.36 %), 10 g/L glucose (3.67 %), as well as lignin-degradation products, and ethyl acetate extracts (4.87 %), exhibited evident inhibition effect on enzymatic hydrolysis. Therefore, removal of soluble sugars and lignin-degradation products could effectively promote the enzymatic hydrolysis performance.

Kinetic analysis of glucoamylase-catalyzed hydrolysis of starch granules from various botanical sources.

PubMed

Tatsumi, Hirosuke; Katano, Hajime; Ikeda, Tokuji

2007-04-01

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k(0)) were determined to be 23.3+/-4.4, 14.8+/-6.0, 6.2+/-1.8, 7.1+/-4.1, 4.6+/-3.0, and 1.6+/-0.6 s(-1) for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k(0) was largely influenced by the type of starch granules. A comparison of the k(0)-values in relation to the crystalline structure of the starch granules suggested that k(0) increases as the crystalline structure becomes dense.

Ultrasound assisted enzyme catalyzed hydrolysis of waste cooking oil under solvent free condition.

PubMed

Waghmare, Govind V; Rathod, Virendra K

2016-09-01

The present work demonstrates the hydrolysis of waste cooking oil (WCO) under solvent free condition using commercial available immobilized lipase (Novozyme 435) under the influence of ultrasound irradiation. The process parameters were optimized using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound intensity. It has been observed that ultrasound-assisted lipase-catalyzed hydrolysis of WCO would be a promising alternative for conventional methods. A maximum conversion of 75.19% was obtained at mild operating parameters: molar ratio of oil to water (buffer pH 7) 3:1, catalyst loading of 1.25% (w/w), lower ultrasound power 100W (ultrasound intensity - 7356.68Wm(-2)), duty cycle 50% and temperature (50°C) in a relatively short reaction time (2h). The activation energy and thermodynamic study shows that the hydrolysis reaction is more feasible when ultrasound is combined with mechanical agitation as compared with the ultrasound alone and simple conventional stirring technique. Application of ultrasound considerably reduced the reaction time as compared to conventional reaction. The successive use of the catalyst for repetitive cycles under the optimum experimental conditions resulted in a loss of enzymatic activity and also minimized the product conversion. Copyright © 2016. Published by Elsevier B.V.

Photo-catalyzed surface hydrolysis of iridium(iii) ions on semiconductors: a facile method for the preparation of semiconductor/IrOx composite photoanodes toward oxygen evolution reaction.

PubMed

Wu, Qingyong; Xu, Di; Xue, Ning; Liu, Tengyi; Xiang, Min; Diao, Peng

2016-12-21

We previously reported that the hydrolysis of Ir 3+ in homogeneous solution could be triggered by irradiation with light whose energy was larger than a threshold value. In this work, we demonstrated that, by introducing Fe 2 O 3 particles into solution, the incident light energy-restriction for the photo-catalyzed hydrolysis could be broken and the hydrolysis occurred at the Fe 2 O 3 /solution interface. The photo-generated holes on the Fe 2 O 3 surface played a key role in oxidizing Ir(iii) to Ir(iv) species and triggered the deposition of IrO x . We showed that this photo-catalyzed surface hydrolysis is a universal phenomenon that takes place on the surface of many n-type semiconductors such as Fe 2 O 3 , TiO 2 , and Ag 3 PO 4 . As IrO x is an efficient catalyst for oxygen evolution reaction, surface hydrolysis is a general, facile and efficient strategy to prepare semiconductor/IrO x composites, which can be used as anodic materials for photoelectrochemical water splitting.

Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics.

PubMed

Majiduddin, Fahd K; Palzkill, Timothy

2003-03-01

Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring beta-lactamases and extended-spectrum beta-lactamases. Four enzymes from the class A group of beta-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these beta-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of beta-lactam antibiotics.

Amino Acid Sequence Requirements at Residues 69 and 238 for the SME-1 β-Lactamase To Confer Resistance to β-Lactam Antibiotics

PubMed Central

Majiduddin, Fahd K.; Palzkill, Timothy

2003-01-01

Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring β-lactamases and extended-spectrum β-lactamases. Four enzymes from the class A group of β-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these β-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of β-lactam antibiotics. PMID:12604542

FeCl3-catalyzed ethanol pretreatment of sugarcane bagasse boosts sugar yields with low enzyme loadings and short hydrolysis time.

PubMed

Zhang, Hongdan; Zhang, Shuaishuai; Yuan, Hongyou; Lyu, Gaojin; Xie, Jun

2018-02-01

An organosolv pretreatment system consisting of 60% ethanol and 0.025 mol·L -1 FeCl 3 under various temperatures was developed in this study. During the pretreatment, the highest xylose yield was 11.4 g/100 g raw material, representing 49.8% of xylose in sugarcane bagasse. Structural features of raw material and pretreated substrates were characterized to better understand how hemicellulose removal and delignification affected subsequent enzymatic hydrolysis. The 160 °C pretreated solid presented a remarkable glucose yield of 93.8% for 72 h. Furthermore, the influence of different additives on the enzymatic hydrolysis of pretreated solid was investigated. The results indicated that the addition of Tween 80 shortened hydrolysis time to 6 h and allowed a 50% reduction of enzyme loading to achieve the same level of glucose yield. This work suggested that FeCl 3 -catalyzed organosolv pretreatment could improve the enzymatic hydrolysis significantly and reduce the hydrolysis time and enzyme dosage with the addition of Tween 80. Copyright © 2017 Elsevier Ltd. All rights reserved.

Behaviors of glucose decomposition during acid-catalyzed hydrothermal hydrolysis of pretreated Gelidium amansii.

PubMed

Jeong, Tae Su; Choi, Chang Ho; Lee, Ji Ye; Oh, Kyeong Keun

2012-07-01

Acid-catalyzed hydrothermal hydrolysis is one path to cellulosic glucose and subsequently to its dehydration end products such as hydroxymethyl furfural (HMF), formic acid and levulinic acid. The effect of sugar decomposition not only lowers the yield of fermentable sugars but also forms decomposition products that inhibit subsequent fermentation. The present experiments were conducted with four different acid catalysts (H(2)SO(4), HNO(3), HCl, and H(3)PO(4)) at various acid normalities (0.5-2.1N) in batch reactors at 180-210 °C. From the results, H(2)SO(4) was the most suitable catalyst for glucose production, but glucose decomposition occurred during the hydrolysis. The glucose production was maximized at 160.7 °C, 2.0% (w/v) H(2)SO(4), and 40 min, but resulted in a low glucan yield of 33.05% due to the decomposition reactions, which generated formic acid and levulinic acid. The highest concentration of levulinic acid, 7.82 g/L, was obtained at 181.2 °C, 2.0% (w/v) H(2)SO(4), and 40 min. Copyright © 2012 Elsevier Ltd. All rights reserved.

A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis.

PubMed

Song, Xiangfei; Zhang, Shujun; Wang, Yefei; Li, Jingwen; He, Chunyan; Yao, Lishan

2016-06-01

One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15-35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme's capability in hydrolyzing the soluble substrate. Copyright © 2016 Elsevier Inc. All rights reserved.

Stability of cefozopran hydrochloride in aqueous solutions.

PubMed

Zalewski, Przemysław; Skibiński, Robert; Paczkowska, Magdalena; Garbacki, Piotr; Talaczyńska, Alicja; Cielecka-Piontek, Judyta; Jelińska, Anna

2016-01-01

The influence of pH on the stability of cefozopran hydrochloride (CZH) was investigated in the pH range of 0.44-13.00. Six degradation products were identified with a hybrid ESI-Q-TOF mass spectrometer. The degradation of CZH as a result of hydrolysis was a pseudo-first-order reaction. As general acid-base hydrolysis of CZH was not occurred in the solutions of hydrochloric acid, sodium hydroxide, acetate, borate and phosphate buffers, kobs = kpH because specific acid-base catalysis was observed. Specific acid-base catalysis of CZH consisted of the following reactions: hydrolysis of CZH catalyzed by hydrogen ions (kH+), hydrolysis of dications (k1H2O), monocations (k2H2O) and zwitter ions (k3H2O) and hydrolysis of zwitter ions (k1OH-) and monoanions (k2OH-) of CZH catalyzed by hydroxide ions. The total rate of the reaction was equal to the sum of partial reactions: [Formula: see text]. CZH similarly like other fourth generation cephalosporin was most stable at slightly acidic and neutral pH and less stable in alkaline pH. The cleavage of the β-lactam ring resulting from a nucleophilic attack on the carbonyl carbon in the β-lactam moiety is the preferred degradation pathway of β-lactam antibiotics in aqueous solutions.

Novel Aspects on the Preparation of Spirocyclic and Fused Unusual β-Lactams

NASA Astrophysics Data System (ADS)

Alcaide, Benito; Almendros, Pedro

β-Lactam antibiotics have occupied a central role in the fight against pathogenic bacteria and the subsequent rise in quality of life for the world population as a whole. However, the extensive use of common β-lactam antibiotics such as penicillins and cephalosporins in medicine has resulted in an increasing number of resistant strains of bacteria through mutation and β-lactamase gene transfer. The resistance of bacteria to the classical β-lactam antibiotics can be overcome, e.g., by using novel β-lactam moieties in drugs, which show much higher stability towards these resistance bacteria. In addition, there are many important nonantibiotic uses of 2-azetidinones in fields ranging from enzyme inhibition to gene activation. These biological activities, combined with the use of these products as starting materials to prepare α- and β-amino acids, alkaloids, heterocycles, taxoids, and other types of compounds of biological and medicinal interest, provide the motivation to explore new methodologies for the synthesis of substances based on the β-lactam core. The aim of this chapter is to provide a survey of the types of reactions used to prepare nonconventional spirocyclic and fused β-lactams, concentrating on the advances that have been made in the last decade, particularly in the last 5 years. We will draw special attention to radical cyclizations, cycloaddition reactions, and transition metal-catalyzed reactions.

Comprehensive analysis of ß-lactam antibiotics including penicillins, cephalosporins, and carbapenems in poultry muscle using liquid chromatography coupled to tandem mass spectrometry.

PubMed

Berendsen, Bjorn J A; Gerritsen, Henk W; Wegh, Robin S; Lameris, Steven; van Sebille, Ralph; Stolker, Alida A M; Nielen, Michel W F

2013-09-01

A comprehensive method for the quantitative residue analysis of trace levels of 22 ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems, in poultry muscle by liquid chromatography in combination with tandem mass spectrometric detection is reported. The samples analyzed for ß-lactam residues are hydrolyzed using piperidine in order to improve compound stability and to include the total residue content of the cephalosporin ceftifour. The reaction procedure was optimized using a full experimental design. Following detailed isotope labeling, tandem mass spectrometry studies and exact mass measurements using high-resolution mass spectrometry reaction schemes could be proposed for all ß-lactams studied. The main reaction occurring is the hydrolysis of the ß-lactam ring under formation of the piperidine substituted amide. For some ß-lactams, multiple isobaric hydrolysis reaction products are obtained, in accordance with expectations, but this did not hamper quantitative analysis. The final method was fully validated as a quantitative confirmatory residue analysis method according to Commission Decision 2002/657/EC and showed satisfactory quantitative performance for all compounds with trueness between 80 and 110% and within-laboratory reproducibility below 22% at target level, except for biapenem. For biapenem, the method proved to be suitable for qualitative analysis only.

Metal-Catalyzed Oxidation of Protein Methionine Residues in Human Parathyroid Hormone (1-34): Formation of Homocysteine and a Novel Methionine-Dependent Hydrolysis Reaction

PubMed Central

Mozziconacci, Olivier; Ji, Junyan A.; Wang, Y. John; Schöneich, Christian

2013-01-01

The oxidation of PTH(1-34) catalyzed by ferrous ethylenediaminetetraacetic acid (EDTA) is site-specific. The oxidation of PTH(1-34) is localized primarily to the residues Met[8] and His[9]. Beyond the transformation of Met[8] and His[9] into methionine sulfoxide and 2-oxo-histidine, respectively, we observed a hydrolytic cleavage between Met[8] and His[9]. This hydrolysis requires the presence of FeII and oxygen and can be prevented by diethylenetriaminepentaacetic acid (DTPA) and phosphate buffer. Conditions leading to this site-specific hydrolysis also promote the transformation of Met[8] into homocysteine, indicating that the hydrolysis and transformation of homocysteine may proceed through a common intermediate. PMID:23289936

Marine fungi Aspergillus sydowii and Trichoderma sp. catalyze the hydrolysis of benzyl glycidyl ether.

PubMed

Martins, Mariana Provedel; Mouad, Ana Maria; Boschini, Letícia; Regali Seleghim, Mirna Helena; Sette, Lara Durães; Meleiro Porto, André Luiz

2011-04-01

Whole cells of the marine fungi Aspergillus sydowii Gc12, Penicillium raistrickii Ce16, P. miczynskii Gc5, and Trichoderma sp. Gc1, isolated from marine sponges of the South Atlantic Ocean (Brazil), have been screened for the enzymatic resolution of (±)-2-(benzyloxymethyl)oxirane (benzyl glycidyl ether; 1). Whole cells of A. sydowii Gc12 catalyzed the enzymatic hydrolysis of (R,S)-1 to yield (R)-1 with an enantiomeric excess (ee) of 24-46% and 3-(benzyloxy)propane-1,2-diol (2) with ee values <10%. In contrast, whole cells of Trichoderma sp. Gc1 afforded (S)-1 with ee values up to 60% and yields up to 39%, together with (R)-2 in 25% yield and an ee of 32%. This is the first published example of the hydrolysis of 1 by whole cells of marine fungi isolated from the South Atlantic Ocean. The hydrolases from the two studied fungi exhibited complementary regioselectivity in opening the epoxide ring of racemic 1, with those of A. sydowii Gc12 showing an (S) preference and those of Trichoderma sp. Gc1 presenting an (R) preference for the substrate.

Hydrolysis of clavulanate by Mycobacterium tuberculosis β-lactamase BlaC harboring a canonical SDN motif.

PubMed

Soroka, Daria; Li de la Sierra-Gallay, Inès; Dubée, Vincent; Triboulet, Sébastien; van Tilbeurgh, Herman; Compain, Fabrice; Ballell, Lluis; Barros, David; Mainardi, Jean-Luc; Hugonnet, Jean-Emmanuel; Arthur, Michel

2015-09-01

Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase BlaMAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G(132)N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10(4)-fold increase in k cat (0.41 s(-1)), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G(132)N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G(132)N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam-clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine β-lactamases and DD-peptidases.

PubMed

Pelto, Ryan B; Pratt, R F

2012-09-28

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl 5 of may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

Efficient Synthesis of γ-Lactams by a Tandem Reductive Amination/Lactamization Sequence

PubMed Central

Nöth, Julica; Frankowski, Kevin J.; Neuenswander, Benjamin; Aubé, Jeffrey; Reiser, Oliver

2009-01-01

A three-component method for synthesizing highly-substituted γ-lactams from readily available maleimides, aldehydes and amines is described. A new reductive amination/intramolecular lactamization sequence provides a straightforward route to the lactam products in a single manipulation. The general utility of this method is demonstrated by the parallel synthesis of a γ-lactam library. PMID:18338857

Efficient synthesis of gamma-lactams by a tandem reductive amination/lactamization sequence.

PubMed

Nöth, Julica; Frankowski, Kevin J; Neuenswander, Benjamin; Aubé, Jeffrey; Reiser, Oliver

2008-01-01

A three-component method for the synthesis of highly substituted gamma-lactams from readily available maleimides, aldehydes, and amines is described. A new reductive amination/intramolecular lactamization sequence provides a straightforward route to the lactam products in a single manipulation. The general utility of this method is demonstrated by the parallel synthesis of a gamma-lactam library.

Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides.

PubMed

Masson, Patrick; Froment, Marie-Thérèse; Gillon, Emilie; Nachon, Florian; Darvesh, Sultan; Schopfer, Lawrence M

2007-09-01

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes

Comparative analyses of laccase-catalyzed amination reactions for production of novel β-lactam antibiotics.

PubMed

Mikolasch, Annett; Manda, Katrin; Schlüter, Rabea; Lalk, Michael; Witt, Sabine; Seefeldt, Simone; Hammer, Elke; Schauer, Frieder; Jülich, Wolf-Dieter; Lindequist, Ulrike

2012-01-01

Seven novel β-lactam antibiotics with activities against Gram-positive bacterial strains, among them methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, were synthesized by amination of 2,5-dihydroxyphenylacetic acid in usable yields (30-60%). These products protected mice against an infection with S. aureus lethal to the control animals. The results show the usefulness of laccase for the synthesis of potential new antibiotics, in addition to the interdependence of the laccase substrates, the amino coupling partners, and the product formation, yield, and activity. The syntheses of β-lactam antibiotics with 2,5-dihydroxyaromatic acid substructures (para-substituted) are then compared with those of 3,4-dihydroxyaromatic acid substructures (ortho-substituted). Para-substituted laccase substrates were better reaction partners in these syntheses than ortho-substituted compounds. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Inactivation of beta-lactam antibiotics by Legionella pneumophila.

PubMed Central

Fu, K P; Neu, H C

1979-01-01

Beta-lactam-inactivating activity has been found in all sero-groups of Legionella pneumophila. The beta-lactamase activity could be detected in intact cells and released by ethylenediaminetetraacetic acid treatment, indicating that it is located in the periplasmic space. The enzyme acted primarily as a cephalosporinase hydrolyzing cefamandole, cephalothin, cephaloridine, and also penicillin G and ampicillin. Cefoxitin and cefuroxime were not hydrolyzed. Clavulanic acid and CP-45,899, beta-lactamase inhibitors, prevented the hydrolysis of cephalosporins and penicillins. The beta-lactamase activity appears to be different from that found in Enterobacteriaceae and Pseudomonas. Images PMID:316686

Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics.

PubMed

Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I; Zhou, Xiaohui

2016-02-09

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics

PubMed Central

Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I.; Zhou, Xiaohui

2016-01-01

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics. PMID:26831117

Platinum-catalyzed hydrolysis etching of SiC in water: A density functional theory study

NASA Astrophysics Data System (ADS)

Van Bui, Pho; Toh, Daisetsu; Isohashi, Ai; Matsuyama, Satoshi; Inagaki, Kouji; Sano, Yasuhisa; Yamauchi, Kazuto; Morikawa, Yoshitada

2018-05-01

A comprehensive study of the physicochemical interactions and the reaction mechanism of SiC etching with water by Pt catalysts can reveal key details about the surface treatment and catalytic phenomena at interfaces. Therefore, density functional theory simulations were performed to study the kinetics of Pt-assisted water dissociation and breaking of a Si–C bond compared to the HF-assisted mechanism. These calculations carefully considered the elastic and chemical interaction energies at the Pt–SiC interface, activation barriers of Si–C bond dissociation, and the catalytic role of Pt. It was found that the Pt-catalyzed etching of SiC in water is initiated via hydrolysis reactions that break the topmost Si–C bonds. The activation barrier strongly depends on the elastic and chemical interactions. However, chemical interactions are a dominant factor and mainly contribute to the lowering of the activation barrier, resulting in an increased rate of reaction.

Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of “hunger hormone” ghrelin

NASA Astrophysics Data System (ADS)

Yao, Jianzhuang; Yuan, Yaxia; Zheng, Fang; Zhan, Chang-Guo

2016-02-01

Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway.

Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif

PubMed Central

Soroka, Daria; Li de la Sierra-Gallay, Inès; Dubée, Vincent; Triboulet, Sébastien; van Tilbeurgh, Herman; Compain, Fabrice; Ballell, Lluis; Barros, David; Mainardi, Jean-Luc

2015-01-01

Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase BlaMAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G132N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 104-fold increase in kcat (0.41 s−1), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G132N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G132N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies. PMID:26149997

Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI.

PubMed

Zhang, Shujun; Ma, Guangcai; Liu, Yongjun; Ling, Baoping

2015-09-01

2-Pyrone-4,6-dicarboxylate lactonase (LigI) is the first identified enzyme from amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. It catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. In this paper, a combined quantum mechanics and molecule mechanics (QM/MM) approach was employed to study the reaction mechanism of LigI from Sphingomonas paucimobilis. According to the results of our calculations, the whole catalytic reaction contains three elementary steps, including the nucleophilic attack, the cleavage of CO of lactone (substrate) and the intramolecular proton transfer. The intermediate has two intramolecular proton transfer pathways, due to which, two final hydrolysis products can be obtained. The energy profile indicates that 4-carboxy-2-hydroxymuconate (CHM) is the main hydrolysis product, therefore, the isomerization between 4-carboxy-2-hydroxymuconate (CHM) and 4-oxalomesaconate (OMA) is suggested to occur in solvent. During the catalytic reaction, residue Asp248 acts as a general base to activate the hydrolytic water molecule. Although His31, His33 and His180 do not directly participate in the chemical process, they play assistant roles by forming electrostatic interactions with the substrate and its involved species in activating the carbonyl group of the substrate and stabilizing the intermediates and transition states. Copyright © 2015 Elsevier Inc. All rights reserved.

The hydrolysis kinetics of monobasic and dibasic aminoalkyl esters of ketorolac.

PubMed

Qandil, Amjad M; Jamhawi, Noor M; Tashtoush, Bassam M; Al-Ajlouni, Ahmad M; Idkaidek, Nasir M; Obaidat, Aiman A

2013-09-01

Six aminoethyl and aminobutyl esters of ketorolac containing 1-methylpiperazine (MPE and MPB), N-acetylpiperazine (APE and APB) or morpholine (ME and MB), were synthesized and their hydrolysis kinetics were studied. The hydrolysis was studied at pH 1 to 9 (for MPE, APE and ME) and pH 1 to 8 (for MPB, APB and MB) in aqueous phosphate buffer (0.16 M) with ionic strength (0.5 M) at 37°C. Calculation of k(obs), construction of the pH-rate profiles and determination of the rate equations were performed using KaleidaGraph® 4.1. The hydrolysis displays pseudo-first order kinetics and the pH-rate profiles shows that the aminobutyl esters, MPE, APB and MB, are the most stable. The hydrolysis of the ethyl esters MPE, APE and ME, depending on the pH, is either fast and catalyzed by the hydroxide anion or slow and uncatalyzed for the diprotonated, monoprotonated and nonprotonated forms. The hydrolysis of the butyl esters showed a similar profile, albeit it was also catalyzed by hydronium cation. In addition, the hydroxide anion is 105 more effective in catalyzing the hydrolysis than the hydronium cation. The hydrolysis pattern of the aminoethyl esters is affected by the number and pKa of its basic nitrogen atoms. The monobasic APE and ME, show a similar hydrolysis pattern that is different than the dibasic MPE. The length of the side chain and the pKa of the basic nitrogen atoms in the aminoethyl moiety affect the mechanism of hydrolysis as the extent of protonation at a given pH is directly related to the pKa.

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

PubMed Central

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao

2017-01-01

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one).

PubMed

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao; Zheng, Guojun

2018-01-01

To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (-)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a �

β-Lactam Antibiotics Renaissance

PubMed Central

Qin, Wenling; Panunzio, Mauro; Biondi, Stefano

2014-01-01

Since the 1940s β-lactam antibiotics have been used to treat bacterial infections. However, emergence and dissemination of β-lactam resistance has reached the point where many marketed β-lactams no longer are clinically effective. The increasing prevalence of multidrug-resistant bacteria and the progressive withdrawal of pharmaceutical companies from antibiotic research have evoked a strong reaction from health authorities, who have implemented initiatives to encourage the discovery of new antibacterials. Despite this gloomy scenario, several novel β-lactam antibiotics and β-lactamase inhibitors have recently progressed into clinical trials, and many more such compounds are being investigated. Here we seek to provide highlights of recent developments relating to the discovery of novel β-lactam antibiotics and β-lactamase inhibitors. PMID:27025744

Regio- and stereoselective synthesis of benzothiazolo-pyrimidinones via an NHC-catalyzed Mannich/lactamization domino reaction† †Electronic supplementary information (ESI) available. CCDC 1029497. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4cc08594a

PubMed Central

Ni, Qijian; Song, Xiaoxiao; Xiong, Jiawen; Raabe, Gerhard

2015-01-01

An NHC-catalyzed regio- and stereoselective Mannich/lactamization domino reaction of N-(benzothiazolyl)imines with α-chloroaldehydes has been developed. This new protocol provides a facile approach for the asymmetric synthesis of benzothiazolo-pyrimidinones and a pyrrolo[1,2-a]indolone in moderate to good yields (34–78%) and excellent stereoselectivities (87–99% ee, up to >20 : 1 d.r.). PMID:25476422

Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition

NASA Astrophysics Data System (ADS)

Brem, Jürgen; van Berkel, Sander S.; Aik, Weishen; Rydzik, Anna M.; Avison, Matthew B.; Pettinati, Ilaria; Umland, Klaus-Daniel; Kawamura, Akane; Spencer, James; Claridge, Timothy D. W.; McDonough, Michael A.; Schofield, Christopher J.

2014-12-01

The use of β-lactam antibiotics is compromised by resistance, which is provided by β-lactamases belonging to both metallo (MBL)- and serine (SBL)-β-lactamase subfamilies. The rhodanines are one of very few compound classes that inhibit penicillin-binding proteins (PBPs), SBLs and, as recently reported, MBLs. Here, we describe crystallographic analyses of the mechanism of inhibition of the clinically relevant VIM-2 MBL by a rhodanine, which reveal that the rhodanine ring undergoes hydrolysis to give a thioenolate. The thioenolate is found to bind via di-zinc chelation, mimicking the binding of intermediates in β-lactam hydrolysis. Crystallization of VIM-2 in the presence of the intact rhodanine led to observation of a ternary complex of MBL, a thioenolate fragment and rhodanine. The crystallographic observations are supported by kinetic and biophysical studies, including 19F NMR analyses, which reveal the rhodanine-derived thioenolate to be a potent broad-spectrum MBL inhibitor and a lead structure for the development of new types of clinically useful MBL inhibitors.

The road to avibactam: the first clinically useful non-β-lactam working somewhat like a β-lactam.

PubMed

Wang, David Yuxin; Abboud, Martine I; Markoulides, Marios S; Brem, Jürgen; Schofield, Christopher J

2016-06-01

Avibactam, which is the first non-β-lactam β-lactamase inhibitor to be introduced for clinical use, is a broad-spectrum serine β-lactamase inhibitor with activity against class A, class C, and, some, class D β-lactamases. We provide an overview of efforts, which extend to the period soon after the discovery of the penicillins, to develop clinically useful non-β-lactam compounds as antibacterials, and, subsequently, penicillin-binding protein and β-lactamase inhibitors. Like the β-lactam inhibitors, avibactam works via a mechanism involving covalent modification of a catalytically important nucleophilic serine residue. However, unlike the β-lactam inhibitors, avibactam reacts reversibly with its β-lactamase targets. We discuss chemical factors that may account for the apparently special nature of β-lactams and related compounds as antibacterials and β-lactamase inhibitors, including with respect to resistance. Avenues for future research including non-β-lactam antibacterials acting similarly to β-lactams are discussed.

ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. II. ACID AND GENERAL BASE CATALYZED HYDROLYSIS

EPA Science Inventory

SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to calculate acid and neutral hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition states of a ...

Aquivion Perfluorosulfonic Superacid as an Efficient Pickering Interfacial Catalyst for the Hydrolysis of Triglycerides.

PubMed

Shi, Hui; Fan, Zhaoyu; Hong, Bing; Pera-Titus, Marc

2017-09-11

Rational design of the surface properties of heterogeneous catalysts can boost the interfacial activity in biphasic reactions through the generation of Pickering emulsions. This concept, termed Pickering interfacial catalysis (PIC), has shown promising credentials in acid-catalyzed transesterification, ester hydrolysis, acetalization, etherification, and alkylation reactions. PIC has now been applied to the efficient, solvent-free hydrolysis of the triglyceride glyceryl trilaurate to lauric acid, catalyzed by Aquivion perfluorosulfonic superacid at mild conditions (100 °C and ambient pressure). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

USDA-ARS?s Scientific Manuscript database

Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabi...

Site- and species-specific hydrolysis rates of heroin.

PubMed

Szöcs, Levente; Orgován, Gábor; Tóth, Gergő; Kraszni, Márta; Gergó, Lajos; Hosztafi, Sándor; Noszál, Béla

2016-06-30

The hydroxide-catalyzed non-enzymatic, simultaneous and consecutive hydrolyses of diacetylmorphine (DAM, heroin) are quantified in terms of 10 site- and species-specific rate constants in connection with also 10 site- and species-specific acid-base equilibrium constants, comprising all the 12 coexisting species in solution. This characterization involves the major and minor decomposition pathways via 6-acetylmorphine and 3-acetylmorphine, respectively, and morphine, the final product. Hydrolysis has been found to be 18-120 times faster at site 3 than at site 6, depending on the status of the amino group and the rest of the molecule. Nitrogen protonation accelerates the hydrolysis 5-6 times at site 3 and slightly less at site 6. Hydrolysis rate constants are interpreted in terms of intramolecular inductive effects and the concomitant local electron densities. Hydrolysis fraction, a new physico-chemical parameter is introduced and determined to quantify the contribution of the individual microspecies to the overall hydrolysis. Hydrolysis fractions are depicted as a function of pH. Copyright © 2016 Elsevier B.V. All rights reserved.

Organosolv pretreatment for enzymatic hydrolysis of poplars: I. enzyme hydrolysis of cellulosic residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chum, H.L.; Johnson, D.K.; Black, S.

1988-01-01

Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H/sub 2/SO/sub 4/ and H/sub 3/PO/sub 4/) such that the cooking liquor pH less than or equal to 3 are easily digested by enzymes. The total yields of hydrolysis residues (pulps) are in the 40-60% range; the acid-catalyzed delignification followed by enzyme hydrolysis can generate 70-88% of the original six-carbon sugars contained in the wood. Glucomannan and arabinogalactan are dissolved in to the pulping liquor in the pH range of 2-4.5. Lowermore » pH (less than or equal to 3) leads to additional solubilization of six-carbon sugars. These sugars may be fermented directly. From the insoluble hydrolysis residues, 36-41% conversions of wood into fermentable sugars were obtained after enzyme hydrolysis; the starting feedstocks contain 50.8 and 46.6% hexosans, respectively, for aspen and black cottonwood. The kinetics of enzymatic hydrolysis of cellulose can be formally treated as two simultaneous pseudo-first-order reactions in which fast and slow hydrolysis of cellulose occur. Correlations between the glucan digestibility and the effect of the pretreatment have been made. The higher residual xylan content reduces the amount of the rapidly hydrolyzable glucan fraction and lowers the glucan digestibility. The proposed simple kinetic treatment is very helpful in assessing the effect of the pretreatment on pulp enzyme hydrolyzability.« less

The Hydrolysis of Carbonyl Sulfide at Low Temperature: A Review

PubMed Central

Zhao, Shunzheng; Yi, Honghong; Tang, Xiaolong; Jiang, Shanxue; Gao, Fengyu; Zhang, Bowen; Zuo, Yanran; Wang, Zhixiang

2013-01-01

Catalytic hydrolysis technology of carbonyl sulfide (COS) at low temperature was reviewed, including the development of catalysts, reaction kinetics, and reaction mechanism of COS hydrolysis. It was indicated that the catalysts are mainly involved metal oxide and activated carbon. The active ingredients which can load on COS hydrolysis catalyst include alkali metal, alkaline earth metal, transition metal oxides, rare earth metal oxides, mixed metal oxides, and nanometal oxides. The catalytic hydrolysis of COS is a first-order reaction with respect to carbonyl sulfide, while the reaction order of water changes as the reaction conditions change. The controlling steps are also different because the reaction conditions such as concentration of carbonyl sulfide, reaction temperature, water-air ratio, and reaction atmosphere are different. The hydrolysis of carbonyl sulfide is base-catalyzed reaction, and the force of the base site has an important effect on the hydrolysis of carbonyl sulfide. PMID:23956697

Desensitization in patients with beta-lactam drug allergy.

PubMed

Yusin, J S; Klaustermeyer, W; Simmons, C W; Baum, M

2013-01-01

Patients with a history of beta-lactam antibiotic allergy are often admitted to the hospital with severe or life-threatening infections requiring beta-lactam antibiotics. Strict avoidance of beta lactams to such patients may prevent them from getting adequate coverage and can lead to an increase in the use of alternative antibiotics, which can predispose to antibiotic resistance. Past studies revealed a lower incidence of pen allergy then patients' histories suggest. Fortunately today, there are three options for patients presenting with a history of beta-lactam allergy. Penicillin skin testing, beta-lactam challenge or beta-lactam desensitization. Recently Pre Pen has been FDA re-approved and when combined with Pen G is a valid way to determine if patients are able to tolerate beta-lactam antibiotic. When these agents are not available one must decide about desensitization or challenge. When a patient has a positive penicillin skin test, desensitization or beta-lactam avoidance are the only options. This paper reviews the safety of beta-lactam desensitization. To perform a chart review on patients desensitised with beta lactam to determine if desensitizations can be performed safely without minimal complications. A retrospective chart review was performed on allergy and immunology inpatient consultations for beta-lactam desensitization between September 2003 and August 2006 at the Cedars-Sinai Medical Centre in Los Angeles. Patient data and outcomes of desensitization were analysed. A total of 13 intravenous desensitizations were performed on 12 patients. The patients consisted of eight females and four males with an average age of 65 years. Age range was 36-92 years old. All 13 intravenous desensitizations were completed without complications. No patient required a slower rate of desensitization or discontinuance of the desensitization. Patients were able to tolerate the initial therapeutic dose of their beta-lactam antibiotic and were then able to complete full

β-Lactam antibiotics. Spectroscopy and molecular orbital (MO) calculations . Part I: IR studies of complexation in penicillin-transition metal ion systems and semi-empirical PM3 calculations on simple model compounds

NASA Astrophysics Data System (ADS)

Kupka, Teobald

1997-12-01

IR studies were preformed to determine possible transition metal ion binding sites of penicillin. the observed changes in spectral position and shape of characteristic IR bands of cloxacillin in the presence of transition metal ions (both in solutions and in the solid state) indicate formation of M-L complexes with engagement of -COO - and/or -CONH- functional groups. The small shift of νCO towards higher frequencies rules out direct M-L interaction via β-lactam carbonyl. PM3 calculations on simple model compounds (substituted formamide, cyclic ketones, lactams and substituted monocyclic β-lactams) have been performed. All structures were fully optimized and the calculated bond lengths, angles, heats of formation and CO stretching frequencies were discussed to determine the β-lactam binding sites and to explain its susceptibility towards nucleophilic attack (hydrolysis in vitro) and biological activity. The relative changes of calculated values were critically compared with available experimental data and same correlation between structural parameters and in vivo activity was shown.

Relationships Between Base-Catalyzed Hydrolysis Rates or Glutathione Reactivity for Acrylates and Methacrylates and Their NMR Spectra or Heat of Formation

PubMed Central

Fujisawa, Seiichiro; Kadoma, Yoshinori

2012-01-01

The NMR chemical shift, i.e., the π-electron density of the double bond, of acrylates and methacrylates is related to the reactivity of their monomers. We investigated quantitative structure-property relationships (QSPRs) between the base-catalyzed hydrolysis rate constants (k1) or the rate constant with glutathione (GSH) (log kGSH) for acrylates and methacrylates and the 13C NMR chemical shifts of their α,β-unsaturated carbonyl groups (δCα and δCβ) or heat of formation (Hf) calculated by the semi-empirical MO method. Reported data for the independent variables were employed. A significant linear relationship between k1 and δCβ, but not δCα, was obtained for methacrylates (r2 = 0.93), but not for acrylates. Also, a significant relationship between k1 and Hf was obtained for both acrylates and methacrylates (r2 = 0.89). By contrast, log kGSH for acrylates and methacrylates was linearly related to their δCβ (r2 = 0.99), but not to Hf. These findings indicate that the 13C NMR chemical shifts and calculated Hf values for acrylates and methacrylates could be valuable for estimating the hydrolysis rate constants and GSH reactivity of these compounds. Also, these data for monomers may be an important tool for examining mechanisms of reactivity. PMID:22754331

Modeling and optimization of phospholipase A₁-catalyzed hydrolysis of phosphatidylcholine using response surface methodology for lysophosphatidylcholine production.

PubMed

Lim, Chang Wan; Kim, Byung Hee; Kim, In-Hwan; Lee, Moon-Won

2015-01-01

Modeling the phospholipase A1 (PLA1 )-catalyzed partial hydrolysis of soy phosphatidylcholine (PC) in hexane for the production of lysophosphatidylcholine (LPC) and optimizing the reaction conditions using response surface methodology were described. The reaction was performed with 4 g of PC in a stirred batch reactor using a commercial PLA1 (Lecitase Ultra) as the biocatalyst. The effects of temperature, reaction time, water content, and enzyme loading on LPC and glycerylphosphorylcholine (GPC) content in the reaction products were elucidated using the models established. Optimal reaction conditions for maximizing the LPC content while suppressing acyl migration, which causes GPC formation, were as follows: temperature, 60°C; reaction time, 3 h; water content, 10% of PC; and enzyme loading, 1% of PC. When the reaction was conducted with 40 g of PC under these conditions, the reaction products contained 83.7 mol % LPC and were free of GPC. LPC had a higher total unsaturated fatty acid content than original PC had and was mainly composed of linoleic acid (78.0 mol % of the total fatty acids). © 2014 American Institute of Chemical Engineers.

Chromophoric spin-labeled β-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C β-lactamases

NASA Astrophysics Data System (ADS)

Mustafi, Devkumar; Hofer, Jennifer E.; Huang, Wanzhi; Palzkill, Timothy; Makinen, Marvin W.

2004-05-01

The chromophoric spin-label substrate 6- N-[3-(2,2,5,5-tetramethyl-1-oxypyrrolin-3-yl)-propen-2-oyl]penicillanic acid (SLPPEN) was synthesized by acylation of 6-aminopenicillanic acid with the acid chloride of 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propenoic acid and characterized by physical methods. By application of angle-selected electron nuclear double resonance (ENDOR), we have determined the molecular structure of SLPPEN in solution. SLPPEN exhibited UV absorption properties that allowed accurate monitoring of the kinetics of its enzyme-catalyzed hydrolysis. The maximum value of the (substrate-product) difference extinction coefficient was 2824 M -1 cm -1 at 275 nm compared to 670 M -1 cm -1 at 232 nm for SLPEN [J. Am. Chem. Soc. 117 (1995) 6739]. For SLPPEN, the steady-state kinetic parameters kcat and kcat/ KM, determined under initial velocity conditions, were 637±36 s -1 and 13.8±1.4×10 6 M -1 s -1, respectively, for hydrolysis catalyzed by TEM-1 β-lactamase of E. coli, and 0.5±0.04 s -1 and 3.9±0.4×10 4 M -1 s -1 for hydrolysis catalyzed by the β-lactamase of Enterobacter cloacae P99. We have also observed "burst kinetics" for the hydrolysis of SLPPEN with P99 β-lactamase, indicative of formation of an acylenzyme reaction intermediate. In DMSO:H 2O (30:70, v:v) cryosolvent mixtures buffered to pH ∗ 7.0, the half-life of the acylenzyme intermediate formed with the P99 enzyme at -5 °C was ≥3 min, suitable for optical characterization. The observation of burst kinetics in the hydrolysis of SLPPEN catalyzed by P99 β-lactamase suggests that this chromophoric spin-labeled substrate is differentially sensitive to active site interactions underlying the cephalosporinase and penicillinase reactivity of this class C enzyme.

Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

PubMed Central

Diaz-Meco, M T; Dominguez, I; Sanz, L; Municio, M M; Berra, E; Cornet, M E; Garcia de Herreros, A; Johansen, T; Moscat, J

1992-01-01

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1. Images PMID:1309592

Part I: RNA hydrolysis catalyzed by imidazole compounds. Part II. Hydrophobic acceleration of reactions and mimics of thiamin-dependent enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kool, E.T.

1988-01-01

Catalysts modeled after the active site groups of the enzyme Ribonuclease A were synthesized and tested for catalysis of the hydrolysis of poly(rU), using a quantitative assay. The most effective of all the catalysts is N,N{prime}-bis-imidazolylmethane, which gave a four-fold rate enhancement as compared to N-methyl-imidazole. The structure/activity relationships are discussed in light of the ribonuclease mechanism. Also examined were reactions catalyzed by the coenzyme thiamine. In an investigation of the effects of restricting conformational freedom, a thiazolium salt was attached in two positions to {beta}-cyclodextrin. Since the catalyst gave about the same rate for tritium exchange from benzaldehyde asmore » singly-attached catalysts, we surmise that any rate enhancement due to the restriction of bond rotations has been lost by forcing the structure into less productive conformations. The benzoin condensation catalyzed by cyanide was also investigated. The reaction was shown to be faster in water than in most organic solvents. Kinetic salt effects and the effects of added {beta}- and {gamma}-cyclodextrin were measured in water; salting-out ions and {gamma}-cyclodextrin increase the rate, while salting-in ions and {beta}-cyclodextrin decrease it. Negative salt effects were observed in formamide, ethylene glycol, and DMSO. All these media effects are discussed in relation to the compact, hydrophobic transition state for the reaction.« less

Salt effects on an ion-molecule reaction--hydroxide-catalyzed hydrolysis of benzocaine.

PubMed

Al-Maaieh, Ahmad; Flanagan, Douglas R

2006-03-01

This work investigates the effect of various salts on the rate of a reaction involving a neutral species (benzocaine alkaline hydrolysis). Benzocaine hydrolysis kinetics in NaOH solutions in the presence of different salts were studied at 25 degrees C. Benzocaine solubility in salt solutions was also determined. Solubility data were used to estimate salt effects on benzocaine activity coefficients, and pH was used to estimate salt effects on hydroxide activity coefficients. Salts either increased or decreased benzocaine solubility. For example, solubility increased with 1.0 M tetraethylammonium chloride (TEAC) approximately 3-fold, whereas solubility decreased approximately 35% with 0.33 M Na2SO4. Salt effects on hydrolysis rates were more complex and depended on the relative magnitudes of the salt effects on the activity coefficients of benzocaine, hydroxide ion, and the transition state. As a result, some salts increased the hydrolysis rate constant, whereas others decreased it. For example, the pseudo-first-order rate constant decreased approximately 45% (to 0.0584 h(-1)) with 1 M TEAC, whereas it increased approximately 8% (to 0.116 h(-1)) with 0.33 M Na2SO4. Different salt effects on degradation kinetics can be demonstrated for a neutral compound reacting with an ion. These salt effects depend on varying effects on activity coefficients of reacting and intermediate species.

Inhibitory effect of soy saponins on the activity of β-lactamases, including New Delhi metallo-β-lactamase 1.

PubMed

Horie, Hitoshi; Chiba, Asuka; Wada, Shigeo

2018-05-01

β-Lactamase-producing bacteria encode enzymes that inactivate β-lactam antibiotics by catalyzing the hydrolysis of the β-lactam ring. Crude soy saponins were observed to have synergistic effects on the antimicrobial activity of β-lactam antibiotics against β-lactamase-producing Staphylococcus aureus strains. Furthermore, the activities of β-lactamases derived from Enterobacter cloacae , Escherichia coli , and S. aureus were decreased significantly in the presence of crude soy saponins. This inhibitory effect was also observed against the New Delhi metallo-β-lactamase 1 (NDM-1), an enzyme whose activity is not inhibited by the current β-lactamase inhibitors. The synergistic effect on the antimicrobial activity of β-lactam antibiotics by crude soy saponins was thought to result from the inhibition the β-lactamase activity. The components of crude soy saponins include several kinds of soyasaponins and soyasapogenols. It was revealed that soyasaponin V has the highest inhibitory activity against NDM-1. The combined use of soy saponins with β-lactam antibiotics is expected to serve as a new therapeutic modality, potentially enhancing the effectiveness of β-lactam antibiotics against infectious diseases caused by β-lactamase-producing bacteria, including those encoding NDM-1.

Beta-lactams against methicillin-resistant Staphylococcus aureus.

PubMed

Guignard, Bertrand; Entenza, José M; Moreillon, Philippe

2005-10-01

Methicillin-resistant Staphylococcus aureus (MRSA) have developed resistance to virtually all non-experimental antibiotics. They are intrinsically resistant to beta-lactams by virtue of newly acquired low-affinity penicillin-binding protein 2A (PBP2A). Because PBP2A can build the wall when other PBPs are blocked by beta-lactams, designing beta-lactams capable of blocking this additional target should help solve the issue. Older molecules including penicillin G, amoxicillin and ampicillin had relatively good PBP2A affinities, and successfully treated experimental endocarditis caused by MRSA, provided that the bacterial penicillinase could be inhibited. Newer anti-PBP2A beta-lactams with over 10-fold greater PBP2A affinities and low minimal inhibitory concentrations were developed, primarily in the cephem and carbapenem classes. They are also very resistant to penicillinase. Most have demonstrated anti-MRSA activity in animal models of infection, and two--the carbapenem CS-023 and the cephalosporin ceftopibrole medocaril--have proceeded to Phase II and Phase III clinical evaluation. Thus, clinically useful anti-MRSA beta-lactams are imminent.

Spirocyclic β-Lactams: Synthesis and Biological Evaluation of Novel Heterocycles

NASA Astrophysics Data System (ADS)

Bari, Shamsher S.; Bhalla, Aman

β-Lactam rings containing compounds are a group of antibiotics of unparalleled importance in chemotherapy. Considerable effort has been reported in the development of novel, more effective β-lactam compounds as well as their biological evaluation. This article reviews the progress made in the stereoselective synthesis of spiro-β-lactams, a unique class of heterocycles, their biological evaluation, and their applications in various related fields. The introductory paragraph highlights the significance of the β-lactam chemistry and is followed by an overview of monocyclic-, bicyclic-, and tricyclic-β-lactams. The other sections of the article deal with the stereoselective synthesis and biological evaluation of spiro-β-lactams, including their use as synthetic intermediates for β-turn mimics and β-turn nucleators. The potential of spiro-β-lactams as cholesterol absorption inhibitors, β-lactamase inhibitors, and antiviral, antibacterial, and antimicrobial agents has also been described.

Gas phase hydrolysis of formaldehyde to form methanediol: impact of formic acid catalysis.

PubMed

Hazra, Montu K; Francisco, Joseph S; Sinha, Amitabha

2013-11-21

We find that formic acid (FA) is very effective at facilitating diol formation through its ability to reduce the barrier for the formaldehyde (HCHO) hydrolysis reaction. The rate limiting step in the mechanism involves the isomerization of a prereactive collision complex formed through either the HCHO···H2O + FA and/or HCHO + FA···H2O pathways. The present study finds that the effective barrier height, defined as the difference between the zero-point vibrational energy (ZPE) corrected energy of the transition state (TS) and the HCHO···H2O + FA and HCHO + FA···H2O starting reagents, are respectively only ∼1 and ∼4 kcal/mol. These barriers are substantially lower than the ∼17 kcal/mol barrier associated with the corresponding step in the hydrolysis of HCHO catalyzed by a single water molecule (HCHO + H2O + H2O). The significantly lower barrier heights for the formic acid catalyzed pathway reveal a new important role that organic acids play in the gas phase hydrolysis of atmospheric carbonyl compounds.

Additives enhancing enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Rocha-Martín, Javier; Martinez-Bernal, Claudio; Pérez-Cobas, Yolanda; Reyes-Sosa, Francisco Manuel; García, Bruno Díez

2017-11-01

Linked to the development of cellulolytic enzyme cocktails from Myceliophthora thermophila, we studied the effect of different additives on the enzymatic hydrolysis yield. The hydrolysis of pretreated corn stover (PCS), sugar cane straw (PSCS) and microcrystalline cellulose (Avicel) was performed under industrial conditions using high solid loadings, limited mixing, and low enzyme dosages. The addition of polyethylene glycol (PEG4000) allowed to increase the glucose yields by 10%, 7.5%, and 32%, respectively in the three materials. PEG4000 did not have significant effect on the stability of the main individual enzymes but increased beta-glucosidase and endoglucanase activity by 20% and 60% respectively. Moreover, the presence of PEG4000 accelerated cellulase-catalyzed hydrolysis reducing up to 25% the liquefaction time. However, a preliminary economical assessment concludes that even with these improvements, a lower contribution of PEG4000 to the 2G bioethanol production costs would be needed to reach commercial feasibility. Copyright © 2017. Published by Elsevier Ltd.

Low frequency ultrasonic-assisted hydrolysis of starch in the presence of α-amylase.

PubMed

Gaquere-Parker, Anne; Taylor, Tamera; Hutson, Raihannah; Rizzo, Ashley; Folds, Aubrey; Crittenden, Shastina; Zahoor, Neelam; Hussein, Bilal; Arruda, Aaron

2018-03-01

Hydrolysis of starch is an important process in the food industry and in the production of bioethanol or smaller carbohydrate molecules that can be used as starting blocks for chemical synthesis. Such hydrolysis can be enhanced by lowering the pH, heating the reaction mixture or catalyzing the reaction with enzymes. This study reports the effect of sonication on the reaction rate of starch hydrolysis at different temperatures, in the presence or absence of alpha-amylase. Starch Azure, a commercially available potato starch covalently linked with Remazol Brilliant Blue, has been chosen since its hydrolysis releases a blue dye, which concentration can be monitored by UV Vis spectroscopy. Ultrasounds, regardless of experimental conditions, provide the highest reaction rate for such hydrolysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Metal-Catalyzed Cleavage of tRNA[superscript Phe

ERIC Educational Resources Information Center

Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.

2008-01-01

This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which students assay metal-catalyzed hydrolysis of tRNA[superscript Phe]…

DFT STUDY OF THE HYDROLYSIS OF SOME S-TRIAZINES

EPA Science Inventory

The acid-catalyzed hydrolysis of atrazine and related 2-chloro-s-triazines to the corresponding 2-hydroxy-s-triazines was investigated using the B3LYP hybrid density functional theory method. Gas-phase calculations were performed at the B3LYP/6-311++G(d,p)//B3LYP/6-31G* level of ...

Recent Approaches Toward Solid Phase Synthesis of β-Lactams

NASA Astrophysics Data System (ADS)

Mandal, Bablee; Ghosh, Pranab; Basu, Basudeb

Since the discovery of penicillin in 1929, β-lactam antibiotics have been recognized as potentially chemotherapeutic drugs of incomparable effectiveness, conjugating a broad spectrum of activity with very low toxicity. The primary motif azetidin-2-one ring (β-lactam) has been considered as specific pharmacophores and scaffolds. With the advent of combinatorial chemistry and automated parallel synthesis coupled with ample interests from the pharmaceutical industries, recent trends have been driven mostly by adopting solid phase techniques and polymer-supported synthesis of β-lactams. The present survey will present an overview of the developments on the polymer-supported and solid phase techniques for the preparation of β-lactam ring or β-lactam containing antibiotics published over the last decade. Both unsubstituted and substitutions with different functional groups at various positions of β-lactams have been synthesized using solid phase technology. However, Wang resin and application of Staudinger [2+2] cycloaddition reaction have remained hitherto the major choice. It may be expected that other solid phase approaches involving different resins would be developed in the coming years.

[3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

NASA Technical Reports Server (NTRS)

Hall, P. J.; Bandurski, R. S.

1986-01-01

[3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

A general catalytic β-C-H carbonylation of aliphatic amines to β-lactams.

PubMed

Willcox, Darren; Chappell, Ben G N; Hogg, Kirsten F; Calleja, Jonas; Smalley, Adam P; Gaunt, Matthew J

2016-11-18

Methods for the synthesis and functionalization of amines are intrinsically important to a variety of chemical applications. We present a general carbon-hydrogen bond activation process that combines readily available aliphatic amines and the feedstock gas carbon monoxide to form synthetically versatile value-added amide products. The operationally straightforward palladium-catalyzed process exploits a distinct reaction pathway, wherein a sterically hindered carboxylate ligand orchestrates an amine attack on a palladium anhydride to transform aliphatic amines into β-lactams. The reaction is successful with a wide range of secondary amines and can be used as a late-stage functionalization tactic to deliver advanced, highly functionalized amine products of utility for pharmaceutical research and other areas. Copyright © 2016, American Association for the Advancement of Science.

Snapshots of the maltose transporter during ATP hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oldham, Michael L.; Chen, Jue

2011-12-05

ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

A quantum mechanics/molecular mechanics study on the hydrolysis mechanism of New Delhi metallo-β-lactamase-1.

PubMed

Zhu, Kongkai; Lu, Junyan; Liang, Zhongjie; Kong, Xiangqian; Ye, Fei; Jin, Lu; Geng, Heji; Chen, Yong; Zheng, Mingyue; Jiang, Hualiang; Li, Jun-Qian; Luo, Cheng

2013-03-01

New Delhi metallo-β-lactamase-1 (NDM-1) has emerged as a major global threat to human health for its rapid rate of dissemination and ability to make pathogenic microbes resistant to almost all known β-lactam antibiotics. In addition, effective NDM-1 inhibitors have not been identified to date. In spite of the plethora of structural and kinetic data available, the accurate molecular characteristics of and details on the enzymatic reaction of NDM-1 hydrolyzing β-lactam antibiotics remain incompletely understood. In this study, a combined computational approach including molecular docking, molecular dynamics simulations and quantum mechanics/molecular mechanics calculations was performed to characterize the catalytic mechanism of meropenem catalyzed by NDM-1. The quantum mechanics/molecular mechanics results indicate that the ionized D124 is beneficial to the cleavage of the C-N bond within the β-lactam ring. Meanwhile, it is energetically favorable to form an intermediate if no water molecule coordinates to Zn2. Moreover, according to the molecular dynamics results, the conserved residue K211 plays a pivotal role in substrate binding and catalysis, which is quite consistent with previous mutagenesis data. Our study provides detailed insights into the catalytic mechanism of NDM-1 hydrolyzing meropenem β-lactam antibiotics and offers clues for the discovery of new antibiotics against NDM-1 positive strains in clinical studies.

Fundamental Reaction Pathway and Free Energy Profile for Butyrylcholinesterase-Catalyzed Hydrolysis of Heroin

PubMed Central

Qiao, Yan; Han, Keli; Zhan, Chang-Guo

2013-01-01

The pharmacological function of heroin requires an activation process which transforms heroin into 6-monoacetylmorphine (6-MAM) which is the most active form. The primary enzyme responsible for this activation process in human plasma is butyrylcholinesterase (BChE). The detailed reaction pathway of the activation process via BChE-catalyzed hydrolysis has been explored computationally, for the first time, in the present study by performing molecular dynamics simulation and first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the whole reaction process includes acylation and deacylation stages. The acylation consists of two reaction steps, i.e. the nucleophilic attack on the carbonyl carbon of 3-acetyl group of heroin by the hydroxyl oxygen of Ser198 side chain and the dissociation of 6-MAM. The deacylation also consists of two reaction steps, i.e. the nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate by a water molecule and the dissociation of the acetic acid from Ser198. The calculated free energy profile reveals that the second transition state (TS2) should be rate-determining. The structural analysis reveals that the oxyanion hole of BChE plays an important role in the stabilization of the rate-determining transition state TS2. The free energy barrier (15.9±0.2 or 16.1±0.2 kcal/mol) calculated for the rate-determining step is in good agreement with the experimentally-derived activation free energy (~16.2 kcal/mol), suggesting that the mechanistic insights obtained from the present computational study are reliable. The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse. PMID:23992153

Selective ultrasound-enhanced enzymatic hydrolysis of oleuropein to its aglycon in olive (Olea europaea L.) leaf extracts.

PubMed

Delgado-Povedano, María Del Mar; Priego-Capote, Feliciano; Luque de Castro, María Dolores

2017-04-01

Hydrolysis of oleuropein, the main phenol in olive (Olea europaea L.) leaf extracts, to oleuropein aglycon and other subsequent products in the hydrolytic pathway can be catalyzed by different enzymes. Three of the most used hydrolases were assayed to catalyze the process, and β-glucosidase from Aspergillus niger was selected. Acceleration of the enzymatic hydrolysis by ultrasound (US) was studied using a Box-Behnken design (duty cycle, amplitude, cycle time) and an oleuropein standard, and the optimum US conditions for achieving maximum yield of oleuropein aglycon were 0.5s/s duty cycle, 50% amplitude and 45s cycle. The method was applied to obtain oleuropein aglycon from commercial and laboratory extracts from olive leaves, which may have a pharmacological use as deduced by its healthy properties. The kinetics of the US-assisted enzymatic hydrolysis was monitored by analysis of the target compounds using liquid chromatography-tandem mass spectrometry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

PubMed Central

Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

2013-01-01

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

Bulk gold catalyzed oxidation reactions of amines and isocyanides and iron porphyrin catalyzed N-H and O-H bond insertion/cyclization reactions of diamines and aminoalcohols

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klobukowski, Erik

2011-01-01

and conditions, it was found that the oxidative dehydrogenation of dibenzylamine to Nbenzylidenebenzylamine, with N-methylmorpholine N-oxide (NMMO), was nearly quantitative (96%) within 24 h. However, the reaction with oxygen was much slower, with only a 52% yield of imine product over the same time period. Moreover, the rate of reaction was found to be influenced by the nature of the amine N-oxide. For example, the use of the weakly basic pyridine N-oxide (PyNO) led to an imine yield of only 6% after 24 h. A comparison of amine N-oxide and O2 was also examined in the oxidation of PhCH{sub 2}OH to PhCHO catalyzed by bulk gold. In this reaction, a 52% yield of the aldehyde was achieved when NMMO was used, while only a 7% product yield was afforded when O{sub 2} was the oxidant after 48 h. The bulk gold-catalyzed oxidative dehydrogenation of cyclic amines generates amidines, which upon treatment with Aerosil and water were found to undergo hydrolysis to produce lactams. Moreover, 5-, 6-, and 7-membered lactams could be prepared through a one-pot reaction of cyclic amines by treatment with oxygen, water, bulk gold, and Aerosil. This method is much more atom economical than industrial processes, does not require corrosive acids, and does not generate undesired byproducts. Additionally, the gold and Aerosil catalysts can be readily separated from the reaction mixture. The second project involved studying iron(III) tetraphenylporphyrin chloride, Fe(TPP)Cl, as a homogeneous catalyst for the generation of carbenes from diazo reagents and their reaction with heteroatom compounds. Fe(TPP)Cl, efficiently catalyzed the insertion of carbenes derived from methyl 2-phenyldiazoacetates into O-H bonds of aliphatic and aromatic alcohols. Fe(TPP)Cl was also found to be an effective catalyst for tandem N-H and O-H insertion/cyclization reactions when 1,2-diamines and 1,2-alcoholamines were treated with diazo reagents. This approach provides a one-pot process for synthesizing

Oligonucleotide synthesis catalyzed by the Zn/2+/ ion

NASA Technical Reports Server (NTRS)

Sawai, H.; Orgel, L. E.

1975-01-01

Results of experiments are reported in which Zn(2+) ion catalyzed the formation of oligonucleotides from nucleoside phosphorimidazolides in aqueous solution, even in the absence of a template. Specifically, the imidazolides (ImpU or ImpA) polymerized to form ImpApA, and pApA, pApApA, and pApApApA, or the analogous uracil compounds. In addition, the expected hydrolysis products of the hydrolysis of ImpA were formed (pA, imidazole). Judging from the ratio of pA(n) over pA (with and without zinc ion), this ion increased the efficiency of phosphodiester-bond formation by up to 10 times. Possible mechanisms for the reaction are tentatively proposed.

Use of the SPARC software program to calculate hydrolysis rate constants for the polymeric brominated flame retardants BC-58 and FR-1025.

PubMed

Rayne, Sierra; Forest, Kaya

2016-01-01

The SPARC software program was used to estimate the acid-catalyzed, neutral, and base-catalyzed hydrolysis rate constants for the polymeric brominated flame retardants BC-58 and FR-1025. Relatively rapid hydrolysis of BC-58, producing 2,4,6-tribromophenol-and ultimately tetrabromobisphenol A-as the hydrolytically stable end products from all potential hydrolysis reactions, is expected in both environmental and biological systems with starting material hydrolytic half-lives (t(1/2,hydr)) ranging from less than 1 h in marine systems, several hours in cellular environments, and up to several weeks in slightly acid fresh waters. Hydrolysis of FR-1025 to give 2,3,4,5,6-pentabromobenzyl alcohol is expected to be slower (t(1/2,hydr) less than 0.5 years in marine systems up to several years in fresh waters) than BC-58, but is also expected to occur at rates that will contribute significantly to environmental and in vivo loadings of this compound.

Activation Energies for an Enzyme-Catalyzed and Acid-Catalyzed Hydrolysis: An Introductory Interdisciplinary Experiment for Chemists and Biochemists.

ERIC Educational Resources Information Center

Adams, K. R.; Meyers, M. B.

1985-01-01

Background information, procedures used, and typical results obtained are provided for an experiment in which students determine and compare the Arrhenius activation energies (Ea) for the hydrolysis of salicin. This reaction is subject to catalysis both by acid and by the enzyme emulsin (beta-d-glucoside glycohydrolase). (JN)

Penicillin and beta-lactam allergy: epidemiology and diagnosis.

PubMed

Macy, Eric

2014-11-01

Penicillin is the most common beta-lactam antibiotic allergy and the most common drug class allergy, reported in about 8% of individuals using health care in the USA. Only about 1% of individuals using health care in the USA have a cephalosporin allergy noted in their medical record, and other specific non-penicillin, non-cephalosporin beta-lactam allergies are even rarer. Most reported penicillin allergy is not associated with clinically significant IgE-mediated reactions after penicillin rechallenge. Un-verified penicillin allergy is a significant and growing public health problem. Clinically significant IgE-mediated penicillin allergy can be safely confirmed or refuted using skin testing with penicilloyl-poly-lysine and native penicillin G and, if skin test is negative, an oral amoxicillin challenge. Acute tolerance of an oral therapeutic dose of a penicillin class antibiotic is the current gold standard test for a lack of clinically significant IgE-mediated penicillin allergy. Cephalosporins and other non-penicillin beta-lactams are widely, safely, and appropriately used in individuals, even with confirmed penicillin allergy. There is little, if any, clinically significant immunologic cross-reactivity between penicillins and other beta-lactams. Routine cephalosporin skin testing should be restricted to research settings. It is rarely needed clinically to safely manage patients and has unclear predictive value at this time. The use of alternative cephalosporins, with different side chains, is acceptable in the setting of a specific cephalosporin allergy. Carbapenems and monobactams are also safely used in individuals with confirmed penicillin allergy. A certain predictable, but low, rate of adverse reactions will occur with all beta-lactam antibiotic use both pre- and post-beta-lactam allergy evaluations.

Switching catalysis from hydrolysis to perhydrolysis in P. fluorescens esterase

PubMed Central

Yin, De Lu (Tyler); Bernhardt, Peter; Morley, Krista L.; Jiang, Yun; Cheeseman, Jeremy D.; Purpero, Vincent; Schrag, Joseph D.; Kazlauskas, Romas J.

2010-01-01

Many serine hydrolases catalyze perhydrolysis – the reversible formation of per-acids from carboxylic acids and hydrogen peroxide. Recently we showed that a single amino acid substitution in the alcohol binding pocket - L29P - in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. Angew. Chem. Intl. Ed. 2005, 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two x-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active-site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of ε-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction – hydrolysis of peracetic acid to acetic acid and hydrogen peroxide – occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed two fold higher kcat, but Km also increased so the specificity constant, kcat/Km, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate), but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of ε-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for

Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol.

PubMed

Shukor, Hafiza; Abdeshahian, Peyman; Al-Shorgani, Najeeb Kaid Nasser; Hamid, Aidil Abdul; Rahman, Norliza A; Kalil, Mohd Sahaid

2016-10-01

Catalytic depolymerization of mannan composition of palm kernel cake (PKC) by mannanase was optimized to enhance the release of mannan-derived monomeric sugars for further application in acetone-butanol-ethanol (ABE) fermentation. Efficiency of enzymatic hydrolysis of PKC was studied by evaluating effects of PKC concentration, mannanase loading, hydrolysis pH value, reaction temperature and hydrolysis time on production of fermentable sugars using one-way analysis of variance (ANOVA). The ANOVA results revealed that all factors studied had highly significant effects on total sugar liberated (P<0.01). The optimum conditions for PKC hydrolysis were 20% (w/v) PKC concentration, 5% (w/w) mannanase loading, hydrolysis pH 4.5, 45°C temperature and 72h hydrolysis time. Enzymatic experiments in optimum conditions revealed total fermentable sugars of 71.54±2.54g/L were produced including 67.47±2.51g/L mannose and 2.94±0.03g/L glucose. ABE fermentation of sugar hydrolysate by Clostridium saccharoperbutylacetonicum N1-4 resulted in 3.27±1.003g/L biobutanol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oxygen isotope effects of enzyme-catalyzed organophosphorus hydrolysis reactions: implications for interpretation of dissolved PO4 δ18O values in natural waters

NASA Astrophysics Data System (ADS)

Liang, Y.; Blake, R. E.

2002-12-01

The geochemical cycling of P in Earth surface environments is controlled largely by biota. It has been recently demonstrated that intracellular cycling of P in microbial cultures and biological turnover of P in natural waters leads to temperature-dependent O isotope equilibrium between dissolved inorganic PO4 (Pi) and ambient water, and that the δ18O of Pi can be a useful tracer of biological reactions and P cycling in aquatic systems/sediments. Oxygen isotope exchange between Pi and water during biological turnover of P is catalyzed by enzymes at low-temperature. Phosphoenzymes play a crucial role in the intracellular functions of all living organisms and also have important extracellular functions in aquatic ecosystems such as regeneration of Pi from organophosphorus compounds (e.g., phosphoesters). Laboratory experiments indicate that extracellular enzyme reactions may result in incomplete Pi turnover and non-equilibrium Pi-water O isotope exchange. Determination of the O isotope effects of phosphoenzyme-catalyzed reactions is fundamental to the understanding of mechanisms of PO4-water O isotope exchange, pathways of biogeochemical P cycling, and interpretation of PO4 δ18O values from natural systems. Here we report on the O isotope fractionation between enzymatically-released Pi and water, in cell-free abiotic systems. Alkaline phosphatase (Apase) is a non-specific phosphohydrolase commonly found in fresh and marine coastal waters that catalyzes the hydrolysis of Pi from phosphomonoesters. We examined the O isotope effects of Apase derived from both microbial and eukaryotic sources and acting on different phosphomonoester substrates (e.g., α-D-Glucose 1-Phosphate, β-Glycerophosphate, AMP) in 18O-labeled waters. Oxygen isotope ratios of Pi released by Apase indicate that only 1 of the 4 O atoms in PO4 is incorporated from water with little or no apparent O isotopic fractionation at the site of incorporation. This observation is consistent with

Coal liquefaction by base-catalyzed hydrolysis with CO.sub.2 capture

DOEpatents

Xiao, Xin

2014-03-18

The one-step hydrolysis of diverse biomaterials including coal, cellulose materials such as lumber and forestry waste, non-food crop waste, lignin, vegetable oils, animal fats and other source materials used for biofuels under mild processing conditions which results in the formation of a liquid fuel product along with the recovery of a high purity CO.sub.2 product is provided.

Hydrolysis of triacetin catalyzed by immobilized lipases: effect of the immobilization protocol and experimental conditions on diacetin yield.

PubMed

Hernandez, Karel; Garcia-Verdugo, Eduardo; Porcar, Raul; Fernandez-Lafuente, Roberto

2011-05-06

The effect of the immobilization protocol and some experimental conditions (pH value and presence of acetonitrile) on the regioselective hydrolysis of triacetin to diacetin catalyzed by lipases has been studied. Lipase B from Candida antarctica (CALB) and lipase from Rhizomucor miehei (RML) were immobilized on Sepabeads (commercial available macroporous acrylic supports) activated with glutaraldehyde (covalent immobilization) or octadecyl groups (adsorption via interfacial activation). All the biocatalysts accumulated diacetin. Covalently immobilized RML was more active towards rac-methyl mandelate than the adsorbed RML. However, this covalent RML preparation presented the lowest activity towards triacetin. For this reason, this preparation was discarded as biocatalyst for this reaction. At pH 7, acyl migration occurred giving a mixture of 1,2 and 1,3 diacetin, but at pH 5.5, only 1,2 diacetin was produced. Yields were improved at acidic pH values and in the presence of 20% acetonitrile (to over 95%). RML immobilized on octadecyl Sepabeads was proposed as optimal preparation, mainly due to its higher specific activity. Each enzyme preparation presented very different properties. Moreover, changes in the reaction conditions affected the various immobilized enzymes in a different way. Copyright © 2011 Elsevier Inc. All rights reserved.

Non-catalytic steam hydrolysis of fats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deibert, M.C.

1992-08-28

Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steammore » mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.« less

Educational case series: β-lactam allergy and cross-reactivity.

PubMed

Atanasković-Marković, Marina

2011-12-01

Penicillins and cephalosporins are the most widely used antibiotics for the treatment of common infections, and they are the two main classes of β-lactams. On the basis of the time of appearance of the reaction after drug intake and for diagnostic purposes, hypersensitivity reactions to β-lactams have been classified as immediate or non-immediate. The diagnostic evaluation of allergic reactions to β-lactams has changed over the last decade, for several reasons. In many countries, major and minor determinants for skin testing are not available. In immediate allergic reactions, the sensitivity of skin testing is decreasing. For non-immediate reactions, skin testing appears to be less sensitive than previously reported. The drug provocation test is still necessary for diagnosis. In this education review series, we described three cases of β-lactam allergy: first, a child with an IgE-mediated allergy to benzyl-penicillin; second, a child with a non-allergic hypersensitivity to amoxicillin; and in the third patient, we will discuss about cross-reactivity between penicillins and cephalosporins. These cases are correlated with the practical management of evaluating β-lactam allergy. © 2011 John Wiley & Sons A/S.

Electrostatic and structural similarity of classical and non-classical lactam compounds

NASA Astrophysics Data System (ADS)

Coll, Miguel; Frau, Juan; Vilanova, Bartolomé; Donoso, Josefa; Muñoz, Francisco

2001-09-01

Various electrostatic and structural parameters for a series of classical and non-classical β-lactams were determined and compared in order to ascertain whether some specific β-lactams possess antibacterial or β-lactamase inhibitory properties. The electrostatic parameters obtained, based on the Distributed Multipole Analysis (DMA) of high-quality wavefunctions for the studied structures, suggest that some non-classical β-lactams effectively inhibit the action of β-lactamases. As shown in this work, such electrostatic parameters provide much more reliable information about the antibacterial and inhibitory properties of β-lactams than do structural parameters.

The conformational preferences of γ-lactam and its role in constraining peptide structure

NASA Astrophysics Data System (ADS)

Paul, P. K. C.; Burney, P. A.; Campbell, M. M.; Osguthorpe, D. J.

1990-09-01

The conformational constraints imposed by γ-lactams in peptides have been studied using valence force field energy calculations and flexible geometry maps. It has been found that while cyclisation restrains the Ψ of the lactam, non-bonded interactions contribute to the constraints on ϕ of the lactam. The γ-lactam also affects the (ϕ,Ψ) of the residue after it in a peptide sequence. For an l-lactam, the ring geometry restricts Ψ to about-120°, and ϕ has two minima, the lowest energy around-140° and a higher minimum (5 kcal/mol higher) at 60°, making an l-γ-lactam more favourably accommodated in a near extended conformation than in position 2 of a type II' β-turn. The energy of the ϕ˜+60° minimum can be lowered substantially until it is more favoured than the-140° minimum by progressive substitution of bulkier groups on the amide N of the l-γ-lactam. The (ϕ,Ψ) maps of the residue succeeding a γ-lactam show subtle differences from those of standard N-methylated residues. The dependence of the constraints on the chirality of γ-lactams and N-substituted γ-lactams, in terms of the formation of secondary structures like β-turns is discussed and the comparison of the theoretical conformations with experimental results is highlighted.

Laser-Based Measurement of Refractive Index Changes: Kinetics of 2,3-Epoxy-1-propanol Hydrolysis.

ERIC Educational Resources Information Center

Spencer, Bert; Zare, Richard N.

1988-01-01

Describes an experiment in which a simple laser-based apparatus is used for measuring the change in refractive index during the acid-catalyzed hydrolysis of glycidol into glycerine. Gives a schematic of the experimental setup and discusses the kinetic analysis. (MVL)

Hydrolysis of amphenicol and macrolide antibiotics: Chloramphenicol, florfenicol, spiramycin, and tylosin.

PubMed

Mitchell, Shannon M; Ullman, Jeffrey L; Teel, Amy L; Watts, Richard J

2015-09-01

Antibiotics that enter the environment can present human and ecological health risks. An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence as biologically active contaminants. In this study, hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for two amphenicol (chloramphenicol and florfenicol) and two macrolide (spiramycin and tylosin) antibiotics. Antibiotic hydrolysis rates in pH 4-9 buffer solutions at 25°C, 50°C, and 60°C were quantified, and degradation products were characterized. All of the antibiotics tested remained stable and exhibited no observable hydrolysis under ambient conditions typical of aquatic ecosystems. Acid- and base-catalyzed hydrolysis occurred at elevated temperatures (50-60°C), and hydrolysis rates increased considerably below pH 5 and above pH 8. Hydrolysis rates also increased approximately 1.5- to 2.9-fold for each 10°C increase in temperature. Based on the degradation product masses found, the functional groups that underwent hydrolysis were alkyl fluoride, amide, and cyclic ester (lactone) moieties; some of the resultant degradation products may remain bioactive, but to a lesser extent than the parent compounds. The results of this research demonstrate that amphenicol and macrolide antibiotics persist in aquatic systems under ambient temperature and pH conditions typical of natural waters. Thus, these antibiotics may present a risk in aquatic ecosystems depending on the concentration present. Copyright © 2015. Published by Elsevier Ltd.

Cellulose pretreatment with 1-n-butyl-3-methylimidazolium chloride for solid acid-catalyzed hydrolysis.

PubMed

Kim, Soo-Jin; Dwiatmoko, Adid Adep; Choi, Jae Wook; Suh, Young-Woong; Suh, Dong Jin; Oh, Moonhyun

2010-11-01

This study has been focused on developing a cellulose pretreatment process using 1-n-butyl-3-methylimidazolium chloride ([bmim]Cl) for subsequent hydrolysis over Nafion(R) NR50. Thus, several pretreatment variables such as the pretreatment period and temperature, and the [bmim]Cl amount were varied. Additionally, the [bmim]Cl-treated cellulose samples were characterized by X-ray diffraction analysis, and their crystallinity index values including CI(XD), CI(XD-CI) and CI(XD-CII) were then calculated. When correlated with these values, the concentrations of total reducing sugars (TRS) obtained by the pretreatment of native cellulose (NC) and glucose produced by the hydrolysis reaction were found to show a distinct relationship with the [CI(NC)-CI(XD)] and CI(XD-CII) values, respectively. Consequently, the cellulose pretreatment step with [bmim]Cl is to loosen a crystalline cellulose through partial transformation of cellulose I to cellulose II and, furthermore, the TRS release, while the subsequent hydrolysis of [bmim]Cl-treated cellulose over Nafion(R) NR50 is effective to convert cellulose II to glucose. Copyright 2010 Elsevier Ltd. All rights reserved.

The computational analysis and modelling of substitution effects on hydrolysis of formanilides in acidic aqueous solutions

NASA Astrophysics Data System (ADS)

Lukeš, Vladimír; Škorňa, Peter; Michalík, Martin; Klein, Erik

2017-11-01

Various para, meta and ortho substituted formanilides have been theoretically studied. For trans and cis-isomers of non-substituted formanilide, the calculated B3LYP vibration normal modes were analyzed. Substituent effect on the selected normal modes was described and the comparison with the available experimental data is presented. The calculated B3LYP proton affinities were correlated with Hammett constants, Fujita-Nishioka equation and the rate constants of the hydrolysis in 1 M HCl. Found linear dependences allow predictions of dissociation constants (pKBH+) and hydrolysis rate constants. Obtained results indicate that protonation of amide group may represent the rate determining step of acid catalyzed hydrolysis.

Comparative Treatment Failure Rates of Respiratory Fluoroquinolones or β-Lactam + Macrolide Versus β-Lactam Alone in the Treatment for Community-Acquired Pneumonia in Adult Outpatients

PubMed Central

Lee, Meng-Tse Gabriel; Lee, Shih-Hao; Chang, Shy-Shin; Chan, Ya-Lan; Pang, Laura; Hsu, Sue-Ming; Lee, Chien-Chang

2015-01-01

Abstract No comparative effectiveness study has been conducted for the following 3 antibiotics: respiratory fluoroquinolone, β-lactam, and β-lactam + advanced macrolide. To gain insights into the real-world clinical effectiveness of these antibiotics for community-acquired pneumonia in adult outpatients, our study investigated the treatment failure rates in 2 million representative participants from the National Health Informatics Project (NHIP) of Taiwan. A new-user cohort design was used to follow NHIP participants from January 2000 until December 2009. Treatment failure was defined by either one of the following events: a second antibiotic prescription, hospitalization due to CAP, an emergency department visit with a diagnosis of CAP, or 30-day nonaccident-related mortality. From 2006 to 2009, we identified 9256 newly diagnosed CAP outpatients, 1602 of whom were prescribed levofloxacin, 2100 were prescribed moxifloxacin, 5049 were prescribed β-lactam alone, and 505 were prescribed advanced macrolide + β-lactam. Compared with the β-lactam-based regimen, the propensity score-matched odds ratio for composite treatment failure was 0.81 (95% CI, 0.67–0.97) for moxifloxacin, 1.10 (95% CI, 0.90–1.35) for levofloxacin, and 0.95 (95% CI, 0.67–1.35) for macrolide +β-lactam. Moxifloxacin was associated with lower treatment failure rates compared with β-lactam alone, or levofloxacin in Taiwanese CAP outpatients. However, due to inherent limitations in our claims database, more randomized controlled trials are required before coming to a conclusion on which antibiotic is more effective for Taiwanese CAP outpatients. More population-based comparative effectiveness studies are also encouraged and should be considered as an integral piece of evidence in local CAP treatment guidelines. PMID:26426664

Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features.

PubMed

Liljeblad, Arto; Kallio, Pauli; Vainio, Marita; Niemi, Jarmo; Kanerva, Liisa T

2010-02-21

Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper.

Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

PubMed Central

Lun, Shichun; Miranda, David; Kubler, Andre; Guo, Haidan; Maiga, Mariama C.; Winglee, Kathryn; Pelly, Shaaretha

2014-01-01

ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. PMID:25227469

Identification of beta-lactam antibiotics in tissue samples containing unknown microbial inhibitors.

PubMed

Moats, W A; Romanowski, R D; Medina, M B

1998-01-01

Antibiotic residues in animal tissues can be detected by various screening tests based on microbial inhibition. In the 7-plate assay used by the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS), penicillinase is incorporated into all but one plate to distinguish beta-lactam antibiotics from other types. However, beta-lactams such as cloxacillin and the cephalosporins are resistant to degradation by penicillinase. They may not be identified as beta-lactams by this procedure, and thus, they may be identified as unidentified microbial inhibitors (UMIs). However, these penicillinase-resistant compounds can be degraded by other beta-lactamases. The present study describes an improved screening protocol to identify beta-lactam antibiotics classified as UMIs. A multiresidue liquid chromatographic procedure based on a method for determining beta-lactams in milk was also used to identify and quantitate residues. The 2 methods were tested with 24 tissue FSIS samples classified as containing UMIs. Of these, 3 contained penicillin G, including one at a violative level, and 5 contained a metabolite of ceftiofur. The others were negative for beta-lactam antibiotics.

An update on adverse drug reactions related to β-lactam antibiotics.

PubMed

Vardakas, Konstantinos Z; Kalimeris, Georgios D; Triarides, Nikolaos A; Falagas, Matthew E

2018-05-01

β-lactams have been consistently associated with the majority of drug-related adverse events. Generally, these are mild under proper dosing and judicious selection. Areas covered: Immediate hypersensitivity reactions are the most feared adverse events encountered after β-lactam administration. Emerging evidence shows that immediate reactions are not as common as previously thought. Specialist consultation and testing seems prudent before a patient is officially declared allergic to β-lactams. The risk of cross-reactions between not only members of the β-lactam super-family but also between specific classes is also lower than previously thought. Newer studies have shown that cross-reactions are not universal and pertain to specific agents with similar side chains or metabolites of the β-lactam core. The frequency of severe kidney or liver toxicity, neurotoxicity, cytopenias and Clostiridium difficile infection following β-lactam administration seem to be agent-specific. Expert opinion: The currently available data denote that in addition to age, gender, co-morbidity, renal or liver function, and co-administered agents, the antibiotic levels rather than the dose itself seem to be associated with the emergence of adverse events. Most of them subside with time after withdrawal of the offending agent, but the number of cases resulting in chronic disabilities or even deaths in not negligible.

Secondary metabolism in simulated microgravity: beta-lactam production by Streptomyces clavuligerus

NASA Technical Reports Server (NTRS)

Fang, A.; Pierson, D. L.; Mishra, S. K.; Koenig, D. W.; Demain, A. L.

1997-01-01

Rotating bioreactors designed at NASA's Johnson Space Center were used to simulate a microgravity environment in which to study secondary metabolism. The system examined was beta-lactam antibiotic production by Streptomyces clavuligerus. Both growth and beta-lactam production occurred in simulated microgravity. Stimulatory effects of phosphate and L-lysine, previously detected in normal gravity, also occurred in simulated microgravity. The degree of beta-lactam antibiotic production was markedly inhibited by simulated microgravity.

Arrhenius analysis of the electrophorus electricus acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

PubMed

Oakes, Jesse; Nguyen, Tina; Britt, B Mark

2003-06-01

Ellman's method was used to determine the Michaelis-Menten parameters for the hydrolysis of acetylthiocholine by Electrophorus electricus acetylcholinesterase from 12 to 37 degrees C. Arrhenius analysis revealed that the activation energy for formation of the enzyme/substrate complex is 22.2 +/- 1.1 kJ/mole. The Arrhenius plot of k(cat) is markedly curved and attributed to comparable rates of acylation and deacylation due to the absence of evidence for a temperature-dependent enzyme conformational change by differential scanning calorimetry.

Phosphoester hydrolysis: the incoming substrate turns the bridging hydroxido nucleophile into a terminal one.

PubMed

Gouré, Eric; Carboni, Michaël; Troussier, Angélique; Lebrun, Colette; Pécaut, Jacques; Jacquot, Jean-François; Dubourdeaux, Patrick; Clémancey, Martin; Blondin, Geneviève; Latour, Jean-Marc

2015-05-26

Identifying the active nucleophile in hydrolysis reactions catalyzed by binuclear hydrolases is a recurrent problem and a matter of intense debate. We report on the phosphate ester hydrolysis by a Fe(III)Fe(II) complex of a binucleating ligand. This complex presents activities in the range of those observed for similar biomimetic compounds in the literature. The specific electronic properties of the Fe(III)Fe(II) complex allowed us to use (1)H NMR and Mössbauer spectroscopies to investigate the nature of the various species present in the solution in the pH range of 5-10. Both techniques showed that the hydrolysis activity is associated to a μ-hydroxido Fe(III)Fe(II) species. Further (1)H NMR experiments show that binding of anions or the substrate changes this bonding mode suggesting that a terminal hydroxide is the likely nucleophile in these hydrolysis reactions. This view is further supported by the structure determination of the hydrolysis product. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulatory link between steryl ester formation and hydrolysis in the yeast Saccharomyces cerevisiae.

PubMed

Ploier, Birgit; Korber, Martina; Schmidt, Claudia; Koch, Barbara; Leitner, Erich; Daum, Günther

2015-07-01

Steryl esters and triacylglycerols are the major storage lipids of the yeast Saccharomyces cerevisiae. Steryl esters are formed in the endoplasmic reticulum by the two acyl-CoA:sterol acyltransferases Are1p and Are2p, whereas steryl ester hydrolysis is catalyzed by the three steryl ester hydrolases Yeh1p, Yeh2p and Tgl1p. To shed light on the regulatory link between steryl ester formation and hydrolysis in the maintenance of cellular sterol and free fatty acid levels we employed yeast mutants which lacked the enzymes catalyzing the degradation of steryl esters. These studies revealed feedback regulation of steryl ester formation by steryl ester hydrolysis although in a Δtgl1Δyeh1Δyeh2 triple mutant the gene expression levels of ARE1 and ARE2 as well as protein levels and stability of Are1p and Are2p were not altered. Nevertheless, the capacity of the triple mutant to synthesize steryl esters was significantly reduced as shown by in vitro and in vivo labeling of lipids with [(14)C]oleic acid and [(14)C]acetate. Enzymatic analysis revealed that inhibition of steryl ester formation occurred at the enzyme level. As the amounts and the formation of sterols and fatty acids were also decreased in the triple mutant we concluded that defects in steryl ester hydrolysis also caused feedback inhibition on the formation of sterols and fatty acids which serve as precursors for steryl ester formation. In summary, this study demonstrates a regulatory link within the steryl ester metabolic network which contributes to non-polar lipid homeostasis in yeast cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds along Two Different Reaction Pathways: Implications for Engineering Bioscavengers

PubMed Central

2015-01-01

Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents (“bioscavengers”) is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P–F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds. PMID:24720808

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase.

PubMed

Wickham, Stephanie; Regan, Nicholas; West, Matthew B; Kumar, Vidya Prasanna; Thai, Justin; Li, Pui Kai; Cook, Paul F; Hanigan, Marie H

2012-08-01

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.

Continuous infusion vs. bolus dosing: implications for beta-lactam antibiotics.

PubMed

Mohd Hafiz, Abdul-Aziz; Staatz, C E; Kirkpatrick, C M J; Lipman, J; Roberts, J A

2012-01-01

Beta-lactam antibiotics display time-dependant pharmacodynamics whereby constant antibiotic concentrations rather than high peak concentrations are most likely to result in effective treatment of infections caused by susceptible bacteria. Continuous administration has been suggested as an alternative strategy, to conventional intermittent dosing, to optimise beta-lactam pharmacokinetic/pharmacodynamic (PK/PD) properties. With the availability of emerging data, we elected to systematically investigate the published literature describing the comparative PK/PD and clinical outcomes of beta-lactam antibiotics administered by continuous or intermittent infusion. We found that the studies have been performed in various patient populations including critically ill, cancer and cystic fibrosis patients. Available in vitro PK/PD data conclusively support the administration of beta-lactams via continuous infusion for maximizing bacterial killing from consistent attainment of pharmacodynamic end-points. In addition, clinical outcome data supports equivalence, even with the use of a lower dose by continuous infusion. However, the present clinical data is limited with small sample sizes common with insufficient power to detect advantages in favour of either dosing strategy. With abundant positive pre-clinical data as well as document in vivo PK/PD advantages, large multi-centre trials are needed to describe whether continuous administration of beta-lactams is truly more effective than intermittent dosing.

On-line characterization using ultrasound of pectin hydrolysis catalyzed by the enzyme pectinmethylesterase

NASA Astrophysics Data System (ADS)

Aparicio, C.; Resa, P.; Sierra, C.; Elvira, L.

2012-12-01

The major problem in the fruit juice industry is associated with juice quality deterioration due to the cloud loss of juice concentrates by the enzymatic reaction of pectinmethylesterase enzyme (PME, EC 3.1.1.11). During pectin hydrolysis, pectin and water are transformed into polygalacturonic acid (pectate) and methanol by the action of PME. In this work, a low-intensity ultrasonic technique is used to monitor this enzymatic reaction, with PME both from orange peel and from Aspergillus niger. Changes in sound velocity during pectin hydrolysis (1% concentration of pectin, T = 30°C and pH = 4.5 and 7) with 0.25 ml of enzyme solution (PME) have been measured using a through-transmission technique. Sound velocity decreases as pectin is transformed into pectate and methanol and at the end of the process, the change in sound velocity reaches 0.3 m/s with PME from orange peel and 0.33 m/s with PME from Aspergillus niger.

Pharmacokinetics and pharmacogenomics of β-lactam-induced neutropenia

PubMed Central

Hahn, Andrea; Fukuda, Tsuyoshi; Hahn, David; Mizuno, Tomoyuki; Frenck, Robert W; Vinks, Alexander A

2016-01-01

Aim: Determine if individuals with β-lactam induced neutropenia have polymorphisms that impair function of MRP4 or OAT1/OAT3. Methods: Subjects with β-lactam induced neutropenia were compared to controls for the presence of MRP4 and OAT1/OAT3 polymorphisms, estimated plasma trough concentrations and area under the curve. Results: Subjects with a homozygous polymorphism at MRP4 3348 A to G were 5.3 times more likely to develop neutropenia (p = 0.171). No statistical differences were noted in pharmacokinetic parameters. Contingency analysis of children greater than 5 years of age showed neutropenia in subjects who were homozygous wild type at MRP4 3348 A to G was significantly associated with standard or high dosing (p = 0.03). Conclusion: MRP4 3348 A to G should be further studied for potential contribution to the development of β-lactam induced neutropenia. PMID:27045542

Hydrolysis of Letrozole catalyzed by macrocyclic Rhodium (I) Schiff-base complexes.

PubMed

Reddy, P Muralidhar; Shanker, K; Srinivas, V; Krishna, E Ravi; Rohini, R; Srikanth, G; Hu, Anren; Ravinder, V

2015-03-15

Ten mononuclear Rhodium (I) complexes were synthesized by macrocyclic ligands having N4 and N2O2 donor sites. Square planar geometry is assigned based on the analytical and spectral properties for all complexes. Rh(I) complexes were investigated as catalysts in hydrolysis of Nitrile group containing pharmaceutical drug Letrozole. A comparative study showed that all the complexes are efficient in the catalysis. The percent yields of all the catalytic reaction products viz. drug impurities were determined by spectrophotometric procedures and characterized by spectral studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds Along Two Different Reaction Pathways: Implica-tions for Engineering Bioscavengers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wymore, Troy W; Langan, Paul; Smith, Jeremy C

Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ( bioscavengers ) is an emerging prophylactic approach to diminishing their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical (QM/MM) umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalentmore » intermediate. P F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily due to the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed -propeller folds for more efficient degradation of OP compounds.« less

Acid-catalyzed autohydrolysis of wheat straw to improve sugar recovery.

PubMed

Ertas, Murat; Han, Qiang; Jameel, Hasan

2014-10-01

A comparison study of autohydrolysis and acid-catalyzed autohydrolysis of wheat straw was performed to understand the impact of acid addition on overall sugar recovery. Autohydrolysis combined with refining is capable of achieving sugar recoveries in the mid 70s. If the addition of a small amount of acid is capable of increasing the sugar recovery even higher it may be economically attractive. Acetic, sulfuric, hydrochloric and sulfurous acids were selected for acid-catalyzed autohydrolysis pretreatments. Autohydrolysis with no acid at 190 °C showed the highest total sugar in the prehydrolyzate. Enzymatic hydrolysis was performed for all the post-treated solids with and without refining at enzyme loadings of 4 and 10 FPU/g for 96 h. Acid-catalyzed autohydrolysis at 190 °C with sulfurous acid showed the highest total sugar recovery of 81.2% at 4 FPU/g enzyme charge compared with 64.3% at 190 °C autohydrolysis without acid. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interactions of β-Lactamases with Sanfetrinem (GV 104326) Compared to Those with Imipenem and with Oral β-Lactams

PubMed Central

Babini, Gioia S.; Yuan, Meifang; Livermore, David M.

1998-01-01

Sanfetrinem is a trinem β-lactam which can be administered orally as a hexatil ester. We examined whether its β-lactamase interactions resembled those of the available carbapenems, i.e., stable to AmpC and extended-spectrum β-lactamases but labile to class B and functional group 2f enzymes. The comparator drugs were imipenem, oral cephalosporins, and amoxicillin. MICs were determined for β-lactamase expression variants, and hydrolysis was examined directly with representative enzymes. Sanfetrinem was a weak inducer of AmpC β-lactamases below the MIC and had slight lability, with a kcat of 0.00033 s−1 for the Enterobacter cloacae enzyme. Its MICs for AmpC-derepressed E. cloacae and Citrobacter freundii were 4 to 8 μg/ml, compared with MICs of 0.12 to 2 μg/ml for AmpC-inducible and -basal strains; MICs for AmpC-derepressed Serratia marcescens and Morganella morganii were not raised. Cefixime and cefpodoxime were more labile than sanfetrinem to the E. cloacae AmpC enzyme, and AmpC-derepressed mutants showed much greater resistance; imipenem was more stable and retained full activity against derepressed mutants. Like imipenem, sanfetrinem was stable to TEM-1 and TEM-10 enzymes and retained full activity against isolates and transconjugants with various extended-spectrum TEM and SHV enzymes, whereas these organisms were resistant to cefixime and cefpodoxime. Sanfetrinem, like imipenem and cefixime but unlike cefpodoxime, also retained activity against Proteus vulgaris and Klebsiella oxytoca strains that hyperproduced potent chromosomal class A β-lactamases. Functional group 2f enzymes, including Sme-1, NMC-A, and an unnamed enzyme from Acinetobacter spp., increased the sanfetrinem MICs by up to 64-fold. These enzymes also compromised the activities of imipenem and amoxicillin but not those of the cephalosporins. The hydrolysis of sanfetrinem was examined with a purified Sme-1 enzyme, and biphasic kinetics were found. Finally, zinc β-lactamases, including IMP

Non-catalytic steam hydrolysis of fats. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deibert, M.C.

1992-08-28

Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steammore » mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.« less

Decomposition of lignin and cellobiose in relation to the enzymatic hydrolysis of cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamanaka, Y.; Carroad, P.A.; Riaz, M.

1977-02-01

Studies are reported on the use of fungal ..beta..-glucosidase in conjunction with Trichoderma viride cellulase and the search for an effective enzyme system for lignin degradation. ..beta..-glucosidase is of potential benefit in cellulose hydrolysis by catalyzing the hydrolysis of cellobiose to glucose thereby reducing product inhibition and producing a higher glucose yield. Removal of lignin from cellulosic material makes the cellulose more accessible to hydrolyzing enzymes. Hydrolysis studies on Solka Floc and newsprint were conducted with T. viride filtrates containing various proportions of B. theobromae filtrates. Significant improvement in hydrolysis rate particularly in glucose content was obtained by thus enrichingmore » the ..beta..-glucosidase content of the cellulase. In the search for a lignin degrading enzyme, major emphasis was given to the fungus Polyporous versicolor. Significant o-diphenol oxidoreductase (catecholase) activity was found in the culture filtrates. Preliminary observations of a surface culture of the fungus in a composting mode suggest that delignification may be obtained in this manner. Work is continuing on this.« less

Beta-lactam antibiotics: newer formulations and newer agents.

PubMed

Martin, Stanley I; Kaye, Kenneth M

2004-09-01

beta-Lactam antibiotics share a common structure and mechanism of action, although they differ in their spectrum of antimicrobial activity and utility in treating different infections. The current classes include the penicillins, the penicillinase-resistant penicillins, the extended- spectrum penicillins, the cephalosporins, the carbapenems, and the monobactams. This article discusses some of the newest beta-lactams available for use in the United States: ertapenem, cefditoren, and cefepime. A new formulation of amoxicillin-clavulanate, which contains higher doses of amoxicillin, is also discussed.

Phospholipids and products of their hydrolysis as dietary preventive factors for civilization diseases.

PubMed

Parchem, Karol; Bartoszek, Agnieszka

2016-12-31

The results of numerous epidemiological studies indicate that phospholipids play an important role in the prevention of chronic diseases faced by contemporary society. Firstly, these compounds are responsible for the proper functioning of cell membranes, by ensuring liquidity and permeability, which is pivotal for normal activity of membrane proteins, including receptors. These mechanisms are at the core of prevention of cancer, autoimmune or neurological disorders. Secondly, structure and properties of phospholipids cause that they are highly available source of biologically active fatty acids. Thirdly, also products of endogenous hydrolysis of phospholipids exhibit biological activity. These include lysophospholipids formed as a result of disconnecting free fatty acid from glycerophospholipids in the reaction catalyzed by phospholipase A, phosphatidic acid and hydrophilic subunits released by the activity of phospholipase D. The bioactive products of hydrolysis also include ceramides liberated from phosphosphingolipids after removal of a hydrophilic unit catalyzed by sphingomyelinase. Phospholipids are supplied to the human body with food. A high content of phospholipids is characteristic for egg yolk, liver, pork and poultry, as well as some soy products. Particularly beneficial are phospholipids derived from seafood because they are a rich source of essential fatty acids of the n-3 family.

Mechanism of 1,4,5,8-naphthalene tetracarboxylic acid dianhydride hydrolysis and formation in aqueous solution.

PubMed

Barros, T C; Cuccovia, I M; Farah, J P S; Masini, J C; Chaimovich, H; Politi, M J

2006-01-07

The study of highly conjugated, carbonyl-containing molecules such as 1,4,5,8-naphthalene tetracarboxylic dianhydride, III, is of interest since reactivity differences and transmission of electronic effects through the conjugated framework can be evidenced. The kinetics of hydrolysis of III in aqueous solution were determined from 5 M acid to pH 10. In basic solution hydrolysis of III yields, sequentially, 1,4,5,8-naphthalene diacid monoanhydride, II, and 1,4,5,8-naphthalene tetracarboxylic acid, I. The second order rate constant for alkaline hydrolysis is 200 fold higher for the first ring opening. The water-catalyzed hydrolysis of III yields a pH-dependent mixture of ionic forms of I and II. The rate constant for water-catalyzed hydrolysis of III is 25 fold higher than that for II. In concentrated acid the rates for reaching equilibrium (I, II and III) increase and III is the major product. The pK(a)s of I (3.24, 5.13 and 6.25) and II (3.05, 5.90) were determined by potentiometric, fluorescence and UV spectroscopy titrations and by quantitative fit of the kinetic and equilibrium data. The apparent, pH-dependent, equilibrium constants, K(EqII), for anhydride formation between I and II were obtained from the UV spectra. The quantitative fit of kinetic and equilibrium data are consistent with the assumption that anhydride formation only proceeds with the fully protonated species for both I and II and permitted the estimation of the equilibrium constants for anhydride formation, K(EqII). The value of K(EqII) (I <==> II) between pH 1 and 6 was ca. 5. Geometry optimization calculations in the gas phase of the reactions of III in alkaline, neutral and acid conditions, at the DFT level of theory, gave electronic distributions that were qualitatively consistent with the experimental results.

Poly(N-vinyl-2-pyrrolidone)-stabilized palladium-platinum nanoparticles-catalyzed hydrolysis of ammonia borane for hydrogen generation

NASA Astrophysics Data System (ADS)

Rakap, Murat

2015-02-01

The catalytic use of highly efficient poly(N-vinyl-2-pyrrolidone)-stabilized palladium-platinum nanoparticles (4.2 ± 1.9 nm) in the hydrolysis of ammonia-borane is reported. The catalyst is prepared by co-reduction of two metal ions in ethanol/water mixture by an alcohol reduction method and characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and UV-Vis spectroscopy. They are recyclable and highly active for hydrogen generation from the hydrolysis of ammonia-borane even at very low concentrations and temperature, providing a record numbers of average turnover frequency value (125 mol H2/mol cat.min-1) and maximum hydrogen generation rate (3468 L H2 min-1 (mol cat)-1). They also provide activation energy of 51.7 ± 2 kJ/mol for the hydrolysis of ammonia borane.

Study of the kinetic parameters for synthesis and hydrolysis of pharmacologically active salicin isomer catalyzed by baker's yeast maltase

NASA Astrophysics Data System (ADS)

Veličković, D. V.; Dimitrijević, A. S.; Bihelović, F. J.; Jankov, R. M.; Milosavić, N.

2011-12-01

One of the key elements for understanding enzyme reactions is determination of its kinetic parameters. Since transglucosylation is kinetically controlled reaction, besides the reaction of synthesis, very important is the reaction of enzymatic hydrolysis of created product. Therefore, in this study, kinetic parameters for synthesis and secondary hydrolysis of pharmacologically active α isosalicin by baker's yeast maltase were calculated, and it was shown that specifity of maltase for hydrolysis is approximately 150 times higher then for synthesis.

Management of allergy to penicillins and other beta-lactams.

PubMed

Mirakian, R; Leech, S C; Krishna, M T; Richter, A G; Huber, P A J; Farooque, S; Khan, N; Pirmohamed, M; Clark, A T; Nasser, S M

2015-02-01

The Standards of Care Committee of the British Society for Allergy and Clinical Immunology (BSACI) and an expert panel have prepared this guidance for the management of immediate and non-immediate allergic reactions to penicillins and other beta-lactams. The guideline is intended for UK specialists in both adult and paediatric allergy and for other clinicians practising allergy in secondary and tertiary care. The recommendations are evidence based, but where evidence is lacking, the panel reached consensus. During the development of the guideline, all BSACI members were consulted using a Web-based process and all comments carefully considered. Included in the guideline are epidemiology of allergic reactions to beta-lactams, molecular structure, formulations available in the UK and a description of known beta-lactam antigenic determinants. Sections on the value and limitations of clinical history, skin testing and laboratory investigations for both penicillins and cephalosporins are included. Cross-reactivity between penicillins and cephalosporins is discussed in detail. Recommendations on oral provocation and desensitization procedures have been made. Guidance for beta-lactam allergy in children is given in a separate section. An algorithm to help the clinician in the diagnosis of patients with a history of penicillin allergy has also been included. © 2015 John Wiley & Sons Ltd.

Oxidation of β-lactam antibiotics by peracetic acid: Reaction kinetics, product and pathway evaluation.

PubMed

Zhang, Kejia; Zhou, Xinyan; Du, Penghui; Zhang, Tuqiao; Cai, Meiquan; Sun, Peizhe; Huang, Ching-Hua

2017-10-15

Peracetic acid (PAA) is a disinfection oxidant used in many industries including wastewater treatment. β-Lactams, a group of widely prescribed antibiotics, are frequently detected in wastewater effluents and surface waters. The reaction kinetics and transformation of seven β-lactams (cefalexin (CFX), cefadroxil (CFR), cefapirin (CFP), cephalothin (CFT), ampicillin (AMP), amoxicillin (AMX) and penicillin G (PG)) toward PAA were investigated to elucidate the behavior of β-lactams during PAA oxidation processes. The reaction follows second-order kinetics and is much faster at pH 5 and 7 than at pH 9 due to speciation of PAA. Reactivity to PAA follows the order of CFR ∼ CFX > AMP ∼ AMX > CFT ∼ CFP ∼ PG and is related to β-lactam's nucleophilicity. The thioether sulfur of β-lactams is attacked by PAA to generate sulfoxide products. Presence of the phenylglycinyl amino group on β-lactams can significantly influence electron distribution and the highest occupied molecular orbital (HOMO) location and energy in ways that enhance the reactivity to PAA. Reaction rate constants obtained in clean water matrix can be used to accurately model the decay of β-lactams by PAA in surface water matrix and only slightly overestimate the decay in wastewater matrix. Results of this study indicate that the oxidative transformation of β-lactams by PAA can be expected under appropriate wastewater treatment conditions. Copyright © 2017. Published by Elsevier Ltd.

N-Thiolated beta-lactam antibacterials: effects of the N-organothio substituent on anti-MRSA activity.

PubMed

Heldreth, Bart; Long, Timothy E; Jang, Seyoung; Reddy, G Suresh Kumar; Turos, Edward; Dickey, Sonja; Lim, Daniel V

2006-06-01

A study on the structure-activity profiles of N-thiolated beta-lactams 1 is reported which demonstrates the importance of the N-organothio moiety on antibacterial activity. Our results indicate that elongation of the N-alkylthio residue beyond two carbons, or extensive branching within the organothio substituent, diminishes antibacterial effects. Of the derivatives we examined, the N-sec-butylthio beta-lactam derivative 5g possesses the strongest growth inhibitory activity against methicillin-resistant Staphylococcus aureus strains. Sulfur oxidation state is important, as the N-sulfenyl and N-sulfinyl groups provide for the best antibacterial activity, while lactams bearing the N-sulfonyl or N-sulfonic acid functionalities have much weaker or no anti-MRSA properties. Stereochemistry within the organothio chain does not seem to be a significant factor, although for N-sec-butylthio beta-lactams 15a-d, the 3R,4S-lactams 15c, d are more active than the 3S,4R-stereoisomers 15a, b in agar diffusion experiments. The N-methylthio lactams are the most sensitive to the presence of glutathione, followed by N-ethylthio and N-sec-butylthio lactams, which indicates that bioactivity and perhaps bacterial selectivity of the lactams may be related to the amount of organothiols in the bacterial cell. These results support the empirical model for the mechanism of action of the compounds in which the lactam transverses the bacterial membrane to deliver the organothio moiety to its cellular target.

By-Product Carrying Humidified Hydrogen: An Underestimated Issue in the Hydrolysis of Sodium Borohydride.

PubMed

Petit, Eddy; Miele, Philippe; Demirci, Umit B

2016-07-21

Catalyzed hydrolysis of sodium borohydride generates up to four molecules of hydrogen, but contrary to what has been reported so far, the humidified evolved gas is not pure hydrogen. Elemental and spectroscopic analyses show, for the first time, that borate by-products pollute the stream as well as the vessel. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dual beta-lactam therapy for serious Gram-negative infections: is it time to revisit?

PubMed

Rahme, Christine; Butterfield, Jill M; Nicasio, Anthony M; Lodise, Thomas P

2014-12-01

We are rapidly approaching a crisis in antibiotic resistance, particularly among Gram-negative pathogens. This, coupled with the slow development of novel antimicrobial agents, underscores the exigency of redeploying existing antimicrobial agents in innovative ways. One therapeutic approach that was heavily studied in the 1980s but abandoned over time is dual beta-lactam therapy. This article reviews the evidence for combination beta-lactam therapy. Overall, in vitro, animal and clinical data are positive and suggest that beta-lactam combinations produce a synergistic effect against Gram-negative pathogens that rivals that of beta-lactam-aminoglycoside or beta-lactam-fluoroquinolone combination therapy. Although the precise mechanism of improved activity is not completely understood, it is likely attributable to an enhanced affinity to the diverse penicillin-binding proteins found among Gram negatives. The collective data indicate that dual beta-lactam therapy should be revisited for serious Gram-negative infections, especially in light of the near availability of potent beta-lactamase inhibitors, which neutralize the effect of problematic beta-lactamases. Copyright © 2014 Elsevier Inc. All rights reserved.

Case Study of Intrapartum Antibiotic Prophylaxis and Subsequent Postpartum Beta-Lactam Anaphylaxis.

PubMed

Stark, Mary Ann; Ross, Mary Frances; Kershner, Wendy; Searing, Kimberly

2015-01-01

Universal screening for maternal group B Streptococcus (GBS) in the prenatal period has led to administration of intrapartum antibiotic prophylaxis (IAP). Although IAP decreased the rate of early neonatal GBS disease, exposure of childbearing women to penicillin and other beta-lactam antibiotics has increased. Beta-lactam-induced anaphylaxis in the breastfeeding woman during the postpartum period illustrates risk factors for beta-lactam allergy and anaphylaxis. Treatment and nursing implications for this adverse reaction are suggested. © 2015 AWHONN, the Association of Women's Health, Obstetric and Neonatal Nurses.

Can penicillins and other beta-lactam antibiotics be used to treat tuberculosis?

PubMed Central

Chambers, H F; Moreau, D; Yajko, D; Miick, C; Wagner, C; Hackbarth, C; Kocagöz, S; Rosenberg, E; Hadley, W K; Nikaido, H

1995-01-01

An increase in the number of tuberculosis cases caused by multiple-drug-resistant strains of Mycobacterium tuberculosis has stimulated search for new antituberculous agents. Beta-lactam antibiotics, traditionally regarded as ineffective against tuberculosis, merit consideration. Four major penicillin-binding proteins (PBPs) with approximate molecular sizes of 94, 82, 52, and 37 kDa were detected by fluorography of [3H]penicillin-radiolabeled membrane proteins prepared from M. tuberculosis H37Ra. The presence of membrane-associated beta-lactamase precluded the use of membranes for assaying the binding affinities of beta-lactam antibiotics. Therefore, ampicillin affinity chromatography was used to purify these four PBPs from crude membranes in order to assay the binding affinities of beta-lactam antibiotics. Ampicillin, amoxicillin, and imipenem, beta-lactam antibiotics previously reported to be active in vitro against M. tuberculosis, bound to M. tuberculosis PBPs at therapeutically achievable concentrations. Binding of the 94-, 82-, and 52-kDa PBPs, but not the 37-kDa PBP, was associated with antibacterial activity, suggesting that these PBPs are the critical targets. Studies of mycobacterial cell wall permeability, which was assayed with a panel of reference cephalosporins and penicillins with different charge positivities, indicated that the rate of penetration of beta-lactam antibiotics to the target PBPs could not account for resistance. Resistance could be reversed with the beta-lactamase inhibitors clavulanate or sulbactam or could be circumvented by the use of a beta-lactamase-stable drug, imipenem, indicating that mycobacterial beta-lactamase, probably in conjunction with slow penetration, is a major determinant of M. tuberculosis resistance to beta-lactam antibiotics. These findings confirm in vitro data that M. tuberculosis is susceptible to some beta-lactam antibiotics. Further evaluation of these drugs for the treatment of tuberculosis in animal models

Review of Antibiotic and Non-Antibiotic Properties of Beta-lactam Molecules.

PubMed

Ochoa-Aguilar, Abraham; Ventura-Martinez, Rosa; Sotomayor-Sobrino, Marco Antonio; Gómez, Claudia; Morales-Espinoza, María del Rosario

2016-01-01

Beta-lactam molecules are a family of drugs commonly used for their antibiotic properties; however, recent research has shown that several members of this group present a large number of other effects such as neuroprotective, antioxidant, analgesic or immunomodulatory capabilities. These properties have been used in both preclinical and clinical studies in different diseases such as hypoxic neuronal damage or acute and chronic pain. The present work briefly reviews the antibiotic effect of these molecules, and will then focus specially on the non-antibiotic effects of three beta-lactam subfamilies: penicillins, cephalosporins and beta lactamase inhibitors, each of which have different molecular structure and pharmacokinetics and therefore have several potential clinical applications. A thorough search of bibliographic databases for peer-reviewed research was performed including only classic experiments or high quality reviews for the antibiotic mechanisms of beta-lactam molecules and only experimental research papers where included when the non-antibiotic properties of these molecules were searched. Only published articles from indexed journals were included. Quality of retrieved papers was assessed using standard tools. The characteristics of screened papers were described and findings of included studies were contextualized to either a mechanistic or a clinical framework. Seventy-eight papers were included in the review; the majority (56) were relative to the non-antibiotic properties of beta-lactam molecules. The non-antibiotic effects reviewed were divided accordingly to the amount of information available for each one. Twelve papers outlined the epileptogenic effects induced by beta-lactam molecules administration; these included both clinical and basic research as well as probable mechanistic explanations. Eighteen papers described a potential neuroprotective effect, mostly in basic in vitro and in vivo experiments. Analgesic properties where identified in

Anti-MRSA beta-lactams in development, with a focus on ceftobiprole: the first anti-MRSA beta-lactam to demonstrate clinical efficacy.

PubMed

Bush, Karen; Heep, Markus; Macielag, Mark J; Noel, Gary J

2007-04-01

Ceftobiprole is the first of the investigational beta-lactam antibiotics with in vitro activity against methicillin-resistant staphylococci to reach and complete Phase III therapeutic trials. Its antibacterial spectrum includes methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, penicillin-resistant streptococci and many Gram-negative pathogens. It has demonstrated in vivo activity against many experimental infections caused by these pathogens. Ceftobiprole has completed Phase III clinical trials for complicated skin and skin structure infections, is being studied in Phase III pneumonia trials and has demonstrated non-inferiority compared with vancomycin in a Phase III complicated skin and skin structure infections trial, resulting in > 90% clinical cures of infections caused by MRSA. Other anti-MRSA beta-lactams in therapeutic clinical trials include the carbapenem CS-023/RO-4908463 and the cephalosporin ceftaroline (PPI-0903). The future of all of these agents will depend on their clinical efficacy, safety and their ability to be accepted as beta-lactams for the reliable treatment of a broad spectrum of infections, including those caused by MRSA.

Increase of β-Lactam-Resistant Invasive Haemophilus influenzae in Sweden, 1997 to 2010

PubMed Central

Resman, Fredrik; Ristovski, Mikael; Forsgren, Arne; Kaijser, Bertil; Kronvall, Göran; Medstrand, Patrik; Melander, Eva; Odenholt, Inga

2012-01-01

The proportions of Haemophilus influenzae resistant to ampicillin and other β-lactam antibiotics have been low in Sweden compared to other countries in the Western world. However, a near-doubled proportion of nasopharyngeal Swedish H. influenzae isolates with resistance to β-lactams has been observed in the last decade. In the present study, the epidemiology and mechanisms of antimicrobial resistance of H. influenzae isolates from blood and cerebrospinal fluid in southern Sweden from 1997 to 2010 (n = 465) were studied. Antimicrobial susceptibility testing was performed using disk diffusion, and isolates with resistance to any tested β-lactam were further analyzed in detail. We identified a significantly increased (P = 0.03) proportion of β-lactam-resistant invasive H. influenzae during the study period, which was mainly attributed to a significant recent increase of β-lactamase-negative β-lactam-resistant isolates (P = 0.04). Furthermore, invasive β-lactamase-negative β-lactam-resistant H. influenzae isolates from 2007 and onwards were found in higher proportions than the corresponding proportions of nasopharyngeal isolates in a national survey. Multiple-locus sequence typing (MLST) of this group of isolates did not completely separate isolates with different resistance phenotypes. However, one cluster of β-lactamase-negative ampicillin-resistant (BLNAR) isolates was identified, and it included isolates from all geographical areas. A truncated variant of a β-lactamase gene with a promoter deletion, blaTEM-1-PΔ dominated among the β-lactamase-positive H. influenzae isolates. Our results show that the proportions of β-lactam-resistant invasive H. influenzae have increased in Sweden in the last decade. PMID:22687505

Theoretical study of the alkaline hydrolysis of an aza-β-lactam derivative of clavulanic acid

NASA Astrophysics Data System (ADS)

Garcías, Rafael C.; Coll, Miguel; Donoso, Josefa; Muñoz, Francisco

2003-04-01

DFT calculations based on the hybrid functional B3LYP/6-31+G * were used to study the alkaline hydrolysis of an aza-clavulanic acid, which results from the substitution of the carbon atom at position 6 in clavulanic acid by a nitrogen atom. The presence of the nitrogen atom endows the compound with special properties; in fact, once formed, the tetrahedral intermediate can evolve with cleavage of the N 4-C 7 or N 6-C 7 bond, which obviously leads to different reaction products. These differential bond cleavages may play a central role in the inactivation of β-lactamases, so the compound may be a powerful inactivator of these enzymes.

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

PubMed

Stevenson, Bradley J; Waller, Christopher C; Ma, Paul; Li, Kunkun; Cawley, Adam T; Ollis, David L; McLeod, Malcolm D

2015-10-01

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation. Copyright © 2015 John Wiley & Sons, Ltd.

Distinct single-cell morphological dynamics under beta-lactam antibiotics

PubMed Central

Yao, Zhizhong; Kahne, Daniel; Kishony, Roy

2012-01-01

Summary The bacterial cell wall is conserved in prokaryotes, stabilizing cells against osmotic stress. Beta-lactams inhibit cell wall synthesis and induce lysis through a bulge-mediated mechanism; however, little is known about the formation dynamics and stability of these bulges. To capture processes of different timescales, we developed an imaging platform combining automated image analysis with live cell microscopy at high time resolution. Beta-lactam killing of Escherichia coli cells proceeded through four stages: elongation, bulge formation, bulge stagnation and lysis. Both the cell wall and outer membrane (OM) affect the observed dynamics; damaging the cell wall with different beta-lactams and compromising OM integrity cause different modes and rates of lysis. Our results show that the bulge formation dynamics is determined by how the cell wall is perturbed. The OM plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows recovery upon drug removal. PMID:23103254

Enhancement of the Rate of Pyrophosphate Hydrolysis by Nonenzymatic Catalysts and by Inorganic Pyrophosphatase*

PubMed Central

Stockbridge, Randy B.; Wolfenden, Richard

2011-01-01

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, 31P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PPi) in the presence and absence of Mg2+ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPPi2− proceeds with a rate constant of 2.8 × 10−10 s−1, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s−1 under the same conditions. The resulting rate enhancement (2 × 1012-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH‡ = −16.6 kcal/mol). The presence of Mg2+ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼106-fold. Transfer to dimethyl sulfoxide accelerated PPi hydrolysis by reducing the enthalpy of activation. Mg2+ increased the rate of PPi hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation. PMID:21460215

Identification and measurement of beta-lactam antibiotic residues in milk: integration of screening kits with liquid chromatography.

PubMed

Harik-Khan, R; Moats, W A

1995-01-01

A procedure for identifying and quantitating violative beta-lactams in milk is described. This procedure integrates beta-lactam residue detection kits with the multiresidue automated liquid chromatographic (LC) cleanup method developed in our laboratory. Spiked milk was deproteinized, extracted, and subjected to reversed-phase LC using a gradient program that concentrated the beta-lactams. Amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin, and penicillin G were, thus, separated into 5 fractions that were subsequently tested for activity by using 4 kits. beta-lactams in the positive fractions were quantitated by analytical LC methods developed in our laboratory. The LC cleanup method separated beta-lactam antibiotics from each other and from interferences in the matrix and also concentrated the antibiotics, thus increasing the sensitivity of the kits to the beta-lactam antibiotics. The procedure facilitated the task of identifying and measuring the beta-lactam antibiotics that may be present in milk samples.

The bactericidal activity of β-lactam antibiotics is increased by metabolizable sugar species.

PubMed

Thorsing, Mette; Bentin, Thomas; Givskov, Michael; Tolker-Nielsen, Tim; Goltermann, Lise

2015-10-01

Here, the influence of metabolizable sugars on the susceptibility of Escherichia coli to β-lactam antibiotics was investigated. Notably, monitoring growth and survival of mono- and combination-treated planktonic cultures showed a 1000- to 10 000-fold higher antibacterial efficacy of carbenicillin and cefuroxime in the presence of certain sugars, whereas other metabolites had no effect on β-lactam sensitivity. This effect was unrelated to changes in growth rate. Light microscopy and flow cytometry profiling revealed that bacterial filaments, formed due to β-lactam-mediated inhibition of cell division, rapidly appeared upon β-lactam mono-treatment and remained stable for up to 18 h. The presence of metabolizable sugars in the medium did not change the rate of filamentation, but led to lysis of the filaments within a few hours. No lysis occurred in E. coli mutants unable to metabolize the sugars, thus establishing sugar metabolism as an important factor influencing the bactericidal outcome of β-lactam treatment. Interestingly, the effect of sugar on β-lactam susceptibility was suppressed in a strain unable to synthesize the nutrient stress alarmone (p)ppGpp. Here, to the best of our knowledge, we demonstrate for the first time a specific and significant increase in β-lactam sensitivity due to sugar metabolism in planktonic, exponentially growing bacteria, unrelated to general nutrient availability or growth rate. Understanding the mechanisms underlying the nutritional influences on antibiotic sensitivity is likely to reveal new proteins or pathways that can be targeted by novel compounds, adding to the list of pharmacodynamic adjuvants that increase the efficiency and lifespan of conventional antibiotics.

A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen β-lactams

NASA Astrophysics Data System (ADS)

Chen, Yanni; Wang, Yongwei; Liu, Liqiang; Wu, Xiaoling; Xu, Liguang; Kuang, Hua; Li, Aike; Xu, Chuanlai

2015-10-01

A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples.A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively

Alkyl gallates, intensifiers of beta-lactam susceptibility in methicillin-resistant Staphylococcus aureus.

PubMed

Shibata, Hirofumi; Kondo, Kyoko; Katsuyama, Ryo; Kawazoe, Kazuyoshi; Sato, Yoichi; Murakami, Kotaro; Takaishi, Yoshihisa; Arakaki, Naokatu; Higuti, Tomihiko

2005-02-01

We found that ethyl gallate purified from a dried pod of tara (Caesalpinia spinosa) intensified beta-lactam susceptibility in methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus (MRSA and MSSA strains, respectively). This compound and several known alkyl gallates were tested with MRSA and MSSA strains to gain new insights into their structural functions in relation to antimicrobial and beta-lactam susceptibility-intensifying activities. The maximum activity of alkyl gallates against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl gallate, with an MIC at which 90% of the isolates tested were inhibited of 15.6 microg/ml. At concentrations lower than the MIC, alkyl gallates synergistically elevated the susceptibility of MRSA and MSSA strains to beta-lactam antibiotics. Such a synergistic activity of the alkyl gallates appears to be specific for beta-lactam antibiotics, because no significant changes were observed in the MICs of other classes of antibiotics examined in this study. The length of the alkyl chain was also associated with the modifying activity of the alkyl gallates, and the optimum length was C5 to C6. The present work clearly demonstrates that the length of the alkyl chain has a key role in the elevation of susceptibility to beta-lactam antibiotics.

First Penicillin-Binding Protein Occupancy Patterns of β-Lactams and β-Lactamase Inhibitors in Klebsiella pneumoniae.

PubMed

Sutaria, Dhruvitkumar S; Moya, Bartolome; Green, Kari B; Kim, Tae Hwan; Tao, Xun; Jiao, Yuanyuan; Louie, Arnold; Drusano, George L; Bulitta, Jürgen B

2018-06-01

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite β-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae , PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using β-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After β-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC 50 ]) were reported as the β-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by β-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC 50 , <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC 50 , <0.0075 mg/liter) and avibactam (IC 50 , 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC 50 , 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC 50 , 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC 50 , 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double β-lactam and β-lactam-β-lactamase inhibitor combinations. Copyright © 2018 American Society for Microbiology.

Designing Predictive Models for Beta-Lactam Allergy Using the Drug Allergy and Hypersensitivity Database.

PubMed

Chiriac, Anca Mirela; Wang, Youna; Schrijvers, Rik; Bousquet, Philippe Jean; Mura, Thibault; Molinari, Nicolas; Demoly, Pascal

Beta-lactam antibiotics represent the main cause of allergic reactions to drugs, inducing both immediate and nonimmediate allergies. The diagnosis is well established, usually based on skin tests and drug provocation tests, but cumbersome. To design predictive models for the diagnosis of beta-lactam allergy, based on the clinical history of patients with suspicions of allergic reactions to beta-lactams. The study included a retrospective phase, in which records of patients explored for a suspicion of beta-lactam allergy (in the Allergy Unit of the University Hospital of Montpellier between September 1996 and September 2012) were used to construct predictive models based on a logistic regression and decision tree method; a prospective phase, in which we performed an external validation of the chosen models in patients with suspicion of beta-lactam allergy recruited from 3 allergy centers (Montpellier, Nîmes, Narbonne) between March and November 2013. Data related to clinical history and allergy evaluation results were retrieved and analyzed. The retrospective and prospective phases included 1991 and 200 patients, respectively, with a different prevalence of confirmed beta-lactam allergy (23.6% vs 31%, P = .02). For the logistic regression method, performances of the models were similar in both samples: sensitivity was 51% (vs 60%), specificity 75% (vs 80%), positive predictive value 40% (vs 57%), and negative predictive value 83% (vs 82%). The decision tree method reached a sensitivity of 29.5% (vs 43.5%), specificity of 96.4% (vs 94.9%), positive predictive value of 71.6% (vs 79.4%), and negative predictive value of 81.6% (vs 81.3%). Two different independent methods using clinical history predictors were unable to accurately predict beta-lactam allergy and replace a conventional allergy evaluation for suspected beta-lactam allergy. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Photoinduced, copper-catalyzed alkylation of amides with unactivated secondary alkyl halides at room temperature.

PubMed

Do, Hien-Quang; Bachman, Shoshana; Bissember, Alex C; Peters, Jonas C; Fu, Gregory C

2014-02-05

The development of a mild and general method for the alkylation of amides with relatively unreactive alkyl halides (i.e., poor substrates for SN2 reactions) is an ongoing challenge in organic synthesis. We describe herein a versatile transition-metal-catalyzed approach: in particular, a photoinduced, copper-catalyzed monoalkylation of primary amides. A broad array of alkyl and aryl amides (as well as a lactam and a 2-oxazolidinone) couple with unactivated secondary (and hindered primary) alkyl bromides and iodides using a single set of comparatively simple and mild conditions: inexpensive CuI as the catalyst, no separate added ligand, and C-N bond formation at room temperature. The method is compatible with a variety of functional groups, such as an olefin, a carbamate, a thiophene, and a pyridine, and it has been applied to the synthesis of an opioid receptor antagonist. A range of mechanistic observations, including reactivity and stereochemical studies, are consistent with a coupling pathway that includes photoexcitation of a copper-amidate complex, followed by electron transfer to form an alkyl radical.

Beta-lactams in continuous infusion for Gram-negative bacilli osteoarticular infections: an easy method for clinical use.

PubMed

Ribera, Alba; Soldevila, Laura; Rigo-Bonnin, Raul; Tubau, Fe; Padullés, Ariadna; Gómez-Junyent, Joan; Ariza, Javier; Murillo, Oscar

2018-04-01

Continuous infusion (CI) of beta-lactams could optimize their pharmacokinetic/pharmacodynamic indices, especially in difficult-to-treat infections. To validate an easy-to-use method to guide beta-lactams dosage in CI (formula). A retrospective analysis was conducted of a prospectively collected cohort (n = 24 patients) with osteoarticular infections caused by Gram-negative bacilli (GNB) managed with beta-lactams in CI. Beta-lactams dose was calculated using a described formula (daily dose = 24 h × beta-lactam clearance × target "steady-state" concentration) to achieve concentrations above the MIC. We correlated the predicted concentration (C pred  = daily dose/24 h × beta-lactam clearance) with the patient's observed concentration (C obs ) measured by UPLC-MS/MS (Spearman's coefficient). The most frequent microorganism treated was P. aeruginosa (21 cases; 9 MDR). Beta-lactams in CI were ceftazidime (n = 14), aztreonam (7), and piperacillin/tazobactam (3), mainly used in combination (12 with colistin, 5 with ciprofloxacin) and administered without notable side effects. The plasma C obs was higher overall than C pred ; the Spearman correlation between both concentrations was rho = 0.6 (IC 95%: 0.2-0.8) for all beta-lactams, and rho = 0.8 (IC 95%: 0.4-1) for those treated with ceftazidime. The formula may be useful in clinical practice for planning the initial dosage of beta-lactams in CI, while we await a systematic therapeutic drug monitoring. The use of beta-lactams in CI was safe.

Synergy between Ursolic and Oleanolic Acids from Vitellaria paradoxa Leaf Extract and β-Lactams against Methicillin-Resistant Staphylococcus aureus: In Vitro and In Vivo Activity and Underlying Mechanisms.

PubMed

Catteau, Lucy; Reichmann, Nathalie T; Olson, Joshua; Pinho, Mariana G; Nizet, Victor; Van Bambeke, Françoise; Quetin-Leclercq, Joëlle

2017-12-16

Combining antibiotics with resistance reversing agents is a key strategy to overcome bacterial resistance. Upon screening antimicrobial activities of plants used in traditional medicine, we found that a leaf dichloromethane extract from the shea butter tree ( Vitellaria paradoxa ) had antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with further evidence of synergy when combined with β-lactams. Using HPLC-MS, we identified ursolic (UA) and oleanolic acids (OA) in leaves and twigs of this species, and quantified them by HPLC-UV as the major constituents in leaf extracts (21% and 6% respectively). Both pure triterpenic acids showed antimicrobial activity against reference and clinical strains of MRSA, with MICs ranging from 8-16 mg/L for UA to 32-128 mg/L for OA. They were highly synergistic with β-lactams (ampicillin and oxacillin) at subMIC concentrations. Reversion of MRSA phenotype was attributed to their capacity to delocalize PBP2 from the septal division site, as observed by fluorescence microscopy, and to disturb thereby peptidoglycan synthesis. Moreover, both compounds also inhibited β-lactamases activity of living bacteria (as assessed by inhibition of nitrocefin hydrolysis), but not in bacterial lysates, suggesting an indirect mechanism for this inhibition. In a murine model of subcutaneous MRSA infection, local administration of UA was synergistic with nafcillin to reduce lesion size and inflammatory cytokine (IL-1β) production. Thus, these data highlight the potential interest of triterpenic acids as resistance reversing agents in combination with β-lactams against MRSA.

Beta-lactam antibiotics during pregnancy: a cross-sectional comparative study Zagreb-Novi Sad.

PubMed

Erić, M; Leppée, M; Sabo, A; Culig, J

2012-01-01

During pregnancy, a number of changes occur in women's body, and some medications are safe and some are not. The aim of our study was to establish the possible correlation between use of beta-lactam antibiotics in pregnancy and occurrence of congenital malformations. The study included 893 pregnant women from Zagreb and 6099 pregnant women from Novi Sad. 527 pregnant women used beta-lactams. First part of the study (one month study) was performed at four maternity hospitals in Zagreb, Croatia. Second part were collected as a part of the study analysing the teratogenicity of drugs used in pregnancy, a longitudinal study performed in Novi Sad district. Pregnant women most frequently used antibacterial agents in the first trimester of pregnancy. They used 15 different antibacterial medications, most often beta-lactams. In Zagreb arm, out of the total number of pregnant women that used medications during pregnancy (859), 231 (26.9%) used beta-lactam antibiotics. Malformations were detected in 8 (3.5%) cases. The prevalence of malformations in newborns whose mothers did not take beta-lactam antibiotics in pregnancy (662) was 2.7% (18 newborns with malformations). In Novi Sad arm, out of the total number of pregnant women that used medications during pregnancy (2013), 296 (14.7%) used beta-lactam antibiotics. Malformations were detected in 14 (4.7%) cases. The prevalence of malformations in newborns whose mothers did not take beta-lactam antibiotics in pregnancy (5803) was 1.7% (99 newborns with malformations). The results show possible teratogenic potential even with those antibacterials which are considered safe (amoxicillin) but as those are usually minor malformations they often pass undetected. International pharmacoepidemiological studies of drug use in pregnancy could substantially contribute to the improvement of pharmacotherapy, and could be of great help in assessing the fetal risks.

Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

PubMed Central

Ah-Seng, Yoan; Rech, Jérôme; Lane, David; Bouet, Jean-Yves

2013-01-01

Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability. PMID:24367270

Beta-lactams and their potential use as novel anticancer chemotherapeutics drugs.

PubMed

Kuhn, Deborah; Coates, Cristina; Daniel, Kenyon; Chen, Di; Bhuiyan, Mohammad; Kazi, Aslamuzzaman; Turos, Edward; Dou, Q Ping

2004-09-01

The discovery of natural and synthetic antibiotics is one of the most important medical breakthroughs in human history. Many diseases, such as bacterial meningitis, pneumonia, and septicemia, are now curable with the use of antibiotics. Antibiotics are efficacious, generally well tolerated in patients, and have a low toxicity level. It is for these reasons antibiotics remain an attractive target for drug discovery. Traditional beta-lactam antibiotics (e.g. penicillins, penems, cephalosporins) have a bicyclic ring structure that is conformationally rigid and functions to inhibit bacterial cell wall synthesis. In addition to the bactericidal action of antibiotics, it has been discovered that many antibiotics are capable of inhibiting tumor cell growth. There are currently many antitumor antibiotics approved for cancer therapy, which work to inhibit tumor cell growth by DNA intercalation. The use of beta-lactams as prodrugs has also met with success by aiding delivery of the chemotherapeutic directly to tumor sites. Recently, a novel class of N-thiolated monobactams, so termed because they possess a monocyclic ring instead of the bicyclic ring, has been found to induce apoptosis potently and specifically in many tumor cell lines but not in normal, non-transformed cell lines. Other beta-lactams, such as the polyaromatics, have been found to slow or inhibit tumor cell growth, and the 4-alkylidene beta-lactams are capable of inhibiting matrix metalloproteinases and leukocyte elactase activity. These data indicate that synthesis and evaluation of beta-lactams are a promising area for further development in anticancer research.

Kinetics study of palm oil hydrolysis using immobilized lipase Candida rugosa in packed bed reactor.

PubMed

Min, C S; Bhatia, S; Kamaruddin, A H

1999-01-01

Continuous hydrolysis of palm oil triglyceride in organic solvent using immobilized Candida rugosa on the Amberlite MB-1 as a source of immobilized lipase was studied in packed bed reactor. The enzymatic kinetics of hydrolysis reaction was studied by changing the substrate concentration, reaction temperature and residence time(tau) in the reactor. At 55 degrees C, the optimum water concentration was found to be 15 % weight per volume of solution (%w/v). The Michaelis-Menten kinetic model was used to obtain the reaction parameters, Km(app) and V max(app). The activation energies were found to be quite low indicating that the lipase-catalyzed process is controlled by diffusion of substrates. The Michaelis-Menten kinetic model was found to be suitable at low water concentration 10-15 %w/v of solution. At higher water concentration, substrate inhibition model was used for data analysis. Reactor operation was found to play an important role in the palm oil hydrolysis kinetic.

Impact of a pharmacist-driven beta-lactam allergy interview on inpatient antimicrobial therapy: A pilot project.

PubMed

Sigona, Nicholas S; Steele, Jeffrey M; Miller, Christopher D

To determine the impact of a pharmacist-driven beta-lactam allergy interview on antimicrobial therapy. Tertiary care academic medical center. Clarification of beta-lactam allergy may expand treatment options for patients and potentially improve outcomes, reduce toxicity, and reduce costs. At our institution, a pilot service using a pharmacy resident and infectious diseases clinical pharmacist was implemented to clarify beta-lactam allergy information and, where appropriate, recommend a change to the patient's antibiotic therapy. Adult patients with a documented beta-lactam allergy who had received non-penicillin antibiotics and who had undergone a beta-lactam allergy interview were identified via pharmacy intervention data. A pharmacist interviewed these patients with the use of an internally developed allergy questionnaire. Recommendations for beta-lactam therapy were made to the patient's primary medical team based on the results of the allergy interview and factors including infection type and culture results. The primary objectives were to determine the percentage of patients successfully switched to beta-lactam therapy as a result of the drug allergy interview, to identify allergy discrepancies between the electronic medical record (EMR) and pharmacist's interview, and to quantify the acceptance rate of the pharmacist's antimicrobial recommendations after drug allergy clarification. Thirty-two patients were interviewed, and 24 were candidates for a beta-lactam recommendation. As a result of the interview, 21 patients (65.6%) were successfully switched from a non-penicillin antibiotic to a cephalosporin, carbapenem, or penicillin. A discrepancy between the EMR-reported allergy and history obtained on interview was identified in 11 patients (34.4%). Medical providers accepted 87.5% of pharmacists' antimicrobial recommendations. A pharmacist-driven beta-lactam allergy interview was effective in switching eligible patients to beta-lactam therapy and identifying

Enantioselective Synthesis of Medium-Sized Lactams via Chiral α,β-Unsaturated Acylammonium Salts.

PubMed

Kang, Guowei; Yamagami, Masaki; Vellalath, Sreekumar; Romo, Daniel

2018-04-06

Medium-sized lactams are important structural motifs found in a variety of bioactive compounds and natural products but are challenging to prepare, especially in optically active form. A Michael addition/proton transfer/lactamization organocascade process is described that delivers medium-sized lactams, including azepanones, benzazepinones, azocanones, and benzazocinones, in high enantiopurity through the intermediacy of chiral α,β-unsaturated acylammonium salts. An unexpected indoline synthesis was also uncovered, and the benzazocinone skeleton was transformed into other complex heterocyclic derivatives, including spiroglutarimides, isoquinolinones, and δ-lactones. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzymatic hydrolysis of esters containing a tetrazole ring.

PubMed

Łukowska-Chojnacka, Edyta; Mierzejewska, Jolanta

2014-12-01

The lipase-catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4-(5-aryl-2H-tetrazol-2yl)butan-2-ol and 1-(5-aryl-2H-tetrazol-2yl)-propan-2-ol and their acetates with the highest optical purities (ee = 95%-99%) and excellent enantioselectivity (E>100). Some of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mixtures of 4-[5-(4-chlorophenyl)-2H-tetrazol-2-yl)butan-2-ol as well as pure enantiomers of this compound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes, and A. niger. © 2014 Wiley Periodicals, Inc.

Automated three-component synthesis of a library of γ-lactams

PubMed Central

Fenster, Erik; Hill, David; Reiser, Oliver

2012-01-01

Summary A three-component method for the synthesis of γ-lactams from commercially available maleimides, aldehydes, and amines was adapted to parallel library synthesis. Improvements to the chemistry over previous efforts include the optimization of the method to a one-pot process, the management of by-products and excess reagents, the development of an automated parallel sequence, and the adaption of the method to permit the preparation of enantiomerically enriched products. These efforts culminated in the preparation of a library of 169 γ-lactams. PMID:23209515

Species differences in the hydrolysis of 2-cyanoethylene oxide, the epoxide metabolite of acrylonitrile.

PubMed

Kedderis, G L; Batra, R

1993-04-01

The carcinogenic effects of acrylonitrile in rats are believed to be mediated by its DNA-reactive epoxide metabolite, 2-cyanoethylene oxide (CEO). Previous studies have shown that conjugation with glutathione is the major detoxication pathway for both acrylonitrile and CEO. This study investigated the role of epoxide hydrolase in the hydrolysis of CEO by HPLC analysis of the products from [2,3-14C]CEO. CEO is a relatively stable epoxide with a half-life of 99 min at 37 degrees C in sodium phosphate buffer (0.1 M), pH 7.3. Incubation with hepatic microsomes or cytosols from male F-344 rats or B6C3F1 mice did not enhance the rate of hydrolysis of CEO (0.69 nmol/min). Human hepatic microsomes significantly increased the rate of hydrolysis of CEO, whereas human hepatic cytosols did not. Human hepatic microsomal hydrolysis activity was heat-sensitive and potently inhibited by 1,1,1-trichloropropene oxide (IC50 of 23 microM), indicating that epoxide hydrolase was the catalyst. The hydrolysis of CEO catalyzed by hepatic microsomes from six individuals exhibited normal saturation kinetics with KM ranging from 0.6 to 3.2 mM and Vmax from 8.3 to 18.8 nmol hydrolysis products/min/mg protein. Pretreatment of rodents with phenobarbital or acetone induced hepatic microsomal hydrolysis activity toward CEO, whereas treatment with beta-naphthoflavone, dexamethasone or acrylonitrile itself was without effect. These data show that humans possess an additional detoxication pathway for CEO that is not active in rodents (but is inducible). The presence of an active epoxide hydrolase hydrolysis activity toward CEO in humans should be considered in assessments of cancer risk from acrylonitrile exposure.

Energetic, Structural, and Antimicrobial Analyses of [beta]-Lactam Side Chain Recognition by [beta]-Lactamases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caselli, E.; Powers, R.A.; Blaszczak, L.C.

2010-03-05

Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these {beta}-lactams, most often through bacterial expression of {beta}-lactamases, threatens public health. To understand how {beta}-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because {beta}-lactams form covalent adducts with {beta}-lactamases. This has complicated functional analyses and inhibitor design. To investigate the contribution to interaction energy of the key amide (R1) side chain of {beta}-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well asmore » four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine {beta}-lactamases. Therefore, binding energies can be calculated directly from K{sub i} values. The K{sub i} values measured span four orders of magnitude against the Group I {beta}-lactamase AmpC and three orders of magnitude against the Group II {beta}-lactamase TEM-1. The acylglycineboronic acids have K{sub i} values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of {beta}-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of {beta}-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to {beta}-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 {angstrom} and 1.75 {angstrom} resolution; these structures suggest interactions that are important to the affinity of the inhibitors. Acylglycineboronic acids allow us to begin to dissect interaction energies between {beta}-lactam side chains and {beta

Reactivity of Dimeric Tetrazirconium(IV) Wells-Dawson Polyoxometalate toward Dipeptide Hydrolysis Studied by a Combined Experimental and Density Functional Theory Approach.

PubMed

Ly, Hong Giang T; Mihaylov, Tzvetan; Absillis, Gregory; Pierloot, Kristine; Parac-Vogt, Tatjana N

2015-12-07

Detailed kinetic studies on the hydrolysis of glycylglycine (Gly-Gly) in the presence of the dimeric tetrazirconium(IV)-substituted Wells-Dawson-type polyoxometalate Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4] · 57H2O (1) were performed by a combination of (1)H, (13)C, and (31)P NMR spectroscopies. The catalyst was shown to be stable under a broad range of reaction conditions. The effect of pD on the hydrolysis of Gly-Gly showed a bell-shaped profile with the fastest hydrolysis observed at pD 7.4. The observed rate constant for the hydrolysis of Gly-Gly at pD 7.4 and 60 °C was 4.67 × 10(-7) s(-1), representing a significant acceleration as compared to the uncatalyzed reaction. (13)C NMR data were indicative for coordination of Gly-Gly to 1 via its amide oxygen and amine nitrogen atoms, resulting in a hydrolytically active complex. Importantly, the effective hydrolysis of a series of Gly-X dipeptides with different X side chain amino acids in the presence of 1 was achieved, and the observed rate constant was shown to be dependent on the volume, chemical nature, and charge of the X amino acid side chain. To give a mechanistic explanation of the observed catalytic hydrolysis of Gly-Gly, a detailed quantum-chemical study was performed. The theoretical results confirmed the nature of the experimentally suggested binding mode in the hydrolytically active complex formed between Gly-Gly and 1. To elucidate the role of 1 in the hydrolytic process, both the uncatalyzed and the polyoxometalate-catalyzed reactions were examined. In the rate-determining step of the uncatalyzed Gly-Gly hydrolysis, a carboxylic oxygen atom abstracts a proton from a solvent water molecule and the nascent OH nucleophile attacks the peptide carbon atom. Analogous general-base activity of the free carboxylic group was found to take place also in the case of polyoxometalate-catalyzed hydrolysis as the main catalytic effect originates from the -C═O···Zr(IV) binding.

Effective, Facile, and Selective Hydrolysis of the Chemical Warfare Agent VX Using Zr6-Based Metal-Organic Frameworks.

PubMed

Moon, Su-Young; Wagner, George W; Mondloch, Joseph E; Peterson, Gregory W; DeCoste, Jared B; Hupp, Joseph T; Farha, Omar K

2015-11-16

The nerve agent VX is among the most toxic chemicals known to mankind, and robust solutions are needed to rapidly and selectively deactivate it. Herein, we demonstrate that three Zr6-based metal-organic frameworks (MOFs), namely, UiO-67, UiO-67-NH2, and UiO-67-N(Me)2, are selective and highly active catalysts for the hydrolysis of VX. Utilizing UiO-67, UiO-67-NH2, and UiO-67-N(Me)2 in a pH 10 buffered solution of N-ethylmorpholine, selective hydrolysis of the P-S bond in VX was observed. In addition, UiO-67-N(Me)2 was found to catalyze VX hydrolysis with an initial half-life of 1.8 min. This half-life is nearly 3 orders of magnitude shorter than that of the only other MOF tested to date for hydrolysis of VX and rivals the activity of the best nonenzymatic materials. Hydrolysis utilizing Zr-based MOFs is also selective and facile in the absence of pH 10 buffer (just water) and for the destruction of the toxic byproduct EA-2192.

Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting β-Lactam Resistance Levels in Streptococcus pneumoniae

PubMed Central

Metcalf, Benjamin J.; Chochua, Sopio; Li, Zhongya; Gertz, Robert E.; Walker, Hollis; Hawkins, Paulina A.; Tran, Theresa; Whitney, Cynthia G.; McGee, Lesley; Beall, Bernard W.

2016-01-01

ABSTRACT β-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of β-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking β-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a “PBP type” based on sequence signatures in the transpeptidase domains (TPDs) of the three critical penicillin-binding proteins (PBPs), PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six β-lactams based on broth microdilution. We found that increased β-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to β-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution) of >98%, category agreement (interpretive results agree) of >94%, a major discrepancy (sensitive isolate predicted as resistant) rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive) rate of <2% for all six β-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different β-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing. PMID:27302760

The rabbit liver microsomal biotransformation of 1,1-dialkylethylenes: enantioface selection of epoxidation and enantioselectivity of epoxide hydrolysis.

PubMed

Bellucci, G; Chiappe, C; Cordoni, A; Marioni, F

1994-01-01

The rabbit liver microsomal biotransformation of alpha-methylstyrene (1a), 2-methyl-1-hexene (1b), 2,4,4-trimethyl-1-pentene (1c), and 1,3,3-trimethyl-1-butene (1d) has been investigated with the aim at establishing the enantioface selection of the cytochrome P-450-promoted epoxidation of the double bond and the enantioselectivity of microsomal epoxide hydrolase(mEH)-catalyzed hydrolysis of the resulting epoxides. GLC on a Chiraldex G-TA (ASTEC) column was used to determine the enantiomeric composition of the products. The epoxides 2 first produced in incubations carried out in the presence of an NADPH regenerating system were not detected, being rapidly hydrolyzed by mEH to diols 3. The enantiomeric composition of the latter showed that no enantioface selection occurred in the epoxidation of 1c and 1d, and a very low (8%) ee of the (R)-epoxide was formed from 1b. Incubation of racemic epoxides 2b-d with the microsomal fraction showed that the mEH-catalyzed hydrolysis of 2c and 2d was practically nonenantioselective, while that of 2b exhibited a selectivity E = 4.9 favoring the hydrolysis of the (S)-enantiomer. A comparison of these results with those previously obtained for linear and branched chain alkyl monosubstituted oxiranes shows that the introduction of the second alkyl substituent suppresses the selectivity of the mEH reaction of the latter and reverses that of the former substrates.

Beta-lactam antibiotics modulate T-cell functions and gene expression via covalent binding to cellular albumin.

PubMed

Mor, Felix; Cohen, Irun R

2013-02-19

Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell-mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in nonobese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin.

Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

PubMed

Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel

2014-11-04

Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar

Exposure to β-lactams results in the alteration of penicillin-binding proteins in Clostridium perfringens.

PubMed

Park, Miseon; Rafii, Fatemeh

2017-06-01

Clostridium perfringens causes a variety of mild to severe infections in humans and other animals. A decrease in the affinity of penicillin-binding protein (PBP) transpeptidases for β-lactams is considered one of the mechanisms of β-lactam resistance in bacteria. Two strains of C. perfringens isolated from bovines and one isolated from a chicken, which had decreased susceptibility to β-lactams, had variations in the amino acid sequences of the central penicillin-binding regions of the PBPs. β-Lactam-resistant mutants of another C. perfringens strain, ATCC 13124, were selected in vitro to determine the effects of exposure to β-lactams on the PBP genes. Cultures of the wild type rapidly developed resistance to penicillin G, cephalothin and ceftriaxone. The susceptibilities of all of the selected mutants to some other β-lactams also decreased. The largest PBP found in C. perfringens, CPF_2395, appeared to be the primary target of all three drugs. Strain resistant to penicillin G had mutation resulting in the substitution of one amino acid within the central penicillin-binding/transpeptidase domain, but the ceftrioxane and cephalothin-resistant strains had mutations resulting in the substitution of two amino acids in this region. The cephalothin-resistant mutant also had additional mutations in the CPF_0340 and CPF_2218 genes in this critical region. No other mutations were observed in the three other PBPs of the in vitro resistant mutants. Resistance development also altered the growth rate and cell morphology of the mutants, so in addition to the PBPs, some other genes, including regulatory genes, may have been affected during the interaction with β-lactam antibiotics. This is the first study showing the effects of β-lactam drugs on the substitution of amino acids in PBPs of C. perfringens and points to the need for studies to detect other unknown alterations affecting the physiology of resistant strains. Published by Elsevier Ltd.

PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli.

PubMed

Sarkar, Sujoy Kumar; Dutta, Mouparna; Chowdhury, Chiranjit; Kumar, Akash; Ghosh, Anindya S

2011-09-01

Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic β-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared β-lactam sensitivity of the mutants and the parent strain. β-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for β-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter β-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored β-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic β-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.

DFT investigations of phosphotriesters hydrolysis in aqueous solution: a model for DNA single strand scission induced by N-nitrosoureas.

PubMed

Liu, Tingting; Zhao, Lijiao; Zhong, Rugang

2013-02-01

DNA phosphotriester adducts are common alkylation products of DNA phosphodiester moiety induced by N-nitrosoureas. The 2-hydroxyethyl phosphotriester was reported to hydrolyze more rapidly than other alkyl phosphotriesters both in neutral and in alkaline conditions, which can cause DNA single strand scission. In this work, DFT calculations have been employed to map out the four lowest activation free-energy profiles for neutral and alkaline hydrolysis of triethyl phosphate (TEP) and diethyl 2-hydroxyethyl phosphate (DEHEP). All the hydrolysis pathways were illuminated to be stepwise involving an acyclic or cyclic phosphorane intermediate for TEP or DEHEP, respectively. The rate-limiting step for all the hydrolysis reactions was found to be the formation of phosphorane intermediate, with the exception of DEHEP hydrolysis in alkaline conditions that the decomposition process turned out to be the rate-limiting step, owing to the extraordinary low formation barrier of cyclic phosphorane intermediate catalyzed by hydroxide. The rate-limiting barriers obtained for the four reactions are all consistent with the available experimental information concerning the corresponding hydrolysis reactions of phosphotriesters. Our calculations performed on the phosphate triesters hydrolysis predict that the lower formation barriers of cyclic phosphorane intermediates compared to its acyclic counter-part should be the dominant factor governing the hydrolysis rate enhancement of DEHEP relative to TEP both in neutral and in alkaline conditions.

QSAR modeling of β-lactam binding to human serum proteins

NASA Astrophysics Data System (ADS)

Hall, L. Mark; Hall, Lowell H.; Kier, Lemont B.

2003-02-01

The binding of beta-lactams to human serum proteins was modeled with topological descriptors of molecular structure. Experimental data was the concentration of protein-bound drug expressed as a percent of the total plasma concentration (percent fraction bound, PFB) for 87 penicillins and for 115 β-lactams. The electrotopological state indices (E-State) and the molecular connectivity chi indices were found to be the basis of two satisfactory models. A data set of 74 penicillins from a drug design series was successfully modeled with statistics: r2=0.80, s = 12.1, q2=0.76, spress=13.4. This model was then used to predict protein binding (PFB) for 13 commercial penicillins, resulting in a very good mean absolute error, MAE = 12.7 and correlation coefficient, q2=0.84. A group of 28 cephalosporins were combined with the penicillin data to create a dataset of 115 beta-lactams that was successfully modeled: r2=0.82, s = 12.7, q2=0.78, spress=13.7. A ten-fold 10% leave-group-out (LGO) cross-validation procedure was implemented, leading to very good statistics: MAE = 10.9, spress=14.0, q2 (or r2 press)=0.78. The models indicate a combination of general and specific structure features that are important for estimating protein binding in this class of antibiotics. For the β-lactams, significant factors that increase binding are presence and electron accessibility of aromatic rings, halogens, methylene groups, and =N- atoms. Significant negative influence on binding comes from amine groups and carbonyl oxygen atoms.

Structural Investigation of a Phosphorylation-Catalyzed, Isoaspartate-Free, Protein Succinimide: Crystallographic Structure of Post-Succinimide His15Asp Histidine-Containing Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Napper, Scott; Prasad, Lata; Delbaere, Louis T.J.

2008-09-08

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through themore » high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.« less

Continuous-Infusion Antipseudomonal Beta-Lactam Therapy in Patients With Cystic Fibrosis

PubMed Central

Prescott, William A.; Gentile, Allison E.; Nagel, Jerod L.; Pettit, Rebecca S.

2011-01-01

Objective: We sought to evaluate the pharmacokinetics, efficacy, safety, stability, pharmacoeconomics, and quality-of-life effects of continuous-infusion antipseudomonal beta-lactam therapy in patients with cystic fibrosis (CF). Data Sources: Literature retrieval was accessed through Medline (from 1950 to December 2010) using the following terms: cystic fibrosis; beta-lactams or piperacillin or ticarcillin or cefepime or ceftazidime or doripenem or meropenem or imipenem/cilastin or aztreonam; continuous infusion or constant infusion; drug stability; economics, pharmaceutical; and quality of life. In addition, reference citations from identified publications were reviewed. Study Selection and Data Extraction: We evaluated all articles in English identified from the data sources. Data Synthesis: Patients with CF often harbor colonies of multidrug-resistant organisms, increasing the risk of suboptimal dosing and failure to meet the time above the minimum inhibitory concentration (T > MIC) pharmacodynamic targets. The pharmacokinetics of continuous-infusion antipseudomonal beta-lactam therapy in CF maintains serum concentrations above the MIC of susceptible strains and is more likely than intermittent infusion to achieve optimal T > MIC targets for some intermediate and resistant strains of Pseudomonas aeruginosa. Three noncomparative and four comparative studies have assessed the efficacy and safety of continuous-infusion antipseudomonal beta-lactam therapy during CF pulmonary exacerbations. Ceftazidime, the most extensively studied antibiotic for continuous infusion in CF, has been shown to improve forced expiratory volume in 1 second (FEV1), to improve forced vital capacity (FVC), and to extend the time between pulmonary exacerbations. Continuous-infusion cefepime has been studied in a small number of patients, and a trend toward improved pulmonary function has been observed. Continuous-infusion antipseudomonal beta-lactam therapy appears to be well tolerated

Selective Transformation of β-Lactam Antibiotics by Peroxymonosulfate: Reaction Kinetics and Nonradical Mechanism.

PubMed

Chen, Jiabin; Fang, Cong; Xia, Wenjun; Huang, Tianyin; Huang, Ching-Hua

2018-02-06

While the β-lactam antibiotics are known to be susceptible to oxidative degradation by sulfate radical (SO 4 •- ), here we report that peroxymonosulfate (PMS) exhibits specific high reactivity toward β-lactam antibiotics without SO 4 •- generation for the first time. Apparent second-order reaction constants (k 2,app ) were determined for the reaction of PMS with three penicillins, five cephalosporins, two carbapenems, and several structurally related chemicals. The pH-dependency of k 2,app could be well modeled based on species-specific reactions. On the basis of reaction kinetics, stoichiometry, and structure-activity assessment, the thioether sulfur, on the six- or five-membered rings (penicillins and cephalosporins) and the side chain (carbapenems), was the main reaction site for PMS oxidation. Cephalosporins were more reactive toward PMS than penicillins and carbapenems, and the presence of a phenylglycine side chain significantly enhanced cephalosporins' reactivity toward PMS. Product analysis indicated oxidation of β-lactam antibiotics to two stereoisomeric sulfoxides. A radical scavenging study and electron paramagnetic resonance (EPR) technique confirmed lack of involvement of radical species (e.g., SO 4 •- ). Thus, the PMS-induced oxidation of β-lactam antibiotics was proposed to proceed through a nonradical mechanism involving direct two-electron transfer along with the heterolytic cleavage of the PMS peroxide bond. The new findings of this study are important for elimination of β-lactam antibiotic contamination, because PMS exhibits specific high reactivity and suffers less interference from the water matrix than the radical process.

Point-of-care Beta-lactam Allergy Skin Testing by Antimicrobial Stewardship Programs: A Pragmatic Multicenter Prospective Evaluation.

PubMed

Leis, Jerome A; Palmay, Lesley; Ho, Grace; Raybardhan, Sumit; Gill, Suzanne; Kan, Tiffany; Campbell, Jackie; Kiss, Alex; McCready, Janine B; Das, Pavani; Minnema, Brian; Powis, Jeff E; Walker, Sandra A N; Ferguson, Heather; Wong, Benny; Weber, Elizabeth

2017-06-01

Beta-lactam allergy skin testing (BLAST) is recommended by antimicrobial stewardship program (ASP) guidelines, yet few studies have systematically evaluated its impact when delivered at point-of-care. We conducted a pragmatic multicenter prospective evaluation of the use of point-of-care BLAST by ASPs. In staggered 3-month intervals, ASP teams at three hospitals received training by allergists to offer BLAST for eligible patients with infectious diseases receiving non-preferred beta-lactam therapy due to severity of their allergy. The primary outcome was the proportion of patients receiving the preferred beta-lactam therapy. Of 827 patients with reported beta-lactam allergy over 15-months, beta-lactam therapy was preferred among 632(76%). During baseline periods, 50% (124/246) received preferred beta-lactam therapy based on history, compared with 60% (232/386) during the intervention periods (p=0.02), which improved further to 81% (313/386) upon provision of BLAST (p<0.001) without any increase in incidence of adverse drug reactions (4% vs. 3%; p=0.4). After adjusting for patient variables and the correlation between hospitals, the intervention period was associated with a 4.5-fold greater odds of receiving preferred beta-lactam therapy (95% CI, 2.4-8.2; p<0.0001). The use of BLAST at the point-of-care across three hospital ASPs resulted in greater use of preferred beta-lactam therapy without increasing the risk of adverse drug reactions. Longer term studies are needed to better assess the safety and clinical impact of this ASP intervention. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Exploring the Scope of Asymmetric Synthesis of β-Hydroxy-γ-lactams via Noyori-type Reductions.

PubMed

Lynch, Denis; Deasy, Rebecca E; Clarke, Leslie-Ann; Slattery, Catherine N; Khandavilli, U B Rao; Lawrence, Simon E; Maguire, Anita R; Magnus, Nicholas A; Moynihan, Humphrey A

2016-10-07

Enantio- and diastereoselective hydrogenation of β-keto-γ-lactams with a ruthenium-BINAP catalyst, involving dynamic kinetic resolution, has been employed to provide a general, asymmetric approach to β-hydroxy-γ-lactams, a structural motif common to several bioactive compounds. Full conversion to the desired β-hydroxy-γ-lactams was achieved with high diastereoselectivity (up to >98% de) by addition of catalytic HCl and LiCl, while β-branching of the ketone substituent demonstrated a pronounced effect on the modest to excellent enantioselectivity (up to 97% ee) obtained.

How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins

PubMed Central

Molitor, Alexander; Bolla, Jean-Michel; Bessonov, Andrey N.; Winterhalter, Mathias; Pagès, Jean-Marie

2009-01-01

Background Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. β-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression. Methodology/Principal Findings Here influx of β-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single β-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of β-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility. Conclusions/Significance We propose the idea of a molecular “passport” that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections. PMID:19434239

β-lactam resistance in gram-negative pathogens isolated from animals.

PubMed

Trott, Darren

2013-01-01

Although β-lactams remain a cornerstone of veterinary therapeutics, only a restricted number are actually approved for use in food-producing livestock in comparison to companion animals and wildlife. Nevertheless, both registered and off-label use of third and fourth-generation cephalosporins in livestock may have influenced the emergence of plasmid-encoded AmpC β-lactamases (pAmpC) (mainly CMY-2) and CTX-M extended-spectrum β-lactamases (ESBLs) in both Gram-negative pathogens and commensals isolated from animals. This presents a public health concern due to the potential risk of transfer of β-lactam-resistant pathogens from livestock to humans through food. The recent detection of pAmpC and ESBLs in multidrug-resistant Enterobacteriaceae isolated from dogs has also confirmed the public health importance of β-lactam resistance in companion animals, though in this case, human-to-animal transmission may be equally as relevant as animal-to-human transmission. Identification of pAmpC and ESBLs in Enterobacteriaceae isolated from wildlife and aquaculture species may be evidence of environmental selection pressure arising from both human and veterinary use of β- lactams. Such selection pressure in animals could be reduced by the availability of reliable alternative control measures such as vaccines, bacteriophage treatments and/or competitive exclusion models for endemic production animal diseases such as colibacillosis. The global emergence and pandemic spread of extraintestinal pathogenic E. coli O25-ST131 strains expressing CTX-M-15 ESBL in humans and its recent detection in livestock, companion animals and wildlife is a major cause for concern and goes against the paradigm that Gramnegative pathogens do not necessarily have to lose virulence in compensation for acquiring resistance.

Subtleties in practical application of prolonged infusion of β-lactam antibiotics.

PubMed

De Waele, Jan J; Lipman, Jeffrey; Carlier, Mieke; Roberts, Jason A

2015-05-01

Prolonged infusion (PI) of β-lactam antibiotics is increasingly used in order to optimise antibiotic exposure in critically ill patients. Physicians are often not aware of a number of subtleties that may jeopardise the treatment. In this clinically based paper, we stress pragmatic issues, such as the importance of a loading dose before PI, and discuss a number of important practicalities that are mandatory to benefit from the pharmacokinetic advantages of prolonged β-lactam antibiotic administration. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Ability of the VITEK 2 Advanced Expert System To Identify β-Lactam Phenotypes in Isolates of Enterobacteriaceae and Pseudomonas aeruginosa

PubMed Central

Sanders, Christine C.; Peyret, Michel; Moland, Ellen Smith; Shubert, Carole; Thomson, Kenneth S.; Boeufgras, Jean-Marc; Sanders, W. Eugene

2000-01-01

The Advanced Expert System (AES) was used in conjunction with the VITEK 2 automated antimicrobial susceptibility test system to ascertain the β-lactam phenotypes of 196 isolates of the family Enterobacteriaceae and the species Pseudomonas aeruginosa. These isolates represented a panel of strains that had been collected from laboratories worldwide and whose β-lactam phenotypes had been characterized by biochemical and molecular techniques. The antimicrobial susceptibility of each isolate was determined with the VITEK 2 instrument, and the results were analyzed with the AES to ascertain the β-lactam phenotype. The results were then compared to the β-lactam resistance mechanism determined by biochemical and molecular techniques. Overall, the AES was able to ascertain a β-lactam phenotype for 183 of the 196 (93.4%) isolates tested. For 111 of these 183 (60.7%) isolates, the correct β-lactam phenotype was identified definitively in a single choice by the AES, while for an additional 46 isolates (25.1%), the AES identified the correct β-lactam phenotype provisionally within two or more choices. For the remaining 26 isolates (14.2%), the β-lactam phenotype identified by the AES was incorrect. However, for a number of these isolates, the error was due to remediable problems. These results suggest that the AES is capable of accurate identification of the β-lactam phenotypes of gram-negative isolates and that certain modifications can improve its performance even further. PMID:10655347

Neutral β-Lactams Inactivate High Molecular Mass Penicillin-Binding Proteins of Class B1, Including PBP2a of MRSA.

PubMed

Dave, Kinjal; Palzkill, Timothy; Pratt, R F

2014-02-13

The targets of β-lactam antibiotics are bacterial DD-peptidases (penicillin-binding proteins). β-Lactam SAR studies over many years have demonstrated the importance of a specifically placed negative charge, usually carboxylate, on these molecules. We show here that neutral analogues of classical β-lactam antibiotics are of comparable activity to the originals against β-lactam-resistant high molecular mass DD-peptidases of the B1 class, a group that includes PBP2a of methicillin-resistant Staphylococcus aureus. These neutral β-lactams may direct new development of antibiotics against certain penicillin-resistant bacteria. These molecules do have antibiotic activity against Gram-positive bacteria.

Is the addition of aminoglycosides to beta-lactams in cancer patients with febrile neutropenia needed?

PubMed

Contreras, Valeria; Sepúlveda, Sebastián; Heredia, Ana

2016-02-24

It is still controversial if the combined use of beta-lactam antibiotics and aminoglycosides has advantages over broad-spectrum beta-lactam monotherapy for the empirical treatment of cancer patients with febrile neutropenia. Searching in Epistemonikos database, which is maintained by screening 30 databases, we identified three systematic reviews including 14 pertinent randomized trials. We combined the evidence using meta-analysis and generated a summary of findings table following the GRADE approach. We concluded the combination of beta-lactam antibiotics and aminoglycosides probably does not lead to a reduced mortality in febrile neutropenic cancer patients and it might increase nephrotoxicity.

A New Pathway for Protein Haptenation by β-Lactams.

PubMed

Pérez-Ruíz, Raúl; Lence, Emilio; Andreu, Inmaculada; Limones-Herrero, Daniel; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

2017-10-09

The covalent binding of β-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of β-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the β-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cyclic Boronates Inhibit All Classes of β-Lactamases

PubMed Central

Cain, Ricky; Wang, David Y.; Lohans, Christopher T.; Wareham, David W.; Oswin, Henry P.; Mohammed, Jabril; Spencer, James; Fishwick, Colin W. G.; McDonough, Michael A.

2017-01-01

ABSTRACT β-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis. PMID:28115348

Phage Conversion for β-Lactam Antibiotic Resistance of Staphylococcus aureus from Foods.

PubMed

Lee, Young-Duck; Park, Jong-Hyun

2016-02-01

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-β-lactamase enzymes that degrade β-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-β-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a β-lactam antibiotic susceptibility test, the transductant was more highly resistant to β-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of β-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.

Hydrolysis of Ketene Catalyzed by Formic Acid: Modification of Reaction Mechanism, Energetics, and Kinetics with Organic Acid Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Louie, Matthew K.; Francisco, Joseph S.; Verdicchio, Marco

2015-05-14

The hydrolysis of ketene (H2C=C=O) to form acetic acid involving two water molecules and also separately in the presence of one to two water molecules and formic acid (FA) was investigated. Our results show that, while the currently accepted indirect mechanism, involving addition of water across the carbonyl C=O bond of ketene to form an ene-diol followed by tautomerization of the ene-diol to form acetic acid, is the preferred pathway when water alone is present, with formic acid as catalyst, addition of water across the ketene C=C double bond to directly produce acetic acid becomes the kinetically favored pathway formore » temperatures below 400 K. We find not only that the overall barrier for ketene hydrolysis involving one water molecule and formic acid (H2C2O + H2O + FA) is significantly lower than that involving two water molecules (H2C2O + 2H(2)O) but also that FA is able to reduce the barrier height for the direct path, involving addition of water across the C=C double bond, so that it is essentially identical with (6.4 kcal/mol) that for the indirect ene-diol formation path involving addition of water across the C=O bond. For the case of ketene hydrolysis involving two water molecules and formic acid (H2C2O + 2H(2)O + FA), the barrier for the direct addition of water across the C=C double bond is reduced even further and is 2.5 kcal/mol lower relative to the ene-diol path involving addition of water across the C=O bond. In fact, the hydrolysis barrier for the H2C2O + 2H(2)O + FA reaction through the direct path is sufficiently low (2.5 kcal/mol) for it to be an energetically accessible pathway for acetic acid formation under atmospheric conditions. Given the structural similarity between acetic and formic acid, our results also have potential implications for aqueous-phase chemistry. Thus, in an aqueous environment, even in the absence of formic acid, though the initial mechanism for ketene hydrolysis is expected to involve addition of water across the

Enzymatic hydrolysis of organic phosphorus in swine manure and soil.

PubMed

He, Zhongqi; Griffin, Timothy S; Honeycutt, C Wayne

2004-01-01

Organic phosphorus (Po) exists in many chemical forms that differ in their susceptibility to hydrolysis and, therefore, bioavailability to plants and microorganisms. Identification and quantification of these forms may significantly contribute to effective agricultural P management. Phosphatases catalyze reactions that release orthophosphate (Pi) from Po compounds. Alkaline phosphatase in tris-HCl buffer (pH 9.0), wheat (Triticum aestivum L.) phytase in potassium acetate buffer (pH 5.0), and nuclease P1 in potassium acetate buffer (pH 5.0) can be used to classify and quantify Po in animal manure. Background error associated with different pH and buffer systems is observed. In this study, we improved the enzymatic hydrolysis approach and tested its applicability for investigating Po in soils, recognizing that soil and manure differ in numerous physicochemical properties. We applied (i) acid phosphatase from potato (Solanum tuberosum L.), (ii) acid phosphatases from both potato and wheat germ, and (iii) both enzymes plus nuclease P1 to identify and quantify simple labile monoester P, phytate (myo-inositol hexakis phosphate)-like P, and DNA-like P, respectively, in a single pH/buffer system (100 mM sodium acetate, pH 5.0). This hydrolysis procedure released Po in sequentially extracted H2O, NaHCO3, and NaOH fractions of swine (Sus scrofa) manure, and of three sandy loam soils. Further refinement of the approach may provide a universal tool for evaluating hydrolyzable Po from a wide range of sources.

Pyrimidine Nucleosides with a Reactive (β-Chlorovinyl)sulfone or (β-Keto)sulfone Group at the C5 Position, Their Reactions with Nucleophiles and Electrophiles, and Their Polymerase-Catalyzed Incorporation into DNA

PubMed Central

2018-01-01

Transition-metal-catalyzed chlorosulfonylation of 5-ethynylpyrimidine nucleosides provided (E)-5-(β-chlorovinyl)sulfones A, which undergo nucleophilic substitution with amines or thiols affording B. The treatment of vinyl sulfones A with ammonia followed by acid-catalyzed hydrolysis of the intermediary β-sulfonylvinylamines gave 5-(β-keto)sulfones C. The latter reacts with electrophiles, yielding α-carbon-alkylated or -sulfanylated analogues D. The 5′-triphosphates of A and C were incorporated into double-stranded DNA, using open and one-nucleotide gap substrates, by human or Escherichia coli DNA-polymerase-catalyzed reactions. PMID:29732453

Magnesium ion catalyzed P-N bond hydrolysis in imidazolide-activated nucleotides - Relevance to template-directed synthesis of polynucleotides

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia; Bernasconi, Claude F.; Doodokyan, Donald L.; Alberas, Diann J.

1989-01-01

Results are presented from a detailed study of the P-N bond hydrolysis in guanosine 5-prime-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-prime-imidazolide (ImpG) in the presence of 0-0.50 M Mg(2+). Pseudo-first-order rate constants of these compounds were obtained as a function of Mg(2+) concentration, for pH values between 6 and 10 and 37 C. It was found that Mg(2+) catalysis was most effective at pH 10, where a 15-fold increase in hydrolysis was achieved in 0.02 M Mg; at 0.2 M, a 115-fold increase was observed. Implication of these results for the mechanism of template-directed oligomerization is discussed.

Catalytic hydrolysis of cellulose into furans

NASA Astrophysics Data System (ADS)

Shi, Chengmei; Tao, Furong; Cui, Yuezhi

2016-12-01

Chromium chloride in 4-(3-methylimidazolium-1-yl)butane-1-sulfonic acid hydrogen sulfate (IL-1) was found to effectively catalyze the hydrolysis of microcrystalline cellulose (MCC) at 150°C for 300 min to achieve 87.8% conversion to a slate of products. With a catalytic amount of CrCl3, the yields of 5-hydroxymethyl furfural (HMF) and furfural were up to 32.4 and 15.2%, respectively, small molecules levulinic acid (LA, 10.8%) and the total reducing sugars (TRS, 10.7%) were also generated. Through LC-MSD analysis and mass spectra, dimer of furan compounds as the main by-products were speculated, and the components of gas products were methane, ethane, CO, CO2, and H2. We suggested that IL-1 and CrCl3 exhibited a coordination interaction; the formation of the intermediate via the hydride shift played a key role in the formation of HMF. The catalyst was recycled and exhibited constant activity for five successive trials.

Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.

PubMed

Chen, Chao; Cao, Ruikai; Shrestha, Ruben; Ward, Christina; Katz, Benjamin B; Fischer, Christopher J; Tomich, John M; Li, Ping

2015-05-15

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.

ATP hydrolysis is critical for induction of conformational changes in GroEL that expose hydrophobic surfaces.

PubMed

Gorovits, B M; Ybarra, J; Horowitz, P M

1997-03-14

The degree of hydrophobic exposure in the molecular chaperone GroEL during its cycle of ATP hydrolysis was analyzed using 1,1'-bis(4-anilino)naphthalene-5,5'disulfonic acid (bisANS), a hydrophobic probe, whose fluorescence is highly sensitive to the environment. In the presence of 10 mM MgCl2 and 10 mM KCl the addition of ATP, but not ADP or AMP-PNP, resulted in a time-dependent, linear increase in the bisANS fluorescence. The rate of the increase in the bisANS fluorescence depended on the concentrations of both GroEL and the probe. The effect could be substantially inhibited by addition of excess ADP or by converting ATP to ADP using hexokinase, showing that the increase in the bisANS fluorescence was correlated with ATP hydrolysis. The rate of ATP hydrolysis catalyzed by GroEL was uncompetitively inhibited in the presence of bisANS. This uncompetitive inhibition suggests that the probe can interact with the GroEL-ATP complex. The inability of the nonhydrolyzable ATP analog, AMP-PNP, to cause a similar effect is explained by the interaction of bisANS with a transient conformational state of GroEL formed consequent to ATP hydrolysis. It is suggested that this short lived hydrophobic exposure reflects a conformational shift in GroEL that results from electrostatic repulsion between the bound products of ATP hydrolysis, and it plays an important role in the mechanism of the chaperonin cycle.

Beta lactam antibiotics residues in cow's milk: comparison of efficacy of three screening tests used in Bosnia and Herzegovina.

PubMed

Fejzic, Nihad; Begagic, Muris; Šerić-Haračić, Sabina; Smajlovic, Muhamed

2014-08-27

Beta lactam antibiotics are widely used in therapy of cattle, particularly for the treatment of mastitis.  Over 95% of residue testing in dairies in Bosnia and Herzegovina is for Beta lactams. The aim of this paper is to compare the efficacy of three most common screening tests for Beta lactam residues in cow's milk in our country. The tests used in the study are SNAP β Lactam test (Idexx), Rosa Charm β Lactam test and Inhibition MRL test. Study samples included: standardized concentrations of penicillin solution (0, 2, 3, 4, 5 and 6 ppb). In addition we tested milk samples from three equal size study groups (not receiving any antibiotic therapy, treated with Beta lactams for mastitis and treated with Beta lactams for diseases other than mastitis). Sensitivity and specificity were determined for each test, using standard penicillin concentrations with threshold value set at concentration of 4 ppb (Maximum residue level - MLR). Additionally we determined proportions of presumably false negative and false positive results for each test using results of filed samples testing. Agreement of test results for each test pair was assessed through Kappa coefficients interpreted by Landis-Koch scale. Detection level of all tests was shown to be well below MRL. This alongside with effects of natural inhibitors in milk contributed to finding of positive results in untreated and treated animals after the withholding period. Screening tests for beta lactam residues are important tools for ensuring that milk for human consumption is free from antibiotics residues.

Consequences of avoiding β-lactams in patients with β-lactam allergies.

PubMed

Jeffres, Meghan N; Narayanan, Prasanna P; Shuster, Jerrica E; Schramm, Garrett E

2016-04-01

The choice of empiric antibiotics for the treatment of gram-negative bacilli (GNB) bloodstream infections (BSIs) in patients presenting with a β-lactam (BL) allergy is often a difficult decision given that these agents are first-line treatment in many guidelines. We sought to compare rates of clinical failure between patients with a history of BL allergy who received either a BL or a non-β-lactam (NBL). Adult patients with a past medical history of BL allergy and receipt of antibiotics for treatment of a GNB BSI were included from 3 academic medical centers. Treatment groups were classified as BL or NBL groups based on the empiric antibiotics received. Clinical failure was assessed 72 to 96 hours after initiation of empiric antibiotics. Hypersensitivity reactions during receipt of antibiotic therapy for the index BSI were recorded. A total of 552 patients were included for analysis: 433 patients in the BL group and 119 patients in the NBL group. Clinical failure was higher in the NBL group compared with the BL group (38.7% vs 27.4%, P = .030). The most common cause of clinical failure was a temperature of greater than 38.0°C 72 to 96 hours after receipt of empiric antibiotics (NBL group: 22.7% vs BL group: 13.9%, P = .016). Hypersensitivity occurred in 16 (2.9%) of 552 patients. Thirteen (2.5%) of 552 patients experiencing hypersensitivity reactions were exposed to a BL during treatment for GNB BSI. Among patients with a BL allergy, use of BL antibiotics is associated with a lower rate of clinical failure. The low rate of hypersensitivity provides further evidence about the risk of cross-reactivity between BL classes. These results support the practice of using a BL from an alternative class for patients in need of gram-negative antibiotic coverage. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Adsorption of acetanilide herbicides on soil and its components. II. Adsorption and catalytic hydrolysis of diethatyl-ethyl on saturated Na(+)-, K(+)-, Ca(2+)-, and Mg(2+)-montmorillonite.

PubMed

Liu, W P; Fang, Z; Liu, H J; Yang, W C

2001-04-01

Adsorption and catalytic hydrolysis of the herbicide diethatyl-ethyl [N-chloroacetyl-N-(2,6-diethylphenyl)glycine ethyl ester] on homoionic Na(+)-, K(+)-, Ca(2+)-, and Mg(2+)-montmorillonite clays were investigated in water solution. The Freundlich adsorption coefficient, Ki, got from isotherms on clay followed the order of Na+ approximately K+ > Mg2+ approximately Ca2+. Analysis of FT-IR spectra of diethatyl-ethyl adsorbed on clay suggests probable bonding at the carboxyl and amide carbonyl groups of the herbicide. The rate of herbicide hydrolysis in homoionic clay suspensions followed the same order as that for adsorption, indicating that adsorption may have preceded and thus caused hydrolysis. Preliminary product identification showed that hydrolysis occurred via nucleophilic substitution at the carboxyl carbon, causing the cleavage of the ester bond and formation of diethatyl and its dechlorinated derivative, and at the amide carbon, yielding an ethyl ester derivative and its acid. These pathways also suggest that hydrolysis of diethatyl-ethyl was catalyzed by adsorption on the clay surface.

Aureoverticillactam, a novel 22-atom macrocyclic lactam from the marine actinomycete Streptomyces aureoverticillatus.

PubMed

Mitchell, Scott S; Nicholson, Benjamin; Teisan, Sy; Lam, Kin S; Potts, Barbara C M

2004-08-01

During the course of our screening program designed to discover novel anticancer and anti-infective agents from marine microorganisms, a strain of Streptomyces aureoverticillatus (NPS001583) isolated from a marine sediment was found to produce a novel macrocyclic lactam with cytotoxicity against various tumor cell lines. Using extensive MS, UV, and NMR spectral analyses, the structure has been established as compound 1, aureoverticillactam, a 22-atom macrocyclic lactam incorporating both triene and tetraene conjugated olefins.

A General LC-MS/MS Method for Monitoring Potential β-Lactam Contamination in Drugs and Drug-Manufacturing Surfaces.

PubMed

Qiu, Chen; Zhu, Hongbin; Ruzicka, Connie; Keire, David; Ye, Hongping

2018-05-15

Penicillins and some non-penicillin β-lactams may cause potentially life-threatening allergic reactions. Thus, possible cross contamination of β-lactams in food or drugs can put people at risk. Therefore, when there is a reasonable possibility that a non-penicillin product could be contaminated by penicillin, the drug products are tested for penicillin contamination. Here, a sensitive and rapid method for simultaneous determination of multiple β-lactam antibiotics using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Mass spectral acquisition was performed on a Q-Exactive HF mass spectrometer in positive ion mode with parallel reaction monitoring (PRM). The method was validated for seven β-lactam antibiotics including one or two from each class and a synthetic intermediate. The quantification precision and accuracy at 200 ppb were in the range of ± 1.84 to ± 4.56 and - 5.20 to 3.44%, respectively. The limit of detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 2 ppb with a linear dynamic range (LDR) of 2-2000 ppb for all eight β-lactams. From various drug products, the recoveries of eight β-lactams at 200 and 2 ppb ranged from 93.8 ± 3.2 to 112.1 ± 4.2% and 89.7 ± 4.6 to 110.6 ± 1.9%, respectively. The application of the method for detecting cross contamination of trace β-lactams (0.2 ppb) and for monitoring facility surface cleaning was also investigated. This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amount of β-lactam contamination, monitoring cross contamination in manufacturing processes, and determining potency for regulatory purposes and for quality control.

Anti-MRSA beta-lactams in development.

PubMed

Page, Malcolm G P

2006-10-01

Ceftobiprole medocaril, the most advanced of the anti-MRSA (methicillin-resistant Staphylococcus aureus) beta-lactams in clinical development, has recently completed its first Phase III clinical trial, and has demonstrated non-inferiority to vancomycin. Phase II clinical trials have been initiated with PPI0903, which is, like ceftobiprole medocaril, an injectable pro-drug of a broad-spectrum cephalosporin with anti-MRSA activity, and with RO4908643, a carbapenem with more modest activity against MRSA.

Hydrolysis of substance P in the presence of the osteosarcoma cell line SaOS-2: release of free amino acids.

PubMed

Cavazza, Antonella; Marini, Mario; Roda, L Giorgio; Tarantino, Umberto; Valenti, Angela

2011-12-01

The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

Antimicrobial Effects of β-Lactams on Imipenem-Resistant Ceftazidime-Susceptible Pseudomonas aeruginosa

PubMed Central

Wi, Yu Mi; Choi, Ji-Young; Lee, Ji-Young; Kang, Cheol-In; Chung, Doo Ryeon; Peck, Kyong Ran; Song, Jae-Hoon

2017-01-01

ABSTRACT We studied the resistance mechanism and antimicrobial effects of β-lactams on imipenem-resistant Pseudomonas aeruginosa isolates that were susceptible to ceftazidime as detected by time-kill curve methods. Among 215 P. aeruginosa isolates from hospitalized patients in eight hospitals in the Republic of Korea, 18 isolates (23.4% of 77 imipenem-resistant isolates) were imipenem resistant and ceftazidime susceptible. Multilocus sequence typing revealed diverse genotypes, which indicated independent emergence. These 18 isolates were negative for carbapenemase genes. All 18 imipenem-resistant ceftazidime-susceptible isolates showed decreased mRNA expression of oprD, and overexpression of mexB was observed in 13 isolates. In contrast, overexpression of ampC, mexD, mexF, or mexY was rarely found. Time-kill curve methods were applied to three selected imipenem-resistant ceftazidime-susceptible isolates at a standard inoculum (5 × 105 CFU/ml) or at a high inoculum (5 × 107 CFU/ml) to evaluate the antimicrobial effects of β-lactams. Inoculum effects were detected for all three β-lactam antibiotics, ceftazidime, cefepime, and piperacillin-tazobactam, against all three isolates. The antibiotics had significant killing effects in the standard inoculum, but no effects in the high inoculum were observed. Our results suggest that β-lactam antibiotics should be used with caution in patients with imipenem-resistant ceftazidime-susceptible P. aeruginosa infection, especially in high-inoculum infections such as endocarditis and osteomyelitis. PMID:28373200

Antimicrobial Effects of β-Lactams on Imipenem-Resistant Ceftazidime-Susceptible Pseudomonas aeruginosa.

PubMed

Wi, Yu Mi; Choi, Ji-Young; Lee, Ji-Young; Kang, Cheol-In; Chung, Doo Ryeon; Peck, Kyong Ran; Song, Jae-Hoon; Ko, Kwan Soo

2017-06-01

We studied the resistance mechanism and antimicrobial effects of β-lactams on imipenem-resistant Pseudomonas aeruginosa isolates that were susceptible to ceftazidime as detected by time-kill curve methods. Among 215 P. aeruginosa isolates from hospitalized patients in eight hospitals in the Republic of Korea, 18 isolates (23.4% of 77 imipenem-resistant isolates) were imipenem resistant and ceftazidime susceptible. Multilocus sequence typing revealed diverse genotypes, which indicated independent emergence. These 18 isolates were negative for carbapenemase genes. All 18 imipenem-resistant ceftazidime-susceptible isolates showed decreased mRNA expression of oprD , and overexpression of mexB was observed in 13 isolates. In contrast, overexpression of ampC , mexD , mexF , or mexY was rarely found. Time-kill curve methods were applied to three selected imipenem-resistant ceftazidime-susceptible isolates at a standard inoculum (5 × 10 5 CFU/ml) or at a high inoculum (5 × 10 7 CFU/ml) to evaluate the antimicrobial effects of β-lactams. Inoculum effects were detected for all three β-lactam antibiotics, ceftazidime, cefepime, and piperacillin-tazobactam, against all three isolates. The antibiotics had significant killing effects in the standard inoculum, but no effects in the high inoculum were observed. Our results suggest that β-lactam antibiotics should be used with caution in patients with imipenem-resistant ceftazidime-susceptible P. aeruginosa infection, especially in high-inoculum infections such as endocarditis and osteomyelitis. Copyright © 2017 American Society for Microbiology.

Validation of the BetaStar® Advanced for Beta-lactams Test Kit for the Screening of Bulk Tank and Tanker Truck Milks for the Presence of Beta-lactam Drug Residues.

PubMed

Denhartigh, Andrew; Reynolds, Lindsay; Palmer, Katherine; Klein, Frank; Rice, Jennifer; Rejman, John J

2018-05-18

A validation study was conducted for an immunochromatographic method (BetaStar ® Advanced for Beta-lactams) for the detection of beta-lactam residues in raw, commingled bovine milk. The assay detected amoxicillin, ampicillin, cloxacillin, penicillin, cephapirin, and ceftiofur below the U.S. Food and Drug Administration tolerance levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. The results of internal and independent laboratory dose-response studies employing spiked samples were in agreement. The test detected all six drugs at the approximate 90/95% sensitivity levels in milk from cows treated with each drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing 1148 control milk samples. Testing the estimated 90/95% sensitivity level for amoxicillin (8.5 ppb), ampicillin (6.9 ppb), cloxacillin (8.9 ppb), penicillin (4.2 ppb), and cephapirin (17.6 ppb), and at 100 ppb for each antibiotic, resulted in 94-100% positive tests for each of the beta-lactam drugs. The results of ruggedness experiments established the operating parameter tolerances for the assay. Cross-reactivity testing established that the assay detects other certain beta-lactam drugs, but it does not cross-react with any of 30 drugs belonging to seven different drug classes. Abnormally high bacterial or somatic cell counts in raw milk produced no assay interference.

Formic acid catalyzed hydrolysis of SO3 in the gas phase: a barrierless mechanism for sulfuric acid production of potential atmospheric importance.

PubMed

Hazra, Montu K; Sinha, Amitabha

2011-11-02

Computational studies at the B3LYP/6-311++G(3df,3pd) and MP2/6-311++G(3df,3pd) levels are performed to explore the changes in reaction barrier height for the gas phase hydrolysis of SO(3) to form H(2)SO(4) in the presence of a single formic acid (FA) molecule. For comparison, we have also performed calculations for the reference reaction involving water assisted hydrolysis of SO(3) at the same level. Our results show that the FA assisted hydrolysis of SO(3) to form H(2)SO(4) is effectively a barrierless process. The barrier heights for the isomerization of the SO(3)···H(2)O···FA prereactive collision complex, which is the rate limiting step in the FA assisted hydrolysis, are found to be respectively 0.59 and 0.08 kcal/mol at the B3LYP/6-311++G(3df,3pd) and MP2/6-311++G(3df,3pd) levels. This is substantially lower than the ~7 kcal/mol barrier for the corresponding step in the hydrolysis of SO(3) by two water molecules--which is currently the accepted mechanism for atmospheric sulfuric acid production. Simple kinetic analysis of the relative rates suggests that the reduction in barrier height facilitated by FA, combined with the greater stability of the prereactive SO(3)···H(2)O···FA collision complex compared to SO(3)···H(2)O···H(2)O and the rather plentiful atmospheric abundance of FA, makes the formic acid mediated hydrolysis reaction a potentially important pathway for atmospheric sulfuric acid production.

Synthesis and anti Methicillin resistant Staphylococcus aureus activity of substituted chalcones alone and in combination with non-beta-lactam antibiotics.

PubMed

Tran, Thanh-Dao; Do, Tuong-Ha; Tran, Ngoc-Chau; Ngo, Trieu-Du; Huynh, Thi-Ngoc-Phuong; Tran, Cat-Dong; Thai, Khac-Minh

2012-07-15

A total of 30 chalcone analogues was synthesized via a base catalyzed Claisen Schmidt condensation and screened for their in vitro antibacterial activity against Methicillin-sensitive Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) alone or in combination with non beta-lactam antibiotics namely ciprofloxacin, chloramphenicol, erythromycin, vancomycin, doxycycline and gentamicin. In the checkerboard technique, fractional inhibitory concentration indices (FICI) show that the following combinations like ciprofloxacin with 25 (4'-bromo-2-hydroxychalcone); doxycycline with 21 (4-hydroxychalcone); doxycycline with 25; and doxycycline with 4 (2',2-dihydroxychalcone) were synergistic against MRSA. In term SAR study, the relationship between chalcone structure and their antibacterial activity against S. aureus and synergy with tested antibiotics were discussed. Possible mechanisms for antibacterial activity of chalcones alone as well as the synergistic effect in combinations were proposed by molecular modeling studies, respectively. Combinations of chalcones with conventional antibiotics could be an effective alternative in the treatment of infection caused by MRSA. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effects of intact glucosinolates and products produced from glucosinolates in myrosinase-catalyzed hydrolysis on the potato cyst nematode (Globodera rostochiensis Cv. Woll).

PubMed

Buskov, S; Serra, B; Rosa, E; Sørensen, H; Sørensen, J C

2002-02-13

The potato cyst nematode (Globodera rostochiensis cv. Woll) is responsible for large yield losses in the potato crop, and opportunities for reducing the attack of these plant nematode species are, therefore, important. This study has been devoted to the testing of the in vitro effects on the potato cyst nematode of eight glucosinolates [prop-2-enyl-, but-3-enyl-, (R)-4-methylsulfinylbut-3-enyl-, benzyl-, phenethyl-, 4-hydroxybenzyl-, (2S)-2-hydroxybut-3-enyl-, and (2R)-2-hydroxy-2-phenylethylglucosinolate] as well as the effects of the products of this myrosinase-catalyzed hydrolysis. The glucosinolates were used at three concentrations, 0.05, 0.3, and 1.0 mg/mL, in the presence or absence of the enzyme myrosinase. The effects of the compounds on the mortality were monitored every 8 h for a 72 h period. No effects were found for any of the intact glucosinolates. However, when active myrosinase was included with 1 mg/mL phenethylglucosinolate at pH 6.5, 100% mortality was observed within just 16 h. A similar effect was achieved at the same concentration of benzyl- and prop-2-enylglucosinolates in the myrosinase-containing solutions, although longer exposures were required (24 and 40 h, respectively). The main aglucone products released from the glucosinolates with pronounced effects on the nematodes were shown to be the corresponding isothiocyanates. The results suggest that mixtures of these specific glucosinolates and active myrosinase or autolysis of plant materials containing these enzymes and glucosinolates might be used to control the potato cyst nematode in the soil.

Hydrolysis optimization and characterization study of preparing fatty acids from Jatropha curcas seed oil.

PubMed

Salimon, Jumat; Abdullah, Bashar Mudhaffar; Salih, Nadia

2011-11-01

Fatty acids (FAs) are important as raw materials for the biotechnology industry. Existing methods of FAs production are based on chemical methods. In this study potassium hydroxide (KOH)-catalyzed reactions were utilized to hydrolysis Jatropha curcas seed oil. The parameters effect of ethanolic KOH concentration, reaction temperature, and reaction time to free fatty acid (FFA%) were investigated using D-Optimal Design. Characterization of the product has been studied using Fourier transforms infrared spectroscopy (FTIR), gas chromatography (GC) and high performance liquid chromatography (HPLC). The optimum conditions for maximum FFA% were achieved at 1.75M of ethanolic KOH concentration, 65°C of reaction temperature and 2.0 h of reaction time. This study showed that ethanolic KOH concentration was significant variable for J. curcas seed oil hydrolysis. In a 18-point experimental design, FFA% of hydrolyzed J. curcas seed oil can be raised from 1.89% to 102.2%, which proved by FTIR and HPLC.

Base-catalyzed hydrolysis and speciation-dependent photolysis of two cephalosporin antibiotics, ceftiofur and cefapirin.

PubMed

Ribeiro, Alyson R; Lutze, Holger V; Schmidt, Torsten C

2018-05-01

Lately, special attention has been given to veterinary cephalosporin antibiotics due to their broad activity spectrum and significant consumption. Indeed, the determination of hydrolytic and photolytic kinetics provides a better comprehension of the undesired persistence of cephalosporins in aqueous matrices. In this work, the two widely used veterinary antibiotics ceftiofur (CEF) and cefapirin (CEPA) showed high instability under alkaline conditions, degrading in few minutes at pH > 11. In buffered solutions at neutral pH and natural temperature (T = 22 ± 1 °C), both drugs presented moderate stability (t½ = 3 d, CEPA and 1.4 d, CEF). Our study also demonstrated that CEPA and CEF speciation did not significantly influence the direct photolysis rates. Using a simulated water disinfection set-up (λ = 254 nm), all ionic species of CEF and CEPA presented fast and similar pseudo-first order degradation rates, k app 0.0095 ± 0.0004 and 0.0092 ± 0.001 cm 2 mJ -1 , respectively. Furthermore, using surface water in hydrolysis experiments, CEF demonstrated significant matrix-dependent stability with a half-life (t½ = 14.7 d) tenfold higher than in buffered solutions. In contrast, CEPA presented a very similar hydrolysis rate in river water (t½ = 4.2 d) and a subtle faster photo-degradation rate in this same matrix (k app 0.0128 ± 0.001 cm 2 mJ -1 ), highlighting the importance of disinfection radiation for cephalosporin depletion in aqueous environments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

PubMed Central

Ruggiero, Melina; Kerff, Frédéric; Herman, Raphaël; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric

2014-01-01

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. PMID:25070104

Synthesis, hydrolysis rates, supercomputer modeling, and antibacterial activity of bicyclic tetrahydropyridazinones.

PubMed

Jungheim, L N; Boyd, D B; Indelicato, J M; Pasini, C E; Preston, D A; Alborn, W E

1991-05-01

Bicyclic tetrahydropyridazinones, such as 13, where X are strongly electron-withdrawing groups, were synthesized to investigate their antibacterial activity. These delta-lactams are homologues of bicyclic pyrazolidinones 15, which were the first non-beta-lactam containing compounds reported to bind to penicillin-binding proteins (PBPs). The delta-lactam compounds exhibit poor antibacterial activity despite having reactivity comparable to the gamma-lactams. Molecular modeling based on semiempirical molecular orbital calculations on a Cray X-MP supercomputer, predicted that the reason for the inactivity is steric bulk hindering high affinity of the compounds to PBPs, as well as high conformational flexibility of the tetrahydropyridazinone ring hampering effective alignment of the molecule in the active site. Subsequent PBP binding experiments confirmed that this class of compound does not bind to PBPs.

Occurrence and Characteristics of a Rapid Exchange of Phosphate Oxygens Catalyzed by Sarcoplasmic Reticulum Vesicles

DOE R&D Accomplishments Database

Kanazawa, T.; Boyer, P. D.

1972-01-01

Sarcoplasmic reticulum vesicles isolated from skeletal muscle actively take up Ca{sup ++} from the medium in the presence of Mg{sup ++} and ATP. This transport is coupled to ATP hydrolysis catalyzed by membrane-bound Ca{sup++}, Mg{sup ++}-ATPase which is activated by concurrent presence of Ca{sup ++} and Mg{sup ++}. Considerable informations have accumulated that give insight into the ATPase and its coupling to the calcium transport. The hydrolysis of ATP by this enzyme occurs through a phosphorylated intermediate. Formation and decomposition of the intermediate show vectorial requirements for Ca{sup ++} and Mg{sup ++}, suggesting an intimate involvement of the intermediate in the transport process. ATP synthesis from P{sub i} and ADP coupled to outflow of Ca{sup ++} from sarcoplasmic reticulum vesicles has recently been demonstrated. This indicates the reversibility of the entire process of calcium transport in sarcoplasmic reticulum vesicles.

Beta lactam antibiotics residues in cow’s milk: comparison of efficacy of three screening tests used in Bosnia and Herzegovina

PubMed Central

Fejzić, Nihad; Begagić, Muris; Šerić-Haračić, Sabina; Smajlović, Muhamed

2014-01-01

Beta lactam antibiotics are widely used in therapy of cattle, particularly for the treatment of mastitis. Over 95% of residue testing in dairies in Bosnia and Herzegovina is for Beta lactams. The aim of this paper is to compare the efficacy of three most common screening tests for Beta lactam residues in cow’s milk in our country. The tests used in the study are SNAP β Lactam test (Idexx), Rosa Charm β Lactam test (Charm Sciences) and Inhibition MRL test (A&M). Study samples included: standardized concentrations of penicillin solution (0, 2, 3, 4, 5 and 6 ppb). In addition we tested milk samples from three equal size study groups (not receiving any antibiotic therapy, treated with Beta lactams for mastitis and treated with Beta lactams for diseases other than mastitis). Sensitivity and specificity were determined for each test, using standard penicillin concentrations with threshold value set at concentration of 4 ppb (Maximum residue level – MLR). Additionally we determined proportions of presumably false negative and false positive results for each test using results of filed samples testing. Agreement of test results for each test pair was assessed through Kappa coefficients interpreted by Landis-Koch scale. Detection level of all tests was shown to be well below MRL. This alongside with effects of natural inhibitors in milk contributed to finding of positive results in untreated and treated animals after the withholding period. Screening tests for beta lactam residues are important tools for ensuring that milk for human consumption is free from antibiotics residues. PMID:25172975

Process development of short-chain polyols synthesis from corn stover by combination of enzymatic hydrolysis and catalytic hydrogenolysis.

PubMed

Fang, Zhen-Hong; Zhang, Jian; Lu, Qi-Ming; Bao, Jie

2014-09-01

Currently short-chain polyols such as ethanediol, propanediol, and butanediol are produced either from the petroleum feedstock or from the starch-based food crop feedstock. In this study, a combinational process of enzymatic hydrolysis with catalytic hydrogenolysis for short-chain polyols production using corn stover as feedstock was developed. The enzymatic hydrolysis of the pretreated corn stover was optimized to produce stover sugars at the minimum cost. Then the stover sugars were purified and hydrogenolyzed into polyols products catalyzed by Raney nickel catalyst. The results show that the yield of short-chain polyols from the stover sugars was comparable to that of the corn-based glucose. The present study provided an important prototype for polyols production from lignocellulose to replace the petroleum- or corn-based polyols for future industrial applications.

Beta-lactam hypersensitivity and cross-reactivity.

PubMed

Terico, Adrienne T; Gallagher, Jason C

2014-12-01

Penicillin is the most frequently reported cause of drug allergy, and cross-reactivity of penicillins with other beta-lactam antibiotics is an area of debate. This review evaluates the available data on immunoglobulin E-mediated penicillin hypersensitivity and cross-reactivity with cephalosporin, carbapenem, and monobactam antibiotics. A MEDLINE search was conducted from 1950 to October 2013, and selected references from review articles were also evaluated. There is a wide variety in reported incidences of cross-reactivity between penicillins and cephalosporins or carbapenems, with early retrospective studies suggesting up to 41.7% and 47.4% cross-reactivity, respectively. Conversely, the use of monobactam antibiotics is frequently employed in the case of a penicillin allergy, as prescribers believe that there is no cross-reactivity between the 2 drug classes. More recent prospective studies suggest that the rates of cross-reactivity with cephalosporins and carbapenems are <5% and <1%, respectively. Similarities in penicillin and cephalosporin side chains may play a role in cross-reactivity between these classes. Cross-reactivity with monobactams is essentially negligible; however, there are some clinical data to support an interaction between ceftazidime and aztreonam, due to the similarity of their side chains. The data reviewed suggest that avoidance of other beta-lactams in patients with type 1 hypersensitivity to penicillins should be reconsidered. © The Author(s) 2014.

Mechanism of Thioesterase-Catalyzed Chain Release in the Biosynthesis of the Polyether Antibiotic Nanchangmycin

PubMed Central

Liu, Tiangang; Lin, Xin; Zhou, Xiufen; Deng, Zixin; Cane, David E.

2008-01-01

Summary The polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates. PMID:18482697

Assays for therapeutic drug monitoring of β-lactam antibiotics: A structured review.

PubMed

Carlier, Mieke; Stove, Veronique; Wallis, Steven C; De Waele, Jan J; Verstraete, Alain G; Lipman, Jeffrey; Roberts, Jason A

2015-10-01

In some patient groups, including critically ill patients, the pharmacokinetics of β-lactam antibiotics may be profoundly disturbed due to pathophysiological changes in distribution and elimination. Therapeutic drug monitoring (TDM) is a strategy that may help to optimise dosing. The aim of this review was to identify and analyse the published literature on the methods used for β-lactam quantification in TDM programmes. Sixteen reports described methods for the simultaneous determination of three or more β-lactam antibiotics in plasma/serum. Measurement of these antibiotics, due to low frequency of usage relative to some other tests, is generally limited to in-house chromatographic methods coupled to ultraviolet or mass spectrometric detection. Although many published methods state they are fit for TDM, they are inconvenient because of intensive sample preparation and/or long run times. Ideally, methods used for routine TDM should have a short turnaround time (fast run-time and fast sample preparation), a low limit of quantification and a sufficiently high upper limit of quantification. The published assays included a median of 6 analytes [interquartile range (IQR) 4-10], with meropenem and piperacillin being the most frequently measured β-lactam antibiotics. The median run time was 8 min (IQR 5.9-21.3 min). There is also a growing number of methods measuring free concentrations. An assay that measures antibiotics without any sample preparation would be the next step towards real-time monitoring; no such method is currently available. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Penicillin skin testing in patients with a history of beta-lactam allergy.

PubMed

del Real, Gonzalo Alvarez; Rose, Mark E; Ramirez-Atamoros, Maria T; Hammel, Jeffrey; Gordon, Steven M; Arroliga, Alejandro C; Arroliga, Mercedes E

2007-04-01

Vancomycin and fluoroquinolones are commonly used in patients with a history of penicillin allergy. To determine the safety and utility of penicillin skin testing (PST). Retrospective study of patients with a history of penicillin allergy between April 1, 1999, and September 30, 2004. Penicillin skin testing was performed by means of standard methods using benzylpenicilloyl-polysine, penicillin G, and histamine and saline controls. Of 596 patients studied, 25.3% were outpatients, 50.3% were inpatients, and 24.3% were intensive care unit patients. The most common antibiotics used during the time of PST were vancomycin and fluoroquinolones. Results of PST were negative in 88.4% of patients, positive in 8.2%, and indeterminate in 3.4%. One patient (0.17%) developed urticaria immediately after PST. Fifty-five percent of patients with negative PST results were changed to a beta-lactam drug, more frequently in the intensive care unit vs the outpatient setting (70.3% vs 8.6%; P < .001) and in adults vs patients younger than 18 years (58.6% vs 8.1%; P < .001). A beta-lactam antibiotic was used in 290 patients with negative PST results. Of the patients given beta-lactam antibiotics, 5 (1.7%) had adverse reactions: 2 had hives after 16 and 20 days of therapy, 1 had a nonspecific rash after 17 days of therapy, 1 had flushing and urticaria 3 hours after a test dose of piperacillin-tazobactam, and 1 had a pruritic rash after 12 hours of therapy. Patients with a history of penicillin allergy can safely use beta-lactam drugs if negative PST results.

[Beta-lactamic antibiotics allergy in cataract surgery. Prevalence and preoperative characteristics of allergic patients].

PubMed

Fernández-Rubio, M E; Cuesta-Rodríguez, T; Urcelay-Segura, J L; Cortés-Valdés, C

2014-03-01

To describe the proportion of patients allergic to β-lactam antibiotics and the prevalence of preoperative conjunctival bacteria among those undergoing cataract surgery in our area. Retrospective cross-sectional study of prevalence of β-lactam allergic patients consecutively scheduled for cataract surgery from 11 July 2005 to November 2012. For studying the prevalence of conjunctival bacteria and clinical characteristics in the patients' preoperative examination, those under 18 years and those with cataract surgery combined with other eye surgeries were excluded. Data from the first preoperative examination of the remaining patients were selected. Clinical data were extracted from the database generated in the evaluation made for anesthetic purposes, and the microbiological data from the laboratory database. Both bases were linked through a patient history code. A comparison was made between the prevalence of conjunctival bacteria and clinical characteristics in allergic and non-allergic patients. From 12,409 adults selected for the bacteriological study, 862 (6.96%) were allergic to β-lactams, their mean age (74.45 years) was higher than that of the non-allergic (P=.005). The proportion of women (71.4%) in the allergic patient group was much higher than that of men. The prevalence of pathogenic bacteria (especially Bacillus spp and Pseudomonas aeruginosa), lung disease and heart failure, was higher in allergic patients. The prevalence of allergy to β-lactams in this study is within the range described in other populations. The higher prevalence of pathogenic bacteria and the predominance of women in those allergic to β-lactams are useful data to guide their surgical prophylaxis. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

β-Lactam Resistance in Haemophilus parasuis Is Mediated by Plasmid pB1000 Bearing blaROB-1▿

PubMed Central

San Millan, Alvaro; Escudero, Jose Antonio; Catalan, Ana; Nieto, Silvia; Farelo, Fidel; Gibert, Magdalena; Moreno, Miguel Angel; Dominguez, Lucas; Gonzalez-Zorn, Bruno

2007-01-01

β-Lactam resistance in Haemophilus parasuis is an emerging phenomenon that has not yet been characterized from a molecular perspective. Clinical high-level β-lactam-resistant isolates from Spain bore a novel plasmid, pB1000, expressing a functionally active ROB-1 β-lactamase. Pulsed-field gel electrophoresis was applied for the first time to H. parasuis and showed that β-lactam resistance is due to clonal spread of a resistant strain, BB1018, bearing pB1000. PMID:17438055

Effective alkaline metal-catalyzed oxidative delignification of hybrid poplar

DOE PAGES

Bhalla, Aditya; Bansal, Namita; Stoklosa, Ryan J.; ...

2016-02-09

Background: Strategies to improve copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment of hybrid poplar were investigated. These improvements included a combination of increasing hydrolysis yields, while simultaneously decreasing process inputs through (i) more efficient utilization of H 2O 2 and (ii) the addition of an alkaline extraction step prior to the metal-catalyzed AHP pretreatment. We hypothesized that utilizing this improved process could substantially lower the chemical inputs needed during pretreatment. Results: Hybrid poplar was pretreated utilizing a modified process in which an alkaline extraction step was incorporated prior to the Cu-AHP treatment step and H 2O 2 was added batch-wise overmore » the course of 10 h. Our results revealed that the alkaline pre-extraction step improved both lignin and xylan solubilization, which ultimately led to improved glucose (86 %) and xylose (95 %) yields following enzymatic hydrolysis. An increase in the lignin solubilization was also observed with fed-batch H 2O 2 addition relative to batch-only addition, which again resulted in increased glucose and xylose yields (77 and 93 % versus 63 and 74 %, respectively). Importantly, combining these strategies led to significantly improved sugar yields (96 % glucose and 94 % xylose) following enzymatic hydrolysis. In addition, we found that we could substantially lower the chemical inputs (enzyme, H 2O 2, and catalyst), while still maintaining high product yields utilizing the improved Cu-AHP process. This pretreatment also provided a relatively pure lignin stream consisting of ≥90 % Klason lignin and only 3 % xylan and 2 % ash following precipitation. Two-dimensional heteronuclear single-quantum coherence (2D HSQC) NMR and size-exclusion chromatography demonstrated that the solubilized lignin was high molecular weight (Mw ≈ 22,000 Da) and only slightly oxidized relative to lignin from untreated poplar. In conclusion: This study demonstrated that the fed

High Surface Area, Thermally Stable, Hydrophobic, Microporous, Rigid Gels Generated at Ambient from MeSi(OEt)3 /(EtO)3 SiCH2 CH2 Si(OEt)3 Mixtures by F- -Catalyzed Hydrolysis.

PubMed

Furgal, Joseph C; Yamane, Honami; Odykirk, Timothy R; Yi, Eongyu; Chujo, Yoshiki; Laine, Richard M

2018-01-02

High surface area materials are of considerable interest for gas storage/capture, molecular sieving, catalyst supports, as well as for slow-release drug-delivery systems. We report here a very simple and fast route to very high surface area, mechanically robust, hydrophobic polymer gels prepared by fluoride-catalyzed hydrolysis of mixtures of MeSi(OEt) 3 and bis-triethoxysilylethane (BTSE) at room temperature. These materials offer specific surface areas up to 1300 m 2  g -1 , peak pore sizes of 0.8 nm and thermal stabilities above 200 °C. The gelation times and surface areas can be controlled by adjusting the solvent volume (dichloromethane), percent fluoride (as nBu 4 NF or TBAF) and the BTSE contents. Polymers with other corners and linkers were also explored. These materials will further expand the materials databank for use in vacuum insulation panels and as thermally stable release and capture media. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic Approaches toward Monocyclic 3‐Amino‐β‐lactams

PubMed Central

Deketelaere, Sari; Van Nguyen, Tuyen; Stevens, Christian V.

2017-01-01

Abstract Due to the emerging resistance against classical β‐lactam‐based antibiotics, a growing number of bacterial infections has become harder to treat. This alarming tendency necessitates continued research on novel antibacterial agents. Many classes of β‐lactam antibiotics are characterized by the presence of the 3‐aminoazetidin‐2‐one core, which resembles the natural substrate of the target penicillin‐binding proteins. In that respect, this Review summarizes the different synthetic pathways toward this key structure for the development of new antibacterial agents. The most extensively applied methods for 3‐amino‐β‐lactam ring formation are discussed, in addition to a few less common strategies. Moreover, approaches to introduce the 3‐amino substituent after ring formation are also covered. PMID:28638759

Stability of Medium-Bridged Twisted Amides in Aqueous Solutions

PubMed Central

Szostak, Michal; Yao, Lei; Aubé, Jeffrey

2012-01-01

“Twisted” amides containing non-standard dihedral angles are typically hypersensitive to hydrolysis, a feature that has stringently limited their utility in water. We have synthesized a series of bridged lactams that contain a twisted amide linkage but which exhibit enhanced stability in aqueous environments. Many of these compounds were extracted unchanged from aqueous mixtures ranging from the strongly basic to the strongly acidic. NMR experiments showed that tricyclic lactams undergo reversible hydrolysis at extreme pH ranges, but that a number of compounds in this structure class are indefinitely stable under physiologically relevant pH conditions; one bicyclic example was additionally water-soluble. We examined the effect of structure on the reversibility of amide bond hydrolysis, which we attributed to the transannular nature of the amino acid analogs. These data suggest that medium-bridged lactams of these types should provide useful platforms for studying the behavior of twisted amides in aqueous systems. PMID:19178141

In Vitro and In Vivo Synergy of the Oxadiazole Class of Antibacterials with β-Lactams.

PubMed

Janardhanan, Jeshina; Meisel, Jayda E; Ding, Derong; Schroeder, Valerie A; Wolter, William R; Mobashery, Shahriar; Chang, Mayland

2016-09-01

The oxadiazole antibacterials target the bacterial cell wall and are bactericidal. We investigated the synergism of ND-421 with the commonly used β-lactams and non-β-lactam antibiotics by the checkerboard method and by time-kill assays. ND-421 synergizes well with β-lactam antibiotics, and it also exhibits a long postantibiotic effect (4.7 h). We also evaluated the in vivo efficacy of ND-421 in a murine neutropenic thigh infection model alone and in combination with oxacillin. ND-421 has in vivo efficacy by itself in a clinically relevant infection model (1.49 log10 bacterial reduction for ND-321 versus 0.36 log10 for linezolid with NRS119) and acts synergistically with β-lactam antibiotics in vitro and in vivo, and the combination of ND-421 with oxacillin is efficacious in a mouse neutropenic thigh methicillin-resistant Staphylococcus aureus (MRSA) infection model (1.60 log10 bacterial reduction). The activity of oxacillin was potentiated in the presence of ND-421, as the strain would have been resistant to oxacillin otherwise. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Reversible Hydrolysis Reaction with the Spore Photoproduct under Alkaline Conditions.

PubMed

Adhikari, Surya; Lin, Gengjie; Li, Lei

2016-09-16

DNA lesions may reduce the electron density at the nucleobases, making them prone to further modifications upon the alkaline treatment. The dominant DNA photolesion found in UV-irradiated bacterial endospores is a thymine dimer, 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP). Here we report a stepwise addition/elimination reaction in the SP hydrolysis product under strong basic conditions where a ureido group is added to the carboxyl moiety to form a cyclic amide, regenerating SP after eliminating a hydroxide ion. Direct amidation of carboxylic acids by reaction with amines in the presence of a catalyst is well documented; however, it is very rare for an amidation reaction to occur without activation. This uncatalyzed SP reverse reaction in aqueous solution is even more surprising because the carboxyl moiety is not a good electrophile due to the negative charge it carries. Examination of the base-catalyzed hydrolyses of two other saturated pyrimidine lesions, 5,6-dihydro-2'-deoxyuridine and pyrimidine (6-4) pyrimidone photoproduct, reveals that neither reaction is reversible even though all three hydrolysis reactions may share the same gem-diol intermediate. Therefore, the SP structure where the two thymine residues maintain a stacked conformation likely provides the needed framework enabling this highly unusual carboxyl addition/elimination reaction.

Towards the design of organocatalysts for nerve agents remediation: The case of the active hydrolysis of DCNP (a Tabun mimic) catalyzed by simple amine-containing derivatives.

PubMed

Barba-Bon, Andrea; Martínez-Máñez, Ramón; Sancenón, Félix; Costero, Ana M; Gil, Salvador; Pérez-Pla, Francisco; Llopis, Elisa

2015-11-15

We report herein a study of the hydrolysis of Tabun mimic DCNP in the presence of different amines, aminoalcohols and glycols as potential suitable organocatalysts for DCNP degradation. Experiments were performed in CD3CN in the presence of 5% D2O, which is a suitable solvent mixture to follow the DCNP hydrolysis. These studies allowed the definition of different DCNP depletion paths, resulting in the formation of diethylphosphoric acid, tetraethylpyrophosphate and phosphoramide species as final products. Without organocatalysts, DCNP hydrolysis occurred mainly via an autocatalysis path. Addition of tertiary amines in sub-stoichiometric amounts largely enhanced DCNP depletion whereas non-tertiary polyamines reacted even faster. Glycols induced very slight increment in the DCNP hydrolysis, whereas DCNP hydrolysis increased sharply in the presence of certain aminoalcohols especially, 2-(2-aminoethylamino)ethanol. For the latter compound, DCNP depletion occurred ca. 80-fold faster than in the absence of organocatalysts. The kinetic studies revealed that DCNP hydrolysis in the presence of 2-(2-aminoethylamino)ethanol occurred via a catalytic process, in which the aminoalcohol was involved. DCNP hydrolysis generally depended strongly on the structure of the amine, and it was found that the presence of the OHCH2CH2N moiety in the organocatalyst structure seems important to induce a fast degradation of DCNP. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel and Recent Synthesis and Applications of β-Lactams

NASA Astrophysics Data System (ADS)

Troisi, Luigino; Granito, Catia; Pindinelli, Emanuela

In this chapter, a comprehensive overview of the most significant and interesting contributions published from 2000 until now, concerning the preparation of novel β-lactam structures is presented. Among the different synthetic strategies available, either novel or already known but efficient and versatile methodologies are covered. The simple modifications of one or more substituents linked to the nitrogen N-1, the C-3, and the C-4 carbon atoms of the β-lactam nucleus were considered as an alternative synthetic protocol of more complex and polyfunctionalized molecules. Indeed, it is well known and extensively reviewed that the biological activity of this strained four-membered heterocycle is strictly dependent on the nature of the substituent groups that affect the reactivity towards the molecular active sites, increasing or lowering the possibility of interaction with the substrates. Finally, a synthetic survey of the most significant biological and pharmacological applications of the 2-azetidinones is reported.

A systematic review on clinical benefits of continuous administration of beta-lactam antibiotics.

PubMed

Roberts, Jason A; Webb, Steven; Paterson, David; Ho, Kwok M; Lipman, Jeffrey

2009-06-01

The clinical benefits of extended infusion or continuous infusion of beta-lactam antibiotics remain controversial. We systematically reviewed the literature to determine whether any clinical benefits exist for administration of beta-lactam antibiotics by extended or continuous infusion. PubMed (January 1950 to November 2007), EMBASE (1966 to November 2007), and the Cochrane Controlled Trial Register were searched (updated November 2007). Randomized controlled trials (RCTs) were meta-analyzed, and observational studies were described by two unblinded reviewers. A total of 846 patients from eligible prospective randomized controlled studies were included in the meta-analysis. Two observational studies were deemed appropriate for description. A meta-analysis of prospective RCTs was undertaken using Review Manager. Among a total of 59 potentially relevant studies, 14 RCTs involving a total of 846 patients from nine countries were deemed appropriate for meta-analysis. The use of continuous infusion of a beta-lactam antibiotic was not associated with an improvement in clinical cure (n = 755 patients; odds ratio: 1.04, 95% confidence interval: 0.74-1.46, p = 0.83, I = 0%) or mortality (n = 541 patients; odds ratio: 1.00, 95% confidence interval: 0.48-2.06, p = 1.00, I = 14.8%). All RCTs except one used a higher antibiotic dose in the bolus administration group. Two observational studies, not pooled because they did not meet the a priori criteria for meta-analysis, showed that beta-lactam administration by extended or continuous infusion was associated with an improvement in clinical cure. The difference in the results between the meta-analysis results and the observational studies could be explained by the bias created by a higher dose of antibiotic in the bolus group in the RCTs and because many of the RCTs only recruited patients with a low acuity of illness. The limited data available suggest that continuous infusion of beta-lactam antibiotics leads to the same

Lactam ergot alkaloids (ergopeptams) as predominant alkaloids in sclerotia of Claviceps purpurea from Norwegian wild grasses.

PubMed

Uhlig, Silvio; Petersen, Dirk

2008-07-01

Four major alkaloids in the extracts from sclerotia of Claviceps purpurea, picked from wild grasses, have been identified as lactam (non-cyclol) ergot alkaloids. The structural information was obtained from ion trap MS and NMR spectroscopy. The data for one of the lactam ergot alkaloids were coinciding with ergocristam [N-(lysergyl-valyl)-cyclo(phenylalanyl-prolyl)]. The structural information of two further lactam alkaloids was suggestive of either alpha- or beta-ergocryptam [N-(lysergyl-valyl)-cyclo(leucyl-prolyl) or N-(lysergyl-valyl)-cyclo(isoleucyl-prolyl)] and ergoannam [N-(lysergyl-leucyl)-cyclo(leucyl-prolyl) or N-(lysergyl-isoleucyl)-cyclo(isoleucyl-prolyl)]. The constitution of the fourth lactam ergot alkaloid corresponded to N-(lysergyl-isoleucyl)-cyclo(phenylalanyl-prolyl), a new ergopeptam, which has not been described before. Additionally, the cyclol-analogue of the new ergopeptam was detected in the extracts and has been identified on the basis of its product ion spectrum from fragmentation of [M+H](+). The study described in this paper shows that lactam ergot alkaloids may not only be minor products of ergopeptine biosynthesis, as has been suggested hitherto, but may be major biosynthetic endproducts for some ergot strains. This is also the first report demonstrating the production of an ergot alkaloid that contains isoleucine as the second amino acid, i.e. the N-(lysergyl-isoleucyl)-moiety, by parasitic, naturally growing C. purpurea. This unusual type of ergot alkaloid has so far only been found in saprophytic cultures of C. purpurea.

Continuous infusion of beta-lactam antibiotics in severe infections: a review of its role.

PubMed

Roberts, Jason A; Paratz, Jennifer; Paratz, Elizabeth; Krueger, Wolfgang A; Lipman, Jeffrey

2007-07-01

Continuous infusion of beta-lactam antibiotics has been widely promoted to optimise their time-dependent activity. Increasing evidence is emerging suggesting potential benefits in patient populations with altered pathophysiology, such as seriously ill patients. From a pharmacokinetic viewpoint, much information supports higher trough concentrations of beta-lactam antibiotics when administered by continuous infusion. This advantage of continuous infusion translates into a superior ability to achieve pharmacodynamic targets, particularly when the minimum inhibitory concentration (MIC) of the pathogen is >or=4 mg/L. One drawback of continuous infusion may be limited physicochemical stability. This issue exists particularly for carbapenem antibiotics whereby prolonged infusions (i.e. >3h) can be used to improve the time above the MIC compared with conventional bolus dosing. Few studies have examined clinical outcomes of bolus and continuous dosing of beta-lactam antibiotics in seriously ill patients. No statistically significant differences have been shown for: mortality; time to normalisation of leukocytosis or pyrexia; or duration of mechanical ventilation, intensive care unit stay or hospital stay. Some evidence suggests improved clinical cure and resolution of illness with continuous infusion in seriously ill patients. Pharmacoeconomic advantages of continuous infusion of beta-lactam antibiotics are well characterised. Available data suggest that seriously ill patients with severe infections requiring significant antibiotic courses (>or=4 days) may be the subgroup that will achieve better outcomes with continuous infusion.

Does prolonged β-lactam infusions improve clinical outcomes compared to intermittent infusions? A meta-analysis and systematic review of randomized, controlled trials

PubMed Central

2011-01-01

Background The emergence of multi-drug resistant Gram-negatives (MDRGNs) coupled with an alarming scarcity of new antibiotics has forced the optimization of the therapeutic potential of available antibiotics. To exploit the time above the minimum inhibitory concentration mechanism of β-lactams, prolonging their infusion may improve outcomes. The primary objective of this meta-analysis was to determine if prolonged β-lactam infusion resulted in decreased mortality and improved clinical cure compared to intermittent β-lactam infusion. Methods Relevant studies were identified from searches of MEDLINE, EMBASE, and CENTRAL. Heterogeneity was assessed qualitatively, in addition to I2 and Chi-square statistics. Pooled relative risks (RR) and 95% confidence intervals (CI) were calculated using Mantel-Haenszel random-effects models. Results Fourteen randomized controlled trials (RCTs) were included. Prolonged infusion β-lactams were not associated with decreased mortality (n= 982; RR 0.92; 95% CI:0.61-1.37) or clinical cure (n = 1380; RR 1.00 95% CI:0.94-1.06) compared to intermittent infusions. Subgroup analysis for β-lactam subclasses and equivalent total daily β-lactam doses yielded similar results. Most studies had notable methodological flaws. Conclusions No clinical advantage was observed for prolonged infusion β-lactams. The limited number of studies with MDRGNs precluded evaluation of prolonged infusion of β-lactams for this subgroup. A large, multicenter RCT with critically ill patients infected with MDRGNs is needed. PMID:21696619

The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1.

PubMed

Malyan, Alexander N

2016-05-01

The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water.

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations.

PubMed

Patelski, J; Pioruńska-Stolzmann, M

1985-01-01

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.

Acid-functionalized nanoparticles for biomass hydrolysis

NASA Astrophysics Data System (ADS)

Pena Duque, Leidy Eugenia

Cellulosic ethanol is a renewable source of energy. Lignocellulosic biomass is a complex material composed mainly of cellulose, hemicellulose, and lignin. Biomass pretreatment is a required step to make sugar polymers liable to hydrolysis. Mineral acids are commonly used for biomass pretreatment. Using acid catalysts that can be recovered and reused could make the process economically more attractive. The overall goal of this dissertation is the development of a recyclable nanocatalyst for the hydrolysis of biomass sugars. Cobalt iron oxide nanoparticles (CoFe2O4) were synthesized to provide a magnetic core that could be separated from reaction using a magnetic field and modified to carry acid functional groups. X-ray diffraction (XRD) confirmed the crystal structure was that of cobalt spinel ferrite. CoFe2O4 were covered with silica which served as linker for the acid functions. Silica-coated nanoparticles were functionalized with three different acid functions: perfluoropropyl-sulfonic acid, carboxylic acid, and propyl-sulfonic acid. Transmission electron microscope (TEM) images were analyzed to obtain particle size distributions of the nanoparticles. Total carbon, nitrogen, and sulfur were quantified using an elemental analyzer. Fourier transform infra-red spectra confirmed the presence of sulfonic and carboxylic acid functions and ion-exchange titrations accounted for the total amount of catalytic acid sites per nanoparticle mass. These nanoparticles were evaluated for their performance to hydrolyze the beta-1,4 glycosidic bond of the cellobiose molecule. Propyl-sulfonic (PS) and perfluoropropyl-sulfonic (PFS) acid functionalized nanoparticles catalyzed the hydrolysis of cellobiose significantly better than the control. PS and PFS were also evaluated for their capacity to solubilize wheat straw hemicelluloses and performed better than the control. Although PFS nanoparticles were stronger acid catalysts, the acid functions leached out of the nanoparticle during

Simple and suitable immunosensor for β-lactam antibiotics analysis in real matrixes: milk, serum, urine.

PubMed

Merola, Giovanni; Martini, Elisabetta; Tomassetti, Mauro; Campanella, Luigi

2015-03-15

The anti-penicillin G was conjugated to avidin-peroxidase and biotin to obtain immunogen and competitor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other β-lactam antibiotics, with Kaff values of the order of 10(8) M(-1). The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of "in situ" analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumental methods reported in the literature and used for the analysis of β-lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amperometric electrode for hydrogen peroxide as transducer, the BSA-penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10(-11)M. Lastly, the new immunosensor displayed low selectivity versus the entire class of β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics. Copyright © 2014 Elsevier B.V. All rights reserved.

A survey of the utilization of anti-pseudomonal beta-lactam therapy in cystic fibrosis patients.

PubMed

Zobell, Jeffery T; Young, David C; Waters, C Dustin; Ampofo, Krow; Cash, Jared; Marshall, Bruce C; Olson, Jared; Chatfield, Barbara A

2011-10-01

The purpose of this study was to characterize the utilization of anti-pseudomonal beta-lactam antibiotics in the treatment of acute pulmonary exacerbations (APE) among Cystic Fibrosis Foundation (CFF)-accredited care centers. An anonymous national cross-sectional survey of CFF-accredited care centers was performed using an electronic survey tool (SurveyMonkey.com®). One hundred and twenty-one of 261 centers completed the survey (46%) representing 56% (14,856/26,740) of patients in the CFF Patient Registry. One hundred and nineteen of 121 (98%) respondents reported using beta-lactams for the treatment of APE. Intermittent dosing regimens constituted 155/167 (93%) reported regimens, while extended infusions were 12/167 (7%). Ceftazidime was the most commonly utilized beta-lactam comprising 74/167 (44%) of all infusions (intermittent and extended) of which 70/74 (95%) were intermittent infusions. The majority of intermittent ceftazidime regimens (56/70; 80%) were at doses lower than CFF and European guidelines recommended doses. In conclusion, a great majority of respondents use intermittent anti-pseudomonal beta-lactam antibiotics, with over half of respondents utilizing lower than guidelines recommended doses. While this is of concern, it is not known if optimization of dosing strategies according to guidelines recommendations will result in clinical benefit. Copyright © 2011 Wiley-Liss, Inc.

Enhanced hydrogen release by catalyzed hydrolysis of sodium borohydride-ammonia borane mixtures: a solution-state 11B NMR study.

PubMed

Hannauer, J; Demirci, U B; Geantet, C; Herrmann, J M; Miele, P

2011-03-07

Hydrolysis of mixtures consisting of sodium borohydride NaBH(4) (SB) and ammonia borane NH(3)BH(3) (AB) was studied in the absence/presence of a Co catalyst. The kinetics of the H(2) evolutions was measured. The reactions were followed in situ by solution-state (11)B NMR and the hydrolysis by-products characterized by NMR, XRD and IR. It is demonstrated that the combination of the two compounds gives a synergetic effect. SB rapidly reduces the Co catalyst precursor and the NH(4)(+) ions from AB contribute in the dispersion of the in situ formed Co nanoparticles. As a result, the kinetics of H(2) evolution is greatly improved. For instance, a hydrogen generation rate of 29.6 L min(-1) g(-1)(Co) was found for a mixture consisting of 81 wt% NH(3)BH(3), 9 wt% NaBH(4) and 10 wt% CoCl(2). By (11)B NMR, it was showed that the reaction mechanisms are quite trivial. As soon as the Co catalyst forms in situ, SB, rather than AB, hydrolyzes until it is totally converted. Then, the overall hydrolysis continues with that of AB. Both reactions follow a bimolecular Langmuir-Hinshelwood mechanism; no reaction intermediates were observed during the process. In fact, SB and AB convert directly into B(OH)(4)(-), which comes in equilibrium with a polyborate compound identified as B(3)O(3)(OH)(4)(-). All of these results are discussed herein.

Chemical and microbiologic aspects of penems, a distinct class of beta-lactams: focus on faropenem.

PubMed

Hamilton-Miller, Jeremy M T

2003-11-01

Many beta-lactam antimicrobials were developed between the 1960s and 1980s, with continuing development driven by the emergence of microbial resistance. Penems form a discrete class of beta-lactams that comprises structural hybrids of penicillins (penams) and cephalosporins (cephems). The chemistry and microbiology of the representative penems MEN 10700, ritipenem, CGP 31608, sulopenem, BRL 42715, and faropenem are reviewed. Particular emphasis is placed on faropenem, which is in late clinical development.

Diastereoselective and enantioselective conjugate addition reactions utilizing α,β-unsaturated amides and lactams

PubMed Central

2015-01-01

Summary The conjugate addition reaction has been a useful tool in the formation of carbon–carbon bonds. The utility of this reaction has been demonstrated in the synthesis of many natural products, materials, and pharmacological agents. In the last three decades, there has been a significant increase in the development of asymmetric variants of this reaction. Unfortunately, conjugate addition reactions using α,β-unsaturated amides and lactams remain underdeveloped due to their inherently low reactivity. This review highlights the work that has been done on both diastereoselective and enantioselective conjugate addition reactions utilizing α,β-unsaturated amides and lactams. PMID:25977728

Investigation of β-lactam antibacterial drugs, β-lactamases, and penicillin-binding proteins with fluorescence polarization and anisotropy: a review

NASA Astrophysics Data System (ADS)

Shapiro, Adam B.

2016-06-01

This review covers the uses of fluorescence polarization and anisotropy for the investigation of bacterial penicillin binding proteins (PBPs), which are the targets of β-lactam antibacterial drugs (penicillins, cephalosporins, carbapenems, and monobactams), and of the β-lactamase enzymes that destroy these drugs and help to render bacterial pathogens resistant to them. Fluorescence polarization and anisotropy-based methods for quantitation of β-lactam drugs are also reviewed. A particular emphasis is on methods for quantitative measurement of the interactions of β-lactams and other inhibitors with PBPs and β-lactamases.

Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline–oxidative pretreatment of hybrid poplar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhalla, Aditya; Fasahati, Peyman; Particka, Chrislyn A.

2018-05-17

When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H2O2) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significant loss inmore » sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified.« less

Stereochemical preference toward oncotarget: Design, synthesis and in vitro anticancer evaluation of diastereomeric β-lactams.

PubMed

Olazarán-Santibáñez, Fabián; Bandyopadhyay, Debasish; Carranza-Rosales, Pilar; Rivera, Gildardo; Balderas-Rentería, Isaías

2017-06-06

In the battle against cancer discovery of new and novel chemotherapeutic agent demands extreme obligation. Development of anticancer compounds with higher potency and reduced side-effects is timely and challenging. A small series of fourteen diastereomeric β-lactams (seven pairs) were synthesized through multi-step process exploring [2+2] ketene-imine cycloaddition as the key step. Comparative stereochemical preferences were studied through computational docking and validated by in vitro evaluation. β-tubulin was considered as possible molecular target and in vitro anticancer evaluation was conducted against SiHa, B16F10, K562 and Chang cell lines. Caspase-3 activation assay and hematoxylin/eosin staining of the cells were also accomplished. Better docking scores of the cis- over the trans-β-lactams indicated favorable β-lactam-β-tubulin interactions in cis-geometry. In vitro (IC50) evaluation confirmed better anticancer activity of the cis-diastereoisomers. Apoptosis-induced cell death was supported by caspase-3 activation study. A cis-β-lactam [(±)-Cis-3-amino-1-phenyl-4-(p-tolyl) azetidin-2-one, 6C] was found to be more active (in vitro) than the marketed natural drug colchicine against SiHa and B16F10 (six times higher potency) cell lines. Reduced toxicity (compared to colchicine) in Chang cells confirmed better site-selectivity (accordingly less side-effects) of 6C than colchicine. Aside from 6C, most of the reported molecules demonstrated good to strong in vitro anticancer activity against SiHa and B16F10 cancer cell lines. Stereochemical preferences of the cis-β-lactams over their trans-counterparts, toward the molecular target β-tubulin, was confirmed by docking studies and in vitro anticancer evaluation. Apoptosis was identified as the cause of cell death. The lead 6C exhibited higher potency and selectivity than the marketed drug colchicine both in silico as well as in vitro.

Elucidation of Mechanisms and Selectivities of Metal-Catalyzed Reactions using Quantum Chemical Methodology.

PubMed

Santoro, Stefano; Kalek, Marcin; Huang, Genping; Himo, Fahmi

2016-05-17

Quantum chemical techniques today are indispensable for the detailed mechanistic understanding of catalytic reactions. The development of modern density functional theory approaches combined with the enormous growth in computer power have made it possible to treat quite large systems at a reasonable level of accuracy. Accordingly, quantum chemistry has been applied extensively to a wide variety of catalytic systems. A huge number of problems have been solved successfully, and vast amounts of chemical insights have been gained. In this Account, we summarize some of our recent work in this field. A number of examples concerned with transition metal-catalyzed reactions are selected, with emphasis on reactions with various kinds of selectivities. The discussed cases are (1) copper-catalyzed C-H bond amidation of indoles, (2) iridium-catalyzed C(sp(3))-H borylation of chlorosilanes, (3) vanadium-catalyzed Meyer-Schuster rearrangement and its combination with aldol- and Mannich-type additions, (4) palladium-catalyzed propargylic substitution with phosphorus nucleophiles, (5) rhodium-catalyzed 1:2 coupling of aldehydes and allenes, and finally (6) copper-catalyzed coupling of nitrones and alkynes to produce β-lactams (Kinugasa reaction). First, the methodology adopted in these studies is presented briefly. The electronic structure method in the great majority of these kinds of mechanistic investigations has for the last two decades been based on density functional theory. In the cases discussed here, mainly the B3LYP functional has been employed in conjunction with Grimme's empirical dispersion correction, which has been shown to improve the calculated energies significantly. The effect of the surrounding solvent is described by implicit solvation techniques, and the thermochemical corrections are included using the rigid-rotor harmonic oscillator approximation. The reviewed examples are chosen to illustrate the usefulness and versatility of the adopted methodology in

Importance of cellulase cocktails favoring hydrolysis of cellulose.

PubMed

Victoria, Juliet; Odaneth, Annamma; Lali, Arvind

2017-07-03

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.

Regio-selective lipase catalyzed hydrolysis of oxanorbornane-based sugar-like amphiphiles at air-water interface: A polarized FT-IRRAS study.

PubMed

Sarangi, Nirod Kumar; Ganesan, M; Muraleedharan, K M; Patnaik, Archita

2017-04-01

Interfacial hydrolysis of oxanorbornane-based amphiphile (Triol C16) by Candida rugosa lipase was investigated using real-time polarized Fourier transform-infrared reflection absorption spectroscopy (FT-IRRAS). The kinetics of hydrolysis was studied by analyzing the ester carbonyl ν(CO) stretching vibration band across the two dimensional (2D) array of molecules at the confined interface. In particular, we demonstrate Triol C16 to form Michaelis-Menten type complex, like that of lipid-substrate analogues, where the Triol C16 head group remained accessible to the catalytic triad of the lipase. The enzyme-induced selective cleavage of the ester bond was spectroscopically monitored by the disappearance of the intense ν(CO) resonance at 1736cm -1 . Consequently, the in situ spectroscopic measurements evidenced selective ester hydrolysis of Triol C16 yielding Tetrol C 2 OH and Palmitic acid, which remained predominantly in the undissociated form at the interface. The conformation sensitive amide I (majorly ν(CO)) and the interfacial water reorganization suggested 2D ordering of the enzyme molecules following which interfacial reactions were employed towards probing the enzyme kinetics at the air/water interface. The investigation demonstrated further the potential of IRRAS spectroscopy for real-time monitoring the hydrolytic product formation and selectivity at biomimetic interfaces. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

PubMed Central

Koch, Miriam; Flür, Sara; Kreutz, Christoph; Ennifar, Eric; Micura, Ronald; Polacek, Norbert

2015-01-01

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases. PMID:25941362

The hydrolysis of polyimides

NASA Technical Reports Server (NTRS)

Hoagland, P. D.; Fox, S. W.

1973-01-01

Thermal polymerization of aspartic acid produces a polysuccinimide (I), a chain of aspartoyl residues. An investigation was made of the alkaline hydrolysis of the imide rings of (I) which converts the polyimide to a polypeptide. The alkaline hydrolysis of polyimides can be expected to be kinetically complex due to increasing negative charge generated by carboxylate groups. For this reason, a diimide, phthaloyl-DL-aspartoyl-beta-alanine (IIA) was synthesized for a progressive study of the hydrolysis of polyimides. In addition, this diimide (IIA) can be related to thalidomide and might be expected to exhibit similar reactivity during hydrolysis of the phthalimide ring.

Enthalpy of phase transitions of lactams

NASA Astrophysics Data System (ADS)

Emel'yanenko, V. N.; Verevkin, S. P.; Ralys, R. V.; Turovtsev, V. V.; Orlov, V. Yu.

2012-10-01

The transpiration method is used to measure the temperature dependences of the vapors pressures of azacyclobutan-2-one (I, CAS 930-21-2) azacyclohexan-2-one (II, CAS 675-20-7); azacyclooctan-2-one (III, CAS 673-66-5); azacyclononan-2-one (IV, CAS 935-30-8) and azacyclotridecan-2-one (V, CAS 947-04-6). Enthalpies of sublimation and vaporisation are determined. The temperatures and enthalpies of fusion of compounds (I, III-V) are found by means of differential scanning calorimetry. The dependences of the enthalpies of vaporisation of lactones, lactams, cycloalkanes, cycloalkanones on the size of a cycle are analyzed.

Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

PubMed Central

2015-01-01

Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance. PMID:25309810

Glycopeptides versus β-lactams for the prevention of surgical site infections in cardiovascular and orthopedic surgery: a meta-analysis.

PubMed

Saleh, Anas; Khanna, Ashish; Chagin, Kevin M; Klika, Alison K; Johnston, Douglas; Barsoum, Wael K

2015-01-01

To compare the efficacy of glycopeptides and β-lactams in preventing surgical site infections (SSIs) in cardiac, vascular, and orthopedic surgery. The cost-effectiveness of switching from β-lactams to glycopeptides for preoperative antibiotic prophylaxis has been controversial. β-Lactams are generally recommended in clean surgical procedures, but they are ineffective against resistant gram-positive bacteria. PubMed, International Pharmaceuticals Abstracts, Scopus, and Cochrane were searched for randomized clinical trials comparing glycopeptides and β-lactams for prophylaxis in adults undergoing cardiac, vascular, or orthopedic surgery. Abstracts and conference proceedings were included. Two independent reviewers performed study selection, data extraction, and assessment of risk of bias. Fourteen studies with a total of 8952 patients were analyzed. No difference was detected in overall SSIs between antibiotic types. However, compared with β-lactams, glycopeptides reduced the risk of resistant staphylococcal SSIs by 48% (relative risk, 0.52; 95% confidence interval, 0.29-0.93; P = 0.03) and enterococcal SSIs by 64% (relative risk, 0.36; 95% confidence interval, 0.16-0.80; P = 0.01), but increased respiratory tract infections by 54% (relative risk, 1.54; 95% confidence interval, 1.19-2.01; P ≤ 0.01). Subgroup analysis of cardiac procedures showed superiority of β-lactams in preventing superficial and deep chest SSIs, susceptible staphylococcal SSIs, and respiratory tract infections. Glycopeptides reduce the risk of resistant staphylococcal SSIs and enterococcal SSIs, but increase the risk of respiratory tract infections. Additional high-quality randomized clinical trials are needed as these results are limited by high risk of bias.

"Click" saccharide/beta-lactam hybrids for lectin inhibition.

PubMed

Palomo, Claudio; Aizpurua, Jesus M; Balentová, Eva; Azcune, Itxaso; Santos, J Ignacio; Jiménez-Barbero, Jesús; Cañada, Javier; Miranda, José Ignacio

2008-06-05

Hybrid glycopeptide beta-lactam mimetics designed to bind lectins or carbohydrate recognition domains in selectins have been prepared according to a "shape-modulating linker" design. This approach was implemented using the azide-alkyne "click" cycloaddition reaction, and as shown by NMR/MD experiments, binding of the resulting mimetics to Ulex Europaeus Lectin-1 (UEL-1) occurred after a "bent-to-extended" conformational change around a partially rotatable triazolylmethylene moiety.

In vitro effects of beta-lactams combined with beta-lactamase inhibitors against methicillin-resistant Staphylococcus aureus.

PubMed Central

Kobayashi, S; Arai, S; Hayashi, S; Sakaguchi, T

1989-01-01

The effects of combinations of beta-lactams with two beta-lactamase inhibitors, sulbactam and clavulanic acid, were determined in vitro against 22 clinical isolates of methicillin-resistant Staphylococcus aureus. Combinations of cefpirome, cefotaxime, and cefazolin with sulbactam (10 micrograms/ml) showed synergistic effects against more than 70% of the strains. Combinations of methicillin and penicillin G with sulbactam also showed synergistic effects against 50 and 68% of the strains, respectively, while cefotiam, moxalactam, flomoxef, and cefmetazole in combination with sulbactam showed such effects against only 40% or fewer. Clavulanic acid was synergistic only when combined with penicillin G, the effect probably being due to the beta-lactamase inhibition by the inhibitor. Sulbactam did not improve the antimicrobial activities of the beta-lactams against methicillin-susceptible S. aureus strains. At 42 degrees C the MICs of cefotaxime, methicillin, and flomoxef alone were markedly decreased from the values at 35 degrees C, and no synergy between these beta-lactams and sulbactam appeared. The resistance to penicillin G was not inhibited by incubation at 42 degrees C, and combinations of penicillin G with sulbactam and clavulanic acid showed synergy. The amounts of beta-lactamase produced were not related to the decreases in the MICs of the beta-lactams, except for penicillin G combined with sulbactam. Clavulanic acid showed slightly stronger beta-lactamase-inhibiting activity than sulbactam did. These results suggest that the synergy between sulbactam and the beta-lactams, except for penicillin G, may not be due to beta-lactamase inhibition but to suppression of the methicillin-resistant S. aureus-specific resistance based on other factors. PMID:2786369

A review on lipase-catalyzed reactions in ultrasound-assisted systems.

PubMed

Lerin, Lindomar A; Loss, Raquel A; Remonatto, Daniela; Zenevicz, Mara Cristina; Balen, Manuela; Netto, Vendelino Oenning; Ninow, Jorge L; Trentin, Cláudia M; Oliveira, J Vladimir; de Oliveira, Débora

2014-12-01

The named "green chemistry" has been receiving increasing prominence due to its environmentally friendly characteristics. The use of enzymes as catalysts in processes of synthesis to replace the traditional use of chemical catalysts present as main advantage the fact of following the principles of the green chemistry. However, processes of enzymatic nature generally provide lower yields when compared to the conventional chemical processes. Therefore, in the last years, the ultrasound has been extensively used in enzymatic processes, such as the production of esters with desirable characteristics for the pharmaceutical, cosmetics, and food industry, for the hydrolysis and glycerolysis of vegetable oils, production of biodiesel, etc. Several works found in the open literature suggest that the energy released by the ultrasound during the cavitation phenomena can be used to enhance mass transfer (substrate/enzyme), hence increasing the rate of products formation, and also contributing to enhance the enzyme catalytic activity. Furthermore, the ultrasound is considered a "green" technology due to its high efficiency, low instrumental requirement and significant reduction of the processing time in comparison to other techniques. The main goal of this review was to summarize studies available to date regarding the application of ultrasound in enzyme-catalyzed esterification, hydrolysis, glycerolysis and transesterification reactions.

Nitric Oxide from IFNγ-Primed Macrophages Modulates the Antimicrobial Activity of β-Lactams against the Intracellular Pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella

PubMed Central

Jones-Carson, Jessica; Zweifel, Adrienne E.; Tapscott, Timothy; Austin, Chad; Brown, Joseph M.; Jones, Kenneth L.; Voskuil, Martin I.; Vázquez-Torres, Andrés

2014-01-01

Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2), while stimulating hydrogen peroxide (H2O2) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics. PMID:25121731

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

PubMed

Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

2018-04-09

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Targeting Wall Teichoic Acid in Situ with Branched Polyethylenimine Potentiates β-Lactam Efficacy against MRSA.

PubMed

Foxley, Melissa A; Wright, Summer N; Lam, Anh K; Friedline, Anthony W; Strange, Stoffel J; Xiao, Min T; Moen, Erika L; Rice, Charles V

2017-10-12

Methicillin-resistant Staphylococcus aureus (MRSA) is a medical concern. Here, we show that branched polyethylenimine (BPEI), a nontoxic, cationic polymer, restores MRSA's susceptibility to β-lactam antibiotics. Checkerboard assays with MRSA demonstrated synergy between BPEI and β-lactam antibiotics. A time-killing curve showed BPEI to be bactericidal in combination with oxacillin. BPEI did not potentiate efficacy with vancomycin, chloramphenicol, or linezolid. When exposed to BPEI, MRSA increased in size and had difficulty forming septa. BPEI electrostatically binds to wall teichoic acid (WTA), a cell wall anionic polymer of Gram-positive bacteria that is important for localization of certain cell wall proteins. Lack of potentiation in a WTA knockout mutant supports the WTA-based mechanism. These data suggest that BPEI may prevent proper localization of cell wall machinery by binding to WTA; leading to cell death when administered in combination with β-lactam antibiotics. Negligible in vitro toxicity suggests the combination could be a viable treatment option.

Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum.

PubMed

Salo, Oleksandr V; Ries, Marco; Medema, Marnix H; Lankhorst, Peter P; Vreeken, Rob J; Bovenberg, Roel A L; Driessen, Arnold J M

2015-11-14

Penicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of β-lactams. The high β-lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved β-lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown. To examine the impact of CSI on secondary metabolism, a comparative genomic analysis of β-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β-lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum. The classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of β-lactams.

Efficient asymmetric hydrolysis of styrene oxide catalyzed by Mung bean epoxide hydrolases in ionic liquid-based biphasic systems.

PubMed

Chen, Wen-Jing; Lou, Wen-Yong; Zong, Min-Hua

2012-07-01

The asymmetric hydrolysis of styrene oxide to (R)-1-phenyl-1,2-ethanediol using Mung bean epoxide hydrolases was, for the first time, successfully conducted in an ionic liquid (IL)-containing biphasic system. Compared to aqueous monophasic system, IL-based biphasic systems could not only dissolve the substrate, but also effectively inhibit the non-enzymatic hydrolysis, and therefore markedly improve the reaction efficiency. Of all the tested ILs, the best results were observed in the biphasic system containing C(4)MIM·PF(6), which exhibited good biocompatibility with the enzyme and was an excellent solvent for the substrate. In the C(4)MIM·PF(6)/buffer biphasic system, it was found that the optimal volume ratio of IL to buffer, reaction temperature, buffer pH and substrate concentration were 1/6, 35°C, 6.5 and 100 mM, respectively, under which the initial reaction rate, the yield and the product e.e. were 18.4 mM/h, 49.4% and 97.0%. The biocatalytic process was shown to be feasible on a 500-mL preparative scale. Copyright © 2011 Elsevier Ltd. All rights reserved.

Reversible formation of intermediates during H/sub 3/O/sup +/-catalyzed hydrolysis of amides. Observation of substantial /sup 18/O exchange accompanying the hydrolysis of acetanilide and N-cyclohexylacetamide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slebocka-Tilk, H.; Brown, R.S.; Olekszyk, J.

1987-07-22

Careful mass spectrometric analysis of the /sup 18/O content of approx. 50% enriched acetanilide (2) and N-cyclohexylacetamide (3) recovered from acidic media during the course of hydrolysis reveals that both species suffer /sup 18/O loss. The percent of /sup 18/O exchange per t/sub 1/2/ of hydrolysis increases as (H/sub 3/O/sup +/) decreases. For 2 at 72/sup 0/C the amount of exchange increases from 0.5 +/- 0.5% (per t/sub 1/2/) in 1 M HCl to 9.4 +/- 0.5% in glycine buffer, (H/sub 3/O/sup +/) = 0.003 M. For 3 at 100/sup 0/C the exchange is 1.05 +/- 0.3% (per t/sub 1/2/)more » at 1 M HCl and 9.0 +/- 0.4% in 0.01 M HCl. When these data are used to compute k/sub ex/ (the exchange rate constant), it shows a first-order dependence on (H/sub 3/O/sup +/) followed by a plateau at high (H/sub 3/O/sup +/) for both 2 and 3.« less

The hydrolysis of epoxides catalyzed by inorganic ammonium salts in water: kinetic evidence for hydrogen bond catalysis.

PubMed

Nozière, B; Fache, F; Maxut, A; Fenet, B; Baudouin, A; Fine, L; Ferronato, C

2018-01-17

Naturally-occurring inorganic ammonium ions have been recently reported as efficient catalysts for some organic reactions in water, which contributes to the understanding of the chemistry in some natural environments (soils, seawater, atmospheric aerosols, …) and biological systems, and is also potentially interesting for green chemistry as many of their salts are cheap and non-toxic. In this work, the effect of NH 4 + ions on the hydrolysis of small epoxides in water was studied kinetically. The presence of NH 4 + increased the hydrolysis rate by a factor of 6 to 25 compared to pure water and these catalytic effects were shown not to result from other ions, counter-ions or from acid or base catalysis, general or specific. The small amounts of amino alcohols produced in the reactions were identified as the actual catalysts by obtaining a strong acceleration of the reactions when adding these compounds directly to the epoxides in water. Replacing the amino alcohols by other strong hydrogen-bond donors, such as trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) gave the same results, demonstrating that the kinetics of these reactions was driven by hydrogen-bond catalysis. Because of the presence of many hydrogen-bond donors in natural environments (for instance amines and hydroxy-containing compounds), hydrogen-bond catalysis is likely to contribute to many reaction rates in these environments.

Effect of γ-lactones and γ-lactams compounds on Streptococcus mutans biofilms

PubMed Central

Sordi, Mariane Beatriz; Moreira, Thaís Altoé; Montero, Juan Felipe Dumes; Barbosa, Luis Cláudio; Benfatti, César Augusto Magalhães; Magini, Ricardo de Souza; Pimenta, Andréa de Lima

2018-01-01

Abstract Considering oral diseases, antibiofilm compounds can decrease the accumulation of pathogenic species such as Streptococcus mutans at micro-areas of teeth, dental restorations or implant-supported prostheses. Objective To assess the effect of thirteen different novel lactam-based compounds on the inhibition of S. mutans biofilm formation. Material and methods We synthesized compounds based on γ-lactones analogues from rubrolides by a mucochloric acid process and converted them into their corresponding γ-hydroxy-γ-lactams by a reaction with isobutylamine and propylamine. Compounds concentrations ranging from 0.17 up to 87.5 μg mL-1 were tested against S. mutans. We diluted the exponential cultures in TSB and incubated them (37°C) in the presence of different γ-lactones or γ-lactams dilutions. Afterwards, we measured the planktonic growth by optical density at 630 nm and therefore assessed the biofilm density by the crystal violet staining method. Results Twelve compounds were active against biofilm formation, showing no effect on bacterial viability. Only one compound was inactive against both planktonic and biofilm growth. The highest biofilm inhibition (inhibition rate above 60%) was obtained for two compounds while three other compounds revealed an inhibition rate above 40%. Conclusions Twelve of the thirteen compounds revealed effective inhibition of S. mutans biofilm formation, with eight of them showing a specific antibiofilm effect. PMID:29489934

Progressing batch hydrolysis process

DOEpatents

Wright, J.D.

1985-01-10

A progressive batch hydrolysis process is disclosed for producing sugar from a lignocellulosic feedstock. It comprises passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with feed stock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feed stock to glucose. The cooled dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, serially fed through a plurality of pre-hydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose. The dilute acid stream containing glucose is cooled after it exits the last prehydrolysis reactor.

Kinetics of Sulbactam Hydrolysis by β-Lactamases, and Kinetics of β-Lactamase Inhibition by Sulbactam

PubMed Central

2017-01-01

ABSTRACT Sulbactam is one of four β-lactamase inhibitors in current clinical use to counteract drug resistance caused by degradation of β-lactam antibiotics by these bacterial enzymes. As a β-lactam itself, sulbactam is susceptible to degradation by β-lactamases. I investigated the Michaelis-Menten kinetics of sulbactam hydrolysis by 14 β-lactamases, representing clinically widespread groups within all four Ambler classes, i.e., CTX-M-15, KPC-2, SHV-5, and TEM-1 for class A; IMP-1, NDM-1, and VIM-1 for class B; Acinetobacter baumannii ADC-7, Pseudomonas aeruginosa AmpC, and Enterobacter cloacae P99 for class C; and OXA-10, OXA-23, OXA-24, and OXA-48 for class D. All of the β-lactamases were able to hydrolyze sulbactam, although they varied widely in their kinetic constants for the reaction, even within each class. I also investigated the inactivation kinetics of the inhibition of these enzymes by sulbactam. The class A β-lactamases varied widely in their susceptibility to inhibition, the class C and D enzymes were very weakly inhibited, and the class B enzymes were essentially or completely unaffected. In addition, we measured the sulbactam turnover number, the sulbactam/enzyme molar ratio required for complete inhibition of each enzyme. Class C enzymes had the lowest turnover numbers, class A enzymes varied widely, and class D enzymes had very high turnover numbers. These results are valuable for understanding which β-lactamases ought to be well inhibited by sulbactam. Moreover, since sulbactam has intrinsic antibacterial activity against Acinetobacter species pathogens, these results contribute to understanding β-lactamase-mediated sulbactam resistance in Acinetobacter, especially due to the action of the widespread class D enzymes. PMID:28971872

Twisted Amides: From Obscurity to Broadly Useful Transition-Metal-Catalyzed Reactions by N-C Amide Bond Activation.

PubMed

Liu, Chengwei; Szostak, Michal

2017-05-29

The concept of using amide bond distortion to modulate amidic resonance has been known for more than 75 years. Two classic twisted amides (bridged lactams) ingeniously designed and synthesized by Kirby and Stoltz to feature fully perpendicular amide bonds, and as a consequence emanate amino-ketone-like reactivity, are now routinely recognized in all organic chemistry textbooks. However, only recently the use of amide bond twist (distortion) has advanced to the general organic chemistry mainstream enabling a host of highly attractive N-C amide bond cross-coupling reactions of broad synthetic relevance. In this Minireview, we discuss recent progress in this area and present a detailed overview of the prominent role of amide bond destabilization as a driving force in the development of transition-metal-catalyzed cross-coupling reactions by N-C bond activation. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In silico analysis of different generation β lactams antibiotics with penicillin binding protein-2 of Neisseria meningitidis for curing meningococcal disease.

PubMed

Tripathi, Vijay; Tripathi, Pooja; Srivastava, Navita; Gupta, Dwijendra

2014-12-01

Neisseria meningitidis is a gram negative, diplococcic pathogen responsible for the meningococcal disease and fulminant septicemia. Penicillin-binding proteins-2 (PBPs) is crucial for the cell wall biosynthesis during cell proliferation of N. meningitidis and these are the target for β-lactam antibiotics. For many years penicillin has been recognized as the antibiotic for meningococcal disease but the meningococcus has seemed to be antibiotic resistance. In the present work we have verified the molecular interaction of Penicillin binding protein-2 N. meningitidis to different generation of β-lactam antibiotics and concluded that the third generation of β-lactam antibiotics shows efficient binding with Penicillin binding protein-2 of N. meningitidis. On the basis of binding efficiency and inhibition constant, ceftazidime emerged as the most efficient antibiotic amongst the other advanced β-lactam antibiotics against Penicillin-binding protein-2 of N. meningitidis.

Enantioselective synthesis of spirooxoindoles via chiral auxiliary (bicyclic lactam) controlled SNAr reactions.

PubMed

Sen, Subhabrata; Potti, Venkata R; Surakanti, Ramu; Murthy, Y L N; Pallepogu, Raghavaiah

2011-01-21

A highly efficient enantioselective S(N)Ar reaction of chiral acyl bicyclic lactam with substituted-2,4-dinitrobenzenes was developed, affording products containing quarternary stereogenic centers. They are further utilized towards an enantioselective synthesis of spirooxoindoles.

Four Decades of β-Lactam Antibiotic Pharmacokinetics in Cystic Fibrosis.

PubMed

Bulitta, Jürgen B; Jiao, Yuanyuan; Drescher, Stefanie K; Oliver, Antonio; Louie, Arnold; Moya, Bartolome; Tao, Xun; Wittau, Mathias; Tsuji, Brian T; Zavascki, Alexandre P; Shin, Beom Soo; Drusano, George L; Sörgel, Fritz; Landersdorfer, Cornelia B

2018-06-23

The pharmacokinetics (PK) of β-lactam antibiotics in cystic fibrosis (CF) patients has been compared with that in healthy volunteers for over four decades; however, no quantitative models exist that explain the PK differences between CF patients and healthy volunteers in older and newer studies. Our aims were to critically evaluate these studies and explain the PK differences between CF patients and healthy volunteers. We reviewed all 16 studies that compared the PK of β-lactams between CF patients and healthy volunteers within the same study. Analysis of covariance (ANCOVA) models were developed. In four early studies that compared adolescent, lean CF patients with adult healthy volunteers, clearance (CL) in CF divided by that in healthy volunteers was 1.72 ± 0.90 (average ± standard deviation); in four additional studies comparing age-matched (primarily adult) CF patients with healthy volunteers, this ratio was 1.46 ± 0.16. The CL ratio was 1.15 ± 0.11 in all eight studies that compared CF patients and healthy volunteers who were matched in age, body size and body composition, or that employed allometric scaling by lean body mass (LBM). Volume of distribution was similar between subject groups after scaling by body size. For highly protein-bound β-lactams, the unbound fraction was up to 2.07-fold higher in older studies that compared presumably sicker CF patients with healthy volunteers. These protein-binding differences explained over half of the variance for the CL ratio (p < 0.0001, ANCOVA). Body size, body composition and lower protein binding in presumably sicker CF patients explained the PK alterations in this population. Dosing CF patients according to LBM seems suitable to achieve antibiotic target exposures.

Hydrolysis of aspartic acid phosphoramidate nucleotides: a comparative quantum chemical study.

PubMed

Michielssens, Servaas; Tien Trung, Nguyen; Froeyen, Matheus; Herdewijn, Piet; Tho Nguyen, Minh; Ceulemans, Arnout

2009-09-07

L-Aspartic acid has recently been found to be a good leaving group during HIV reverse transcriptase catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. This showed that L-Asp is a good mimic for the pyrophosphate moiety of deoxyadenosine triphosphate. The present work explores the thermochemistry and mechanism for hydrolysis of several models for L-aspartic-dAMP using B3LYP/DGDZVP, MP2/6-311++G** and G3MP2 level of theory. The effect of the new compound is gradually investigated: starting from a simple methyl amine leaving group up to the aspartic acid leaving group. The enzymatic environment was mimicked by involving two Mg(2+) ions and some important active site residues in the reaction. All reactions are compared to the corresponding O-coupled leaving group, which is methanol for methyl amine and malic acid for aspartic acid. With methyl amine as a leaving group a tautomeric associative or tautomeric dissociative mechanism is preferred and the barrier is lower than the comparable mechanism with methanol as a leaving group. The calculations on the aspartic acid in the enzymatic environment show that qualitatively the mechanism is the same as for triphosphate but the barrier for hydrolysis by the associative mechanism is higher for L-aspartic-dAMP than for L-malic-dAMP and pyrophosphate.

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

PubMed Central

Otero, Lisandro H.; Rojas-Altuve, Alzoray; Llarrull, Leticia I.; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W.; Fisher, Jed F.; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A.

2013-01-01

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd-transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design. PMID:24085846

How similar is the electronic structures of β-lactam and alanine?

NASA Astrophysics Data System (ADS)

Chatterjee, Subhojyoti; Ahmed, Marawan; Wang, Feng

2016-02-01

The C1s spectra of β-lactam i.e. 2-azetidinone (C3H5NO), a drug and L-alanine (C3H7NO2), an amino acid, exhibit striking similarities, which may be responsible for the competition between 2-azetidinone and the alanyl-alanine moiety in biochemistry. The present study is to reveal the degree of similarities and differences between their electronic structures of the two model molecular pairs. It is found that the similarities in C1s and inner valence binding energy spectra are due to their bonding connections but other properties such as ring structure (in 2-azetidinone) and chiral carbon (alanine) can be very different. Further, the inner valence region of ionization potential greater than 18 eV for 2-azetidinone and alanine is also significantly similar. Finally the strained lactam ring exhibits more chemical reactivity measured at all non-hydrogen atoms by Fukui functions with respect to alanine.

Pilot-scale study on the acid-catalyzed steam explosion of rice straw using a continuous pretreatment system.

PubMed

Chen, Wen-Hua; Tsai, Chia-Chin; Lin, Chih-Feng; Tsai, Pei-Yuan; Hwang, Wen-Song

2013-01-01

A continuous acid-catalyzed steam explosion pretreatment process and system to produce cellulosic ethanol was developed at the pilot-scale. The effects of the following parameters on the pretreatment efficiency of rice straw feedstocks were investigated: the acid concentration, the reaction temperature, the residence time, the feedstock size, the explosion pressure and the screw speed. The optimal presteaming horizontal reactor conditions for the pretreatment process are as follows: 1.7 rpm and 100-110 °C with an acid concentration of 1.3% (w/w). An acid-catalyzed steam explosion is then performed in the vertical reactor at 185 °C for 2 min. Approximately 73% of the total saccharification yield was obtained after the rice straw was pretreated under optimal conditions and subsequent enzymatic hydrolysis at a combined severity factor of 0.4-0.7. Moreover, good long-term stability and durability of the pretreatment system under continuous operation was observed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Confirmatory and quantitative analysis of beta-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Fagerquist, Clifton K; Lightfield, Alan R; Lehotay, Steven J

2005-03-01

A simple, rapid, rugged, sensitive, and specific method for the confirmation and quantitation of 10 beta-lactam antibiotics in fortified and incurred bovine kidney tissue has been developed. The method uses a simple solvent extraction, dispersive solid-phase extraction (dispersive-SPE) cleanup, and liquid chromatography-tandem mass spectrometry (LC/MS/MS) for confirmation and quantitation. Dispersive-SPE greatly simplifies and accelerates sample cleanup and improves overall recoveries compared with conventional SPE cleanup. The beta-lactam antibiotics tested were as follows: deacetylcephapirin (an antimicrobial metabolite of cephapirin), amoxicillin, desfuroylceftiofur cysteine disulfide (DCCD, an antimicrobial metabolite of ceftiofur), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, naficillin, and dicloxacillin. Average recoveries of fortified samples were 70% or better for all beta-lactams except DCCD, which had an average recovery of 58%. The LC/MS/MS method was able to demonstrate quantitative recoveries at established tolerance levels and provide confirmatory data for unambiguous analyte identification. The method was also tested on 30 incurred bovine kidney samples obtained from the USDA Food Safety and Inspection Service, which had previously tested the samples using the approved semiquantitative microbial assay. The results from the quantitative LC/MS/MS analysis were in general agreement with the microbial assay for 23 samples although the LC/MS/MS method was superior in that it could specifically identify which beta-lactam was present and quantitate its concentration, whereas the microbial assay could only identify the type of beta-lactam present and report a concentration with respect to the microbial inhibition of a penicillin G standard. In addition, for 6 of the 23 samples, LC/MS/MS analysis detected a penicillin and a cephalosporin beta-lactam, whereas the microbial assay detected only a penicillin beta-lactam. For samples that do not

Cordioxime: a new dioxime gamma-lactam from Cordia platythyrsa.

PubMed

Christelle, Tsague Dongmo; Hussainb, Hidayat; Dongo, Etienne; Julius, Oben Enyong; Hussain, Javid

2011-08-01

Cordia platythyrsa Baker is known for its medicinal value. This paper deals with a phytochemical investigation of this species, from which cordioxime (1), a new dioxime y-lactam has been isolated. Its structure was determined by comprehensive analyses of its 1H and 13C NMR, COSY, HMQC, and HMBC spectroscopic, and HREIMS data. The remaining two known compounds were identified as beta-sitosterol, and beta-sitosterol glucopyranoside.

Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii.

PubMed

Pratap, Shivendra; Katiki, Madhusudhanarao; Gill, Preet; Kumar, Pravindra; Golemi-Kotra, Dasantila

2016-01-01

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products.

PubMed

Lee, Yujeong; Klauser, Paul C; Brandsen, Benjamin M; Zhou, Cong; Li, Xinyi; Silverman, Scott K

2017-01-11

We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N 40 or N 100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O 2 -• or HOO • ) and hydrogen peroxide (H 2 O 2 ) and leads to the same set of products (3'-phosphoglycolate, 5'-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4' radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H 2 O 2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H 2 O 2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn 2+ and Mg 2+ , although including Mn 2+ increases the activity, and Mn 2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions.

High-Level Resistance of Staphylococcus aureus to β-Lactam Antibiotics Mediated by Penicillin-Binding Protein 4 (PBP4).

PubMed

Hamilton, Stephanie M; Alexander, J Andrew N; Choo, Eun Ju; Basuino, Li; da Costa, Thaina M; Severin, Anatoly; Chung, Marilyn; Aedo, Sandra; Strynadka, Natalie C J; Tomasz, Alexander; Chatterjee, Som S; Chambers, Henry F

2017-06-01

Penicillin-binding protein 4 (PBP4), a nonessential, low-molecular-weight penicillin-binding protein of Staphylococcus aureus , has been implicated in low-level resistance to β-lactam antibiotics, although the mechanism is unknown. Mutations in PBP4 and its promoter were identified in a laboratory-generated mutant strain, CRB, which expresses high-level resistance to β-lactams, including resistance to the new-generation cephalosporins active against methicillin-resistant strains of S. aureus These mutations did not appreciably alter the β-lactam antibiotic binding affinity of purified recombinant mutant PBP4 compared to that of wild-type PBP4. Compared to the susceptible parent strain, COLnex, the CRB strain produces a highly cross-linked cell wall peptidoglycan, indicative of increased transpeptidase activity. The pbp4 promoter mutation of CRB was associated with greatly increased amounts of PBP4 in membranes compared to those in the COLnex parent. Replacement of the native promoter of COLnex with the mutant promoter of CRB resulted in increased amounts of PBP4 in membranes and a highly cross-linked cell wall. PBP4 can be repurposed to provide essential transpeptidase activity in vivo and confer high-level resistance to β-lactam antibiotics, such as ceftobiprole and ceftaroline. Copyright © 2017 American Society for Microbiology.

High-Level Resistance of Staphylococcus aureus to β-Lactam Antibiotics Mediated by Penicillin-Binding Protein 4 (PBP4)

PubMed Central

Hamilton, Stephanie M.; Alexander, J. Andrew N.; Choo, Eun Ju; Basuino, Li; da Costa, Thaina M.; Severin, Anatoly; Chung, Marilyn; Aedo, Sandra; Strynadka, Natalie C. J.; Tomasz, Alexander; Chatterjee, Som S.

2017-01-01

ABSTRACT Penicillin-binding protein 4 (PBP4), a nonessential, low-molecular-weight penicillin-binding protein of Staphylococcus aureus, has been implicated in low-level resistance to β-lactam antibiotics, although the mechanism is unknown. Mutations in PBP4 and its promoter were identified in a laboratory-generated mutant strain, CRB, which expresses high-level resistance to β-lactams, including resistance to the new-generation cephalosporins active against methicillin-resistant strains of S. aureus. These mutations did not appreciably alter the β-lactam antibiotic binding affinity of purified recombinant mutant PBP4 compared to that of wild-type PBP4. Compared to the susceptible parent strain, COLnex, the CRB strain produces a highly cross-linked cell wall peptidoglycan, indicative of increased transpeptidase activity. The pbp4 promoter mutation of CRB was associated with greatly increased amounts of PBP4 in membranes compared to those in the COLnex parent. Replacement of the native promoter of COLnex with the mutant promoter of CRB resulted in increased amounts of PBP4 in membranes and a highly cross-linked cell wall. PBP4 can be repurposed to provide essential transpeptidase activity in vivo and confer high-level resistance to β-lactam antibiotics, such as ceftobiprole and ceftaroline. PMID:28373193

Trans-Cinnamaldehyde and Eugenol Increase Acinetobacter baumannii Sensitivity to Beta-Lactam Antibiotics.

PubMed

Karumathil, Deepti P; Nair, Meera Surendran; Gaffney, James; Kollanoor-Johny, Anup; Venkitanarayanan, Kumar

2018-01-01

Multi-drug resistant (MDR) Acinetobacter baumannii is a major nosocomial pathogen causing a wide range of clinical conditions with significant mortality rates. A. baumannii strains are equipped with a multitude of antibiotic resistance mechanisms, rendering them resistant to most of the currently available antibiotics. Thus, there is a critical need to explore novel strategies for controlling antibiotic resistance in A. baumannii . This study investigated the efficacy of two food-grade, plant-derived antimicrobials (PDAs), namely trans -cinnamaldehyde (TC) and eugenol (EG) in decreasing A. baumannii 's resistance to seven β-lactam antibiotics, including ampicillin, methicillin, meropenem, penicillin, aztreonam, amoxicillin, and piperacillin. Two MDR A. baumannii isolates (ATCC 17978 and AB 251847) were separately cultured in tryptic soy broth (∼6 log CFU/ml) containing the minimum inhibitory concentration (MIC) of TC or EG with or without the MIC of each antibiotic at 37°C for 18 h. A. baumannii strains not exposed to the PDAs or antibiotics served as controls. Following incubation, A. baumannii counts were determined by broth dilution assay. In addition, the effect of PDAs on the permeability of outer membrane and efflux pumps in A. baumannii was measured. Further, the effect of TC and EG on the expression of A. baumannii genes encoding resistance to β-lactam antibiotics ( blaP ), efflux pumps ( adeABC ), and multi-drug resistant protein ( mdrp ) was studied using real-time quantitative PCR (RT-qPCR). The experiment was replicated three times with duplicate samples of each treatment and control. The results from broth dilution assay indicated that both TC and EG in combination with antibiotics increased the sensitivity of A. baumannii to all the tested antibiotics ( P < 0.05). The two PDAs inhibited the function of A. baumannii efflux pump, (AdeABC), but did not increase the permeability of its outer membrane. Moreover, RT-qPCR data revealed that TC and EG

On the electrostatic and steric similarity of lactam compounds and the natural substrate for bacterial cell-wall biosynthesis

NASA Astrophysics Data System (ADS)

Frau, J.; Price, S. L.

1996-04-01

Electrostatic and structural properties of a set of β-lactam, γ-lactam and nonlactam compounds have been analyzed and compared with those of a model of the natural substrate d-alanyl- d-alanine for the carboxy- and transpeptidase enzymes. This first comparison of the electrostatic properties has been based on a distributed multipole analysis of high-quality ab initio wave functions of the substrate and potential antibiotics. The electrostatic similarity of the substrate and active compounds is apparent, and contrasts with the electrostatic properties of the noninhibitors. This has been quantified to give a reasonable correlation with the MIC (Minimum Concentration for Inhibition) and with kinetic data (k2/K) in accordance with the model for interaction of the lactam compounds with dd-peptidase. These correlations provide a better prediction of antibacterial activity than purely structural criteria.

The Lysis of Pathogenic Escherichia coli by Bacteriophages Releases Less Endotoxin Than by β-Lactams.

PubMed

Dufour, Nicolas; Delattre, Raphaëlle; Ricard, Jean-Damien; Debarbieux, Laurent

2017-06-01

Other than numerous experimental data assessing phage therapy efficacy, questions regarding safety of this approach are not sufficiently addressed. In particular, as phages can kill bacterial cells within <10 minutes, the associated endotoxin release (ER) in severe infections caused by gram-negative bacteria could be a matter of concern. Two therapeutic virulent phages and 4 reference antibiotics were studied in vitro for their ability to kill 2 pathogenic strains of Escherichia coli and generate an ER. The early interaction (first 3 hours) between these actors was assessed over time by studying the instantaneous cell viability, the colony-forming unit count, the concentration of free endotoxin released, and the cell morphology under light microscope. While β-lactams have a relatively slow effect, both tested phages, as well as amikacin, were able to rapidly abolish the bacterial growth. Even when considering the fastest phage (cell lysis in 9 minutes), the concentrations of phage-induced ER never reached the highest values, which were recorded with antibiotic treatments. Cumulative concentrations of endotoxin over time in phage-treated conditions were lower than those observed with β-lactams and close to those observed with amikacin. Whereas β-lactams were responsible for strong cell morphology changes (spheroplast with imipenem, filamentous cells with cefoxitin and ceftriaxone), amikacin and phages did not modify cell shape but produced intracellular inclusion bodies. This work provides important and comforting data regarding the safety of phage therapy. Therapeutically relevant phages, with their low endotoxin release profile and fast bactericidal effect, are not inferior to β-lactams. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Ugi 4-CR Synthesis of γ- and δ-Lactams providing new access to diverse enzyme interactions, a PDB analysis.

PubMed

Boltjes, André; Liao, George P; Zhao, Ting; Herdtweck, Eberhardt; Dömling, Alexander

2014-07-01

A three step synthesis of N -unsubstituted tetrazolo γ- and δ-lactams involving a key Ugi-4CR is presented. The compounds, otherwise difficult to access, are conveniently synthesized in overall good yields by our route. PDB analysis of the N -unsubstituted γ- and δ-lactam fragment reveals a strongly tri-directional hydrogen bond donor acceptor interaction with the amino acids of the binding sites.

Dynamics induced by β-lactam antibiotics in the active site of Bacillus subtilis L,D-transpeptidase.

PubMed

Lecoq, Lauriane; Bougault, Catherine; Hugonnet, Jean-Emmanuel; Veckerlé, Carole; Pessey, Ombeline; Arthur, Michel; Simorre, Jean-Pierre

2012-05-09

β-lactams inhibit peptidoglycan polymerization by acting as suicide substrates of essential d,d-transpeptidases. Bypass of these enzymes by unrelated l,d-transpeptidases results in β-lactam resistance, although carbapenems remain unexpectedly active. To gain insight into carbapenem specificity of l,d-transpeptidases (Ldts), we solved the nuclear magnetic resonance (NMR) structures of apo and imipenem-acylated Bacillus subtilis Ldt and show that the cysteine nucleophile is present as a neutral imidazole-sulfhydryl pair in the substrate-free enzyme. NMR relaxation dispersion does not reveal any preexisting conformational exchange in the apoenzyme, and change in flexibility is not observed upon noncovalent binding of β-lactams (K(D) > 37.5 mM). In contrast, covalent modification of active cysteine by both carbapenems and 2-nitro-5-thiobenzoate induces backbone flexibility that does not result from disruption of the imidazole-sulfhydryl proton interaction or steric hindrance. The chemical step of the reaction determines enzyme specificity since no differences in drug affinity were observed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Esculin hydrolysis by Enterobacteriaceae.

PubMed

Edberg, S C; Pittman, S; Singer, J M

1977-08-01

Literature reports disagree concerning esculin hydrolysis in the family Enterobacteriaceae. A total of 2,490 strains of the family were investigated for esculin hydrolysis by two methods, the esculin spot test and the PathoTec incubation strip, which measures constitutive enzyme, and five growth-supporting methods, which determine both constitutive and inducible enzymes. The five growth-supporting media studied were: Vaughn-Levine, the standard esculin hydrolysis medium (P. R. Edwards and W. H. Ewing, Identification of Enterobacteriaceae, 3rd ed., 1972); Vaughn-Levine without iron; Vaughn-Levine without Andrade's indicator; and bile-esculin medium. Growth media were incubated at 35 degrees C and checked every 24 h for 120 h. On growth media, 0.3% of Escherichia coli were positive in 24 h, 34% in 48 h, and 61% in 120 h. No strains were positive on the "nongrowth" tests. It appeared that the esculin hydrolysis enzyme(s) of E. coli was inducible rather than constitutive. All esculin hydrolyzers, which yielded positive tests on "constitutive tests" and 24-h tests, were limited to the genera Klebsiella, Enterobacter, and Serratia and species of Proteus vulgaris, Proteus rettgeri, and Citrobacter diversus. When used with standardized inoculum size and incubation time, the esculin hydrolysis test is very useful for differentiation within the family Enterobacteriaceae.

Esculin hydrolysis by Enterobacteriaceae.

PubMed Central

Edberg, S C; Pittman, S; Singer, J M

1977-01-01

Literature reports disagree concerning esculin hydrolysis in the family Enterobacteriaceae. A total of 2,490 strains of the family were investigated for esculin hydrolysis by two methods, the esculin spot test and the PathoTec incubation strip, which measures constitutive enzyme, and five growth-supporting methods, which determine both constitutive and inducible enzymes. The five growth-supporting media studied were: Vaughn-Levine, the standard esculin hydrolysis medium (P. R. Edwards and W. H. Ewing, Identification of Enterobacteriaceae, 3rd ed., 1972); Vaughn-Levine without iron; Vaughn-Levine without Andrade's indicator; and bile-esculin medium. Growth media were incubated at 35 degrees C and checked every 24 h for 120 h. On growth media, 0.3% of Escherichia coli were positive in 24 h, 34% in 48 h, and 61% in 120 h. No strains were positive on the "nongrowth" tests. It appeared that the esculin hydrolysis enzyme(s) of E. coli was inducible rather than constitutive. All esculin hydrolyzers, which yielded positive tests on "constitutive tests" and 24-h tests, were limited to the genera Klebsiella, Enterobacter, and Serratia and species of Proteus vulgaris, Proteus rettgeri, and Citrobacter diversus. When used with standardized inoculum size and incubation time, the esculin hydrolysis test is very useful for differentiation within the family Enterobacteriaceae. PMID:330558

A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

PubMed

Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

2012-12-01

A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recyclable Thermoresponsive Polymer-β-Glucosidase Conjugate with Intact Hydrolysis Activity.

PubMed

Mukherjee, Ishita; Sinha, Sushant K; Datta, Supratim; De, Priyadarsi

2018-06-11

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic β-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of β-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for β-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

[Mechanisms of beta-lactam and quinolone resistance in Haemophilus influenzae].

PubMed

Ubukata, Kimiko

2012-02-01

Haemophilus influenzae is one of the important pathogens causing respiratory tract infections, pneumonia, and meningitis. Genotypic(g) beta-lactamase-nonproducing ampicillin resistance (gBLNAR) H. influenzae has rapidly increased since 2000 years in Japan. The resistant percentage exceeded 60% in Hib isolates from meningitis in 2009. The affinity of beta-lactam antibiotics for penicillin-binding proteins-3 (PBP3) that involved in septal peptidoglycan synthesis deceased in the resistant strains. Three amino acid substitutions, Ser385Thr, Asn526Lys and Arg517His in PBP3 encoded by ftsI gene are especially responsible for beta-lactam resistance in the gBLNAR. Susceptibilities of cephalosporin agents including cefotaxime for gBLNAR were apparently decreased than the ampicillin and carbapenem antibiotics. Though fluoroquinolone resistant isolates are rare (< 1%) in H. influenzae, strains of levofloxacin and ciprofloxacin MIC with > or = 8 microg/mL were isolated from elderly patients with CAP. These strains possessed amino acid substitutions of Ser84Phe and Asp88Asn in GyrA and Glu88Lys in ParC. It is important to practice rapidly identification of these resistant strains at routine work.

Efficient production of fermentable sugars from oil palm empty fruit bunch by combined use of acid and whole cell culture-catalyzed hydrolyses.

PubMed

Li, Qingxin; Ng, Wei Ting; Puah, Sze Min; Bhaskar, Ravindran Vijay; Soh, Loon Siong; MacBeath, Calum; Parakattil, Pius; Green, Phil; Wu, Jin Chuan

2014-01-01

Empty fruit bunch (EFB) of oil palm trees was converted to fermentable sugars by the combined use of dilute acids and whole fungal cell culture-catalyzed hydrolyses. EFB (5%, w/v) was hydrolyzed in the presence of 0.5% H2 SO4 and 0.2% H3 PO4 at 160 °C for 10 Min. The solid fraction was separated from the acid hydrolysate by filtration and subjected to enzymatic hydrolysis at 50 °C using the whole cell culture of Trichoderma reesei RUT-C30 (2%, w/v), which was prepared by cultivation at 30 °C for 7 days to reach its maximal cellulase activity. The combined hydrolyses of EFB gave a total sugar yield of 82.0%. When used as carbon sources for cultivating Escherichia coli in M9 medium at 37 °C, the combined EFB hydrolysates were shown to be more favorable or at least as good as pure glucose for cell growth in terms of the higher (1.1 times) optical density of E. coli cells. The by-products generated during the acid-catalyzed hydrolysis did not seem to obviously affect cell growth. The combined use of acid and whole cell culture hydrolyses might be a commercially promising method for pretreatment of lignocellulose to get fermentable sugars. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Multicomponent ternary cocrystals of the sulfonamide group with pyridine-amides and lactams.

PubMed

Bolla, Geetha; Nangia, Ashwini

2015-11-04

SMBA was selected as a bifunctional sulfa drug to design ternary cocrystals with pyridine amides and lactam coformers. Supramolecular assembly of five ternary cocrystals of p-sulfonamide benzoic acid with nicotinamide and 2-pyridone is demonstrated and reproducible heterosynthons are identified for crystal engineering.

Enzymatic saccharification of pretreated wheat straw: comparison of solids-recycling, sequential hydrolysis and batch hydrolysis.

PubMed

Pihlajaniemi, Ville; Sipponen, Satu; Sipponen, Mika H; Pastinen, Ossi; Laakso, Simo

2014-02-01

In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series. Copyright © 2013 Elsevier Ltd. All rights reserved.

Response of Legionella pneumophila to beta-lactam antibiotics.

PubMed Central

Weisholtz, S; Tomasz, A

1985-01-01

Legionella pneumophila Philadelphia strain 1 grown in vitro contained five penicillin-binding proteins that were accessible to the antibiotic in membrane preparations and in live cells as well. The bacterium had reasonably low MICs of several beta-lactam antibiotics and was susceptible to both the bactericidal and the lytic activity of these drugs. An unusual feature of the response of this bacterium to penicillin treatment was that cell lysis as determined by decrease in culture turbidity and release of intracellular macromolecules was not accompanied by degradation of the peptidoglycan. Images PMID:2409915

The order of administration of macrolides and beta-lactams may impact the outcomes of hospitalized patients with community-acquired pneumonia: results from the community-acquired pneumonia organization.

PubMed

Peyrani, Paula; Wiemken, Timothy L; Metersky, Mark L; Arnold, Forest W; Mattingly, William A; Feldman, Charles; Cavallazzi, Rodrigo; Fernandez-Botran, Rafael; Bordon, Jose; Ramirez, Julio A

2018-01-01

The beneficial effect of macrolides for the treatment of community-acquired pneumonia (CAP) in combination with beta-lactams may be due to their anti-inflammatory activity. In patients with pneumococcal meningitis, the use of steroids improves outcomes only if they are administered before beta-lactams. The objective of this study was to compare outcomes in hospitalized patients with CAP when macrolides were administered before, simultaneously with, or after beta-lactams. Secondary data analysis of the Community-Acquired Pneumonia Organization (CAPO) International Cohort Study database. Study groups were defined based on the sequence of administration of macrolides and beta-lactams. The study outcomes were time to clinical stability (TCS), length of stay (LOS) and in-hospital mortality. Accelerated failure time models were used to evaluate the adjusted impact of sequential antibiotic administration and time-to-event outcomes, while a logistic regression model was used to evaluate their adjusted impact on mortality. A total of 99 patients were included in the macrolide before group and 305 in the macrolide after group. Administration of a macrolide before a beta-lactam compared to after a beta-lactam reduced TCS (3 vs. 4 days, p = .011), LOS (6 vs. 7 days, p = .002) and mortality (3 vs. 7.2%, p = .228). The administration of macrolides before beta-lactams was associated with a statistically significant decrease in TCS and LOS and a non-statistically significant decrease in mortality. The beneficial effect of macrolides in hospitalized patient with CAP may occur only if administered before beta-lactams.

Acylsugar Acylhydrolases: Carboxylesterase-Catalyzed Hydrolysis of Acylsugars in Tomato Trichomes1[OPEN

PubMed Central

Gilgallon, Karin; Ghosh, Banibrata

2016-01-01

Glandular trichomes of cultivated tomato (Solanum lycopersicum) and many other species throughout the Solanaceae produce and secrete mixtures of sugar esters (acylsugars) on the plant aerial surfaces. In wild and cultivated tomato, these metabolites consist of a sugar backbone, typically glucose or sucrose, and two to five acyl chains esterified to various positions on the sugar core. The aliphatic acyl chains vary in length and branching and are transferred to the sugar by a series of reactions catalyzed by acylsugar acyltransferases. A phenotypic screen of a set of S. lycopersicum M82 × Solanum pennellii LA0716 introgression lines identified a dominant genetic locus on chromosome 5 from the wild relative that affected total acylsugar levels. Genetic mapping revealed that the reduction in acylsugar levels was consistent with the presence and increased expression of two S. pennellii genes (Sopen05g030120 and Sopen05g030130) encoding putative carboxylesterase enzymes of the α/β-hydrolase superfamily. These two enzymes, named ACYLSUGAR ACYLHYDROLASE1 (ASH1) and ASH2, were shown to remove acyl chains from specific positions of certain types of acylsugars in vitro. A survey of related genes in M82 and LA0716 identified another trichome-expressed ASH gene on chromosome 9 (M82, Solyc09g075710; LA0716, Sopen09g030520) encoding a protein with similar activity. Characterization of the in vitro activities of the SpASH enzymes showed reduced activities with acylsugars produced by LA0716, presumably contributing to the high-level production of acylsugars in the presence of highly expressed SpASH genes. PMID:26811191

SYNTHESIS AND EVALUATION OF NEW PHTHALAZINE SUBSTITUTED β-LACTAM DERIVATIVES AS CARBONIC ANHYDRASE INHIBITORS.

PubMed

Berber, Nurcan; Arslan, Mustafa; Bilen, Çiğdem; Sackes, Zübeyde; Gençer, Nahit; Arslan, Oktay

2015-01-01

A new series of phthalazine substituted β-lactam derivatives were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA I and II) were evaluated. 2H-Indazolo[2,1-b]phthala- zine-trione derivative was prepared with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF, and the nitro group was reduced to 13-(4-aminophenyl)-3,3-dimethyl-3,4-dihydro- 2H-indazolo[1,2-b]phthalazine-1,6,11(13H)-trione with SnCl2 · 2H2O. The reduced compound was re- acted with different aromatic aldehydes, and phthalazine substituted imines were synthesized. The imine compounds undergo (2+2) cycloaddition reactions with ketenes to produce 2H-indazolo[2,1-b]phthala-zine-trione substituted β-lactam derivatives. The β-lactam compounds were tested as inhibitors of the CA isoenzyme activity. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. 1-(4-(3,3-dimethyl- 1,6,1 1-trioxo-2,3,4,6,11,13-hexahydro-1H-indazolo[1,2-b]phthalazin-13- yl)phenyl)-2-oxo-4-p-tolylazetidin-3-yl acetate (IC50 = 6.97 µM for hCA I and 8.48 µM for hCA II) had the most inhibitory effect.

[Importance of quality control for the detection of β-lactam antibiotic resistance in Enterobacteriaceae].

PubMed

Rivera, Alba; Larrosa, Nieves; Mirelis, Beatriz; Navarro, Ferran

2014-02-01

β-lactam antimicrobial agents are frequently used to treat infections caused by Enterobacteriaceae. The main mechanism of resistance to these antibiotics is the production of certain enzymes, collectively named β-lactamases. Due to their substrate profile and their epidemiological implications, the most clinically important β-lactamases are extended-spectrum β-lactamases, class C β-lactamases and carbapenemases. Phenotypic detection of these enzymes may be complicated and is based on the use of specific inhibitors of each β-lactamase and on the loss of activity on some β-lactam indicators. Various international committees postulate that it is no longer necessary to interpret the susceptibility results or determine the mechanism of resistance. Several critics disagree, however, and consider that susceptibility results should be interpreted until more data are available on the clinical efficacy of treatment with β-lactams. Given these methodological difficulties and constant changes in the interpretation criteria, we consider that training and external quality controls are essential to keep updated in this field. For learning purposes, these external quality controls should always be accompanied by a review of the results and methodology used, and the analysis of errors. In this paper we review and contextualize all the aspects related to the detection and interpretation of these β-lactamases. Copyright © 2014 Elsevier España, S.L. All rights reserved.

Progressing batch hydrolysis process

DOEpatents

Wright, John D.

1986-01-01

A progressive batch hydrolysis process for producing sugar from a lignocellulosic feedstock, comprising passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feedstock to glucose; cooling said dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, then feeding said dilute acid stream serially through a plurality of prehydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose; and cooling the dilute acid stream containing glucose after it exits the last prehydrolysis reactor.

Hydrolysis of tannic acid catalyzed by immobilized-stabilized derivatives of Tannase from Lactobacillus plantarum.

PubMed

Curiel, Jose Antonio; Betancor, Lorena; de las Rivas, Blanca; Muñoz, Rosario; Guisan, Jose M; Fernández-Lorente, Gloria

2010-05-26

A recombinant tannase from Lactobacillus plantarum , overexpressed in Escherichia coli , was purified in a single step by metal chelate affinity chromatography on poorly activated nickel supports. It was possible to obtain 0.9 g of a pure enzyme by using only 20 mL of chromatographic support. The pure enzyme was immobilized and stabilized by multipoint covalent immobilization on highly activated glyoxyl agarose. Derivatives obtained by multipoint and multisubunit immobilization were 500- and 1000-fold more stable than both the soluble enzyme and the one-point-immobilized enzyme in experiments of thermal and cosolvent inactivation, respectively. In addition, up to 70 mg of pure enzyme was immobilized on 1 g of wet support. The hydrolysis of tannic acid was optimized by using the new immobilized tannase derivative. The optimal reaction conditions were 30% diglyme at pH 5.0 and 4 degrees C. Under these conditions, it was possible to obtain 47.5 mM gallic acid from 5 mM tannic acid as substrate. The product was pure as proved by HPLC. On the other hand, the immobilized biocatalyst preserved >95% of its initial activity after 1 month of incubation under the optimal reaction conditions.

Muon Catalyzed Fusion

NASA Technical Reports Server (NTRS)

Armour, Edward A.G.

2007-01-01

Muon catalyzed fusion is a process in which a negatively charged muon combines with two nuclei of isotopes of hydrogen, e.g, a proton and a deuteron or a deuteron and a triton, to form a muonic molecular ion in which the binding is so tight that nuclear fusion occurs. The muon is normally released after fusion has taken place and so can catalyze further fusions. As the muon has a mean lifetime of 2.2 microseconds, this is the maximum period over which a muon can participate in this process. This article gives an outline of the history of muon catalyzed fusion from 1947, when it was first realised that such a process might occur, to the present day. It includes a description of the contribution that Drachrnan has made to the theory of muon catalyzed fusion and the influence this has had on the author's research.

Lipase-catalyzed kinetic resolution of novel antitubercular benzoxazole derivatives.

PubMed

Łukowska-Chojnacka, Edyta; Kowalkowska, Anna; Napiórkowska, Agnieszka

2018-04-01

Novel benzoxazole derivatives were synthesized, and their antitubercular activity against sensitive and drug-resistant Mycobacterium tuberculosis strains (M. tuberculosis H 37 Rv, M. tuberculosis sp. 210, M. tuberculosis sp. 192, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, and Mycobacterium kansasii) was evaluated. The chemical step included preparation of ketones, alcohols, and esters bearing benzoxazole moiety. All racemic mixtures of alcohols and esters were separated in Novozyme SP 435-catalyzed transesterification and hydrolysis, respectively. The transesterification reactions were carried out in various organic solvents (tert-butyl methyl ether, toluene, diethyl ether, and diisopropyl ether), and depending on the solvent, the enantioselectivity of the reactions ranged from 4 to >100. The enzymatic hydrolysis of esters was performed in 2 phase tert-butyl methyl ether/phosphate buffer (pH = 7.2) system and provided also enantiomerically enriched products (ee 88-99%). The antitubercular activity assay has shown that synthesized compounds exhibit an interesting antitubercular activity. Racemic mixtures of alcohols, (±)-4-(1,3-benzoxazol-2-ylsulfanyl)butan-2-ol ((±)-3a), (±)-4-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]butan-2-ol ((±)-3b), and (±)-4-[(5,7-dibromo-1,3-benzoxazol-2-yl)sulfanyl]butan-2-ol ((±)-3c), displayed as high activity against M. scrofulaceum, M. intracellulare, M. fortuitum, and M. kansasii as commercially available antituberculosis drug-Isoniazid. Moreover, these compounds exhibited twice higher activity toward M. avium (MIC 12.5) compared with Isoniazid (MIC 50). © 2017 Wiley Periodicals, Inc.

Modulation of cellulase activity by charged lipid bilayers with different acyl chain properties for efficient hydrolysis of ionic liquid-pretreated cellulose.

PubMed

Mihono, Kai; Ohtsu, Takeshi; Ohtani, Mai; Yoshimoto, Makoto; Kamimura, Akio

2016-10-01

The stability of cellulase activity in the presence of ionic liquids (ILs) is critical for the enzymatic hydrolysis of insoluble cellulose pretreated with ILs. In this work, cellulase was incorporated in the liposomes composed of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic phosphatidylcholines (PCs) with different length and degree of unsaturation of the acyl chains. The liposomal cellulase-catalyzed reaction was performed at 45°C in the acetate buffer solution (pH 4.8) with 2.0g/L CC31 as cellulosic substrate. The crystallinity of CC31 was reduced by treating with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at 120°C for 30min. The liposomal cellulase continuously catalyzed hydrolysis of the pretreated CC31 for 48h producing glucose in the presence of 15wt% [Bmim]Cl. The charged lipid membranes were interactive with [Bmim](+), as elucidated by the [Bmim]Cl-induced alterations in fluorescence polarization of the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules. The charged membranes offered the microenvironment where inhibitory effects of [Bmim]Cl on the cellulase activity was relieved. The maximum glucose productivity GP of 10.8 mmol-glucose/(hmol-lipid) was obtained at the reaction time of 48h with the cellulase incorporated in the liposomes ([lipid]=5.0mM) composed of 50mol% POPG and 1,2-dilauroyl-sn-glycero-3-phosohocholine (DLPC) with relatively short and saturated acyl chains. Copyright © 2016 Elsevier B.V. All rights reserved.

Enzymatic hydrolysis of 1-monoacyl-SN-glycerol-3-phosphoryl-choline (1-lysolecithin) by phospholipases from peanut seeds.

PubMed

Strauss, H; Leibovitz-Ben Gershon, Z; Heller, M

1976-06-01

Hydrolysis of 1-lysolecithin (1-acyl glycerophosphorylcholine [1-acyl GPC]) by preparations of phospholipase D from peanut seeds was investigated. 1-Lysolecithin was hydrolyzed at a much slower rate than phosphatidylcholine (lecithin). Although Ca+2 ions are required for the cleavage of lecithin by the enzyme, their effect on the hydrolysis of lysolecithin depended upon the concentration of the substrate: at 0.2 mM 1-lysolecithin, Ca+2 ions increased the reaction rates, whereas at concentrations of the substrate lower than 0.1 mM, Ca+2 ions were inhibitory. A broad pH activity curve between 5 and 8 was obtained with higher rates in the alkaline range, both in the absence and presence of Ca+2 ions. The increased hydrolysis of lysolecithin due to Ca+2 was noticed over the entire pH range. Upon storage of the enzyme solutions at 4 C, decreased rates of hydrolysis of lecithin were observed, with t 1/2 values of ca. 50 and 100 days depending on the purity of the preparation. During the same period, no reduction occurred in the activity of these preparations on lysolecithin as substrate. The effects of Ca+2 ions and the analysis of the products of 1-acyl GPC cleavage by the enzyme preparations revealed the presence of more than one enzyme and the formation of the following compounds: lysophosphatidic acids (1 acyl glycerophosphoric acids), free fatty acids, glycerophosphorylcholine, and choline. The possible pathways leading to the degradation of lysolecithin and the formation of these products include reactions catalyzed by lysophospholipase A1 (lysophosphatidylcholine 1-acyl hydrolase, E.C. 3.1.1.5) and a phosphodiesterase (L-3-glycerylphosphorylcholine glycerophosphohydrolase, E.C.3.1.4.2), in addition to phospholipase D (phosphatidyl-choline phosphatidohydrolase, E.C. 3.1.4.4).

New synthesis of artepillin C, a prenylated phenol, utilizing lipase-catalyzed regioselective deacetylation as the key step.

PubMed

Yashiro, Kazuki; Hanaya, Kengo; Shoji, Mitsuru; Sugai, Takeshi

2015-01-01

We have synthesized artepillin C, a diprenylated p-hydroxycinnamate originally isolated from Brazilian propolis and exhibiting antioxidant and antitumor activities, from 2,6-diallylphenol. Replacement of the terminal vinyl with 2,2-dimethylvinyl group by olefin cross-metathesis and subsequent transformation yielded 2,6-diprenyl-1,4-hydroquinone diacetate. Candida antarctica lipase B-catalyzed deacetylation in 2-propanol regioselectively removed the less hindered acetyl group to give 2,6-diprenyl-1,4-hydroquinone 1-monoacetate. After triflation of the liberated 4-hydroxy group, a three-carbon side chain was introduced by palladium-mediated alkenylation with methyl acrylate. Final hydrolysis of the esters furnished artepillin C.

Efficacy of β-lactam/β-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis.

PubMed

Mishra, Saurabh; Shukla, Prashant; Bhaskar, Ashima; Anand, Kushi; Baloni, Priyanka; Jha, Rajiv Kumar; Mohan, Abhilash; Rajmani, Raju S; Nagaraja, Valakunja; Chandra, Nagasuma; Singh, Amit

2017-05-26

Mycobacterium tuberculosis ( Mtb ) expresses a broad-spectrum β-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, β-lactams. Nonetheless, β-lactams showed mycobactericidal activity in combination with β-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to β-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of β-lactam resistance (e.g. β-lactamase) and redox potential in Mtb . We also describe the role of WhiB4 in coordinating the activity of β-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb . Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions.

Metal-containing Complexes of Lactams, Imidazoles, and Benzimidazoles and Their Biological Activity

NASA Astrophysics Data System (ADS)

Kukalenko, S. S.; Bovykin, B. A.; Shestakova, S. I.; Omel'chenko, A. M.

1985-07-01

The results of the latest investigations of the problem of the synthesis of metal-containing complexes of lactams, imidazoles, and benzimidazoles, their structure, and their stability in solutions are surveyed. Some data on their biological activity (pesticide and pharmacological) and the mechanism of their physiological action are presented. The bibliography includes 190 references.

Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nomata, Jiro; Terauchi, Kazuki; Fujita, Yuichi, E-mail: fujita@agr.nagoya-u.ac.jp

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN–BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e{sup –}) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratiomore » of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase. - Highlights: • The stoichiometry of nitrogenase-like protochlorophyllide reductase was determined. • The minimal ATP/2e{sup –} ratio was 4, which coincides with that of nitrogenase. • The ATP/2e{sup –} ratio increases with increasing L-protein/NB-protein molar ratio. • DPOR has an intrinsic property, but retains features shared with nitrogenase.« less

The hydrolysis of proteins by microwave energy

PubMed Central

Margolis, Sam A.; Jassie, Lois; Kingston, H. M.

1991-01-01

Microwave energy, at manually-adjusted, partial power settings has been used to hydrolyse bovine serum albumin at 125 °C. Hydrolysis was complete within 2 h, except for valine and isoleucine which were completely liberated within 4 h. The aminoacid destruction was less than that observed at similar hydrolysis conditions with other methods and complete hydrolysis was achieved more rapidly. These results provide a basis for automating the process of amino-acid hydrolysis. PMID:18924889

The Use of Noncarbapenem β-Lactams for the Treatment of Extended-Spectrum β-Lactamase Infections

PubMed Central

Rodriguez-Baňo, Jesus

2017-01-01

Abstract The continued rise in infections caused by extended-spectrum β-lactamase (ESBL)–producing pathogens is recognized globally as one of the most pressing concerns facing the healthcare community. Carbapenems are widely regarded as the antibiotics of choice for the treatment of ESBL-producing infections, even when in vitro activity to other β-lactams has been demonstrated. However, indiscriminant carbapenem use is not without consequence, and carbapenem overuse has contributed to the emergence of carbapenem-resistant Enterobacteriaceae. The use of non-carbapenem β-lactams for the treatment of ESBL infections has yielded conflicting results. In this review, we discuss the available data for the use of cephamycins, cefepime, piperacillin-tazobactam, ceftolozane-tazobactam, and ceftazidime-avibactam for the treatment of ESBL infections. PMID:28362938

Bio-inspired synthesis yields a tricyclic indoline that selectively resensitizes methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics

PubMed Central

Podoll, Jessica D.; Liu, Yongxiang; Chang, Le; Walls, Shane; Wang, Wei; Wang, Xiang

2013-01-01

The continuous emergence of resistant bacteria has become a major worldwide health threat. The current development of new antibacterials has lagged far behind. To discover reagents to fight against resistant bacteria, we initiated a chemical approach by synthesizing and screening a small molecule library, reminiscent of the polycyclic indole alkaloids. Indole alkaloids are a class of structurally diverse natural products, many of which were isolated from plants that have been used as traditional medicine for millennia. Specifically, we adapted an evolutionarily conserved biosynthetic strategy and developed a concise and unified diversity synthesis pathway. Using this pathway, we synthesized 120 polycyclic indolines that contain 26 distinct skeletons and a wide variety of functional groups. A tricyclic indoline, Of1, was discovered to selectively potentiate the activity of β-lactam antibiotics in multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA), but not in methicillin-sensitive S. aureus. In addition, we found that Of1 itself does not have antiproliferative activity but can resensitize several MRSA strains to the β-lactam antibiotics that are widely used in the clinic, such as an extended-spectrum β-lactam antibiotic amoxicillin/clavulanic acid and a first-generation cephalosporin cefazolin. These data suggest that Of1 is a unique selective resistance-modifying agent for β-lactam antibiotics, and it may be further developed to fight against resistant bacteria in the clinic. PMID:24019472

Real-time activity monitoring of New Delhi metallo-β-lactamase-1 in living bacterial cells by UV-Vis spectroscopy.

PubMed

Yang, Ke-Wu; Zhou, Yajun; Ge, Ying; Zhang, Yuejuan

2017-07-13

We report an UV-Vis method for monitoring the hydrolysis of the β-lactam antibiotics inside living bacterial cells. Cell-based studies demonstrated that the hydrolysis of cefazolin was inhibited by three known NDM-1 inhibitors. This approach can be applied to the monitoring of reactions in a complex biological system, for instance in medical testing.

Impacts of microalgae pre-treatments for improved anaerobic digestion: thermal treatment, thermal hydrolysis, ultrasound and enzymatic hydrolysis.

PubMed

Ometto, Francesco; Quiroga, Gerardo; Pšenička, Pavel; Whitton, Rachel; Jefferson, Bruce; Villa, Raffaella

2014-11-15

Anaerobic digestion (AD) of microalgae is primarily inhibited by the chemical composition of their cell walls containing biopolymers able to resist bacterial degradation. Adoption of pre-treatments such as thermal, thermal hydrolysis, ultrasound and enzymatic hydrolysis have the potential to remove these inhibitory compounds and enhance biogas yields by degrading the cell wall, and releasing the intracellular algogenic organic matter (AOM). This work investigated the effect of four pre-treatments on three microalgae species, and their impact on the quantity of soluble biomass released in the media and thus on the digestion process yields. The analysis of the composition of the soluble COD released and of the TEM images of the cells showed two main degradation actions associated with the processes: (1) cell wall damage with the release of intracellular AOM (thermal, thermal hydrolysis and ultrasound) and (2) degradation of the cell wall constituents with the release of intracellular AOM and the solubilisation of the cell wall biopolymers (enzymatic hydrolysis). As a result of this, enzymatic hydrolysis showed the greatest biogas yield increments (>270%) followed by thermal hydrolysis (60-100%) and ultrasounds (30-60%). Copyright © 2014 Elsevier Ltd. All rights reserved.

Analysis of profitability in the diagnosis of allergy to beta-lactam antibiotics.

PubMed

Ferré-Ybarz, L; Salinas Argente, R; Gómez Galán, C; Duocastella Selvas, P; Nevot Falcó, S

2015-01-01

Drug allergy is the third most common reason for allergy consultations. There is a tendency to call any adverse drug reaction (ADR) allergic, even without confirmatory allergy study. (1) Evaluate time of resolution allergy to beta-lactam's study in a sample of 100 patients. (2) Analyse cost-effectiveness of current diagnostic study (skin tests, specific IgE and drug provocation test (DPT)). (3) Describe type and frequency of ADRs in adult/paediatric patients. (4) Compare cost of complete study with DPT. (5) Assess the need to restructure current study methodology according to results obtained. The study is part of a strategic plan of the allergy department (2005-2010). Patients with suspected allergy to beta-lactams were included. Procedures performed: medical history, specific IgE, skin tests and DPT. Cost/patient analysis. Cost of protocol analysis for current diagnostic/direct DPT. 100 patients were studied, 52 females/48 males; 43 children/57 adults. 89 cutaneous, 4 anaphylaxis, 3 vasovagal reactions, 6 non-specific symptoms and 4 not recalled. Allergy was confirmed in six patients (only one child). Complete-study cost: 149.3 Euros/patient. DPT-study cost: 97.19 Euros/patient (34.9% less). Resolution time 9-13 months, absenteeism 28.04%. In the series studied, diagnosis of allergy to beta-lactams was confirmed in 6% of patients (2.3% of paediatric patients). After analysing results and cost of the study we believe that we should propose a specific diagnostic algorithm in those paediatric patients without suspected IgE-mediated ADR, and for those patients direct DPT should be conducted. This will reduce cost/patient (-34.9%), time of resolution and absenteeism. Copyright © 2014 SEICAP. Published by Elsevier Espana. All rights reserved.

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

Analysis of different beta-lactams antibiotics in pharmaceutical preparations using micellar electrokinetic capillary chromatography.

PubMed

Pérez, M I Bailón; Rodríguez, L Cuadros; Cruces-Blanco, C

2007-01-17

The potential of micellar electrokinetic capillary chromatography (MEKC) for analyzing nine beta-lactams antibiotics (cloxacillin, dicloxacillin, oxacillin, penicillin G, penicillin V, ampicillin, nafcillin, piperacillin, amoxicillin) in different pharmaceutical preparations, have been demonstrated. An experimental design strategy has been applied to optimize the main variables: pH and buffer concentration, concentration of the micellar medium, separation voltage and capillary temperature. Borate buffer (26mM) at pH 8.5 containing 100mM sodium dodecyl sulphate (SDS) was used as the background electrolyte. The method was validated. Linearity, limit of detection and quantitation and precision were established for each compound. The analysis of some of the beta-lactams in Orbenin capsules, Britapen tables and in Veterin-Micipen injectable, all used in human and veterinary medicine, have demonstrated the applicability of these technique for quality control in the pharmaceutical industry.

Kinetic mechanism and structural requirements of the amine-catalyzed decarboxylation of oxaloacetic acid.

PubMed

Thalji, Nabil K; Crowe, William E; Waldrop, Grover L

2009-01-02

The kinetic and chemical mechanism of amine-catalyzed decarboxylation of oxaloacetic acid at pH 8.0 has been reevaluated using a new and versatile assay. Amine-catalyzed decarboxylation of oxaloacetic acid proceeds via the formation of an imine intermediate, followed by decarboxylation of the intermediate and hydrolysis to yield pyruvate. The decrease in oxaloacetic acid was coupled to NADH formation by malate dehydrogenase, which allowed the rates of both initial carbinolamine formation (as part of the imination step) and decarboxylation to be determined. By comparing the rates observed for a variety of amines and, in particular, diamines, the structural and electronic requirements for diamine-catalyzed decarboxylation at pH 8.0 were identified. At pH 8.0, monoamines were found to be very poor catalysts, whereas some diamines, most notably ethylenediamine, were excellent catalysts. The results indicate that the second amino group of diamines enhances the rate of imine formation by acting as a proton shuttle during the carbinolamine formation step, which enables diamines to overcome high levels of solvation that would otherwise inhibit carbinolamine, and thus imine, formation. The presence of the second amino group may also enhance the rate of the carbinolamine dehydration step. In contrast to the findings of previous reports, the second amino group participates in the reaction by enhancing the rate of decarboxylation via hydrogen-bonding to the imine nitrogen to either stabilize the negative charge that develops on the imine during decarboxylation or preferentially stabilize the reactive imine over the unreactive enamine tautomer. These results provide insight into the precise catalytic mechanism of several enzymes whose reactions are known to proceed via an imine intermediate.

Cross-class resistance to non-beta-lactam antimicrobials in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae.

PubMed

Procop, Gary W; Tuohy, Marion J; Wilson, Deborah A; Williams, Delisa; Hadziyannis, Emilia; Hall, Gerri S

2003-08-01

Extended spectrum beta-lactamases are modified beta-lactamase enzymes that impart resistance to third-generation cephalosporins and make all beta-lactam antibiotics and cephalosporins useless for therapy. We compared the antimicrobial susceptibility profiles of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing isolates of Klebsiella pneumoniae. The ESBL producers had significantly diminished susceptibility compared with the non-ESBL producers for gentamicin (P < .001), tobramycin (P < .001), amikacin (P < .005), trimethoprim-sulfamethoxazole (P < .01), ciprofloxacin (P < .001), and nitrofurantoin (P < .001). All isolates were susceptible to imipenem. ESBL-producing K pneumoniae may also be resistant to non-beta-lactam antibiotics. Therefore, susceptibility testing of these isolates is critical for guiding therapy.

Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.

PubMed

Park, Kyung-Eui; Kim, Jun-Dal; Nagashima, Yusuke; Kako, Koichiro; Daitoku, Hiroaki; Matsui, Motoki; Park, Gwi Gun; Fukamizu, Akiyoshi

2014-01-01

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.

Isomerization of 1-O-indol-3-ylacetyl-beta-D-glucose. Enzymatic hydrolysis of 1-O, 4-O, and 6-O-indol-3-ylacetyl-beta-D-glucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex

NASA Technical Reports Server (NTRS)

Kowalczyk, S.; Bandurski, R. S.

1990-01-01

The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-beta-D-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzyme-catalyzed hydrolysis of 4-O and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

PubMed Central

Fouhy, Fiona; O’Connell Motherway, Mary; Fitzgerald, Gerald F.; Ross, R. Paul; Stanton, Catherine; van Sinderen, Douwe; Cotter, Paul D.

2013-01-01

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria. PMID:24324818

Role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins in AmpC expression, β-lactam resistance, and peptidoglycan structure.

PubMed

Ropy, Alaa; Cabot, Gabriel; Sánchez-Diener, Irina; Aguilera, Cristian; Moya, Bartolome; Ayala, Juan A; Oliver, Antonio

2015-07-01

This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of β-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major dd-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as dd-carboxypeptidase only if PBP5 is absent, and their dd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the β-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic β-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, β-lactam

Stereochemical preference toward oncotarget: Design, synthesis and in vitro anticancer evaluation of diastereomeric β-lactams

PubMed Central

Olazarán-Santibáñez, Fabián; Bandyopadhyay, Debasish; Carranza-Rosales, Pilar; Rivera, Gildardo; Balderas-Rentería, Isaías

2017-01-01

Purpose In the battle against cancer discovery of new and novel chemotherapeutic agent demands extreme obligation. Development of anticancer compounds with higher potency and reduced side-effects is timely and challenging. Experimental Design A small series of fourteen diastereomeric β-lactams (seven pairs) were synthesized through multi-step process exploring [2+2] ketene-imine cycloaddition as the key step. Comparative stereochemical preferences were studied through computational docking and validated by in vitro evaluation. β-tubulin was considered as possible molecular target and in vitro anticancer evaluation was conducted against SiHa, B16F10, K562 and Chang cell lines. Caspase-3 activation assay and hematoxylin/eosin staining of the cells were also accomplished. Results Better docking scores of the cis- over the trans-β-lactams indicated favorable β-lactam—β-tubulin interactions in cis-geometry. In vitro (IC50) evaluation confirmed better anticancer activity of the cis-diastereoisomers. Apoptosis-induced cell death was supported by caspase-3 activation study. A cis-β-lactam [(±)-Cis-3-amino-1-phenyl-4-(p-tolyl) azetidin-2-one, 6C] was found to be more active (in vitro) than the marketed natural drug colchicine against SiHa and B16F10 (six times higher potency) cell lines. Reduced toxicity (compared to colchicine) in Chang cells confirmed better site-selectivity (accordingly less side-effects) of 6C than colchicine. Aside from 6C, most of the reported molecules demonstrated good to strong in vitro anticancer activity against SiHa and B16F10 cancer cell lines. Conclusions Stereochemical preferences of the cis-β-lactams over their trans-counterparts, toward the molecular target β-tubulin, was confirmed by docking studies and in vitro anticancer evaluation. Apoptosis was identified as the cause of cell death. The lead 6C exhibited higher potency and selectivity than the marketed drug colchicine both in silico as well as in vitro. PMID:28562328

Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with β-Lactam-Containing Regimens

PubMed Central

Viswanathan, Gopinath; Yadav, Sangya

2017-01-01

ABSTRACT In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the β-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing β-lactams for tuberculosis treatment. PMID:28438925

CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (-) γ-lactam production.

PubMed

Wang, Jianjun; Zhu, Junge; Min, Cong; Wu, Sheng

2014-05-13

γ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened. We prepared two fusion constructs of the γ-lactamase gene gla, which was from Sulfolobus solfataricus P2. These two constructs had Cbd (cellulose binding domain from Clostridium thermocellum) fused at amino or carboxyl terminus of the γ-lactamase. These two constructs were heterogeneously expressed in E. coli rosetta (DE3) as two fusion proteins. Both of them were immobilized well on Avicel (microcrystalline cellulose matrix). The apparent kinetic parameters revealed that carboxyl terminus fused protein (Gla-linker-Cbd) was a better catalyst. The V(max) and k(cat) value of Avicel immobilized Gla-linker-Cbd were 381 U mg⁻¹ and 4.7 × 10⁵ s⁻¹ respectively. And the values of the free Gla-linker-Cbd were 151 U mg⁻¹ and 1.8 × 10⁵ s⁻¹ respectively. These data indicated that the catalytic efficiency of the enzyme was upgraded after immobilization. The immobilized Gla-linker-Cbd had a 10-degree temperature optimum dropping from 80°C to 70°C but it was stable when incubated at 60°C for 48 h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200 mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60 mM. Finally a 10 mL-volume transformation was conducted, in which 0.6 M substrate was hydrolyzed and the resolution was completed within 9 h with a 99.5% ee value. Cellulose is the most

The antioxidant effect of derivatives pyroglutamic lactam

NASA Astrophysics Data System (ADS)

Rohadi, Atisya; Lazim, Azwani Mat; Hasbullah, Siti Aishah

2013-11-01

Diphenylpicrylhydrazyl (DPPH) is widely used for quickly accessing the ability of polyphenols to transfer labile H atoms to radicals. The antioxidant activity of all the synthesized compounds was screened by DPPH method. Compound (4) showed 54% antioxidant potential while all other compounds were found to have moderate to have moderate to mild antioxidant activity ranging from 47-52%. Pyroglutamic lactams have been synthesized stereoselectively in racemic form from levulinic acid as bifunctional adduct using convertible isocyanide in one-pot Ugi 4-center-3-component condensation reaction (U-4C-3CR). The product formed provides biologically interesting products in excellent yields in a short reaction time. The structures of the synthesized compounds were elucidated using spectroscopic data and elemental analysis.

The antioxidant effect of derivatives pyroglutamic lactam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rohadi, Atisya; Lazim, Azwani Mat; Hasbullah, Siti Aishah

Diphenylpicrylhydrazyl (DPPH) is widely used for quickly accessing the ability of polyphenols to transfer labile H atoms to radicals. The antioxidant activity of all the synthesized compounds was screened by DPPH method. Compound (4) showed 54% antioxidant potential while all other compounds were found to have moderate to have moderate to mild antioxidant activity ranging from 47–52%. Pyroglutamic lactams have been synthesized stereoselectively in racemic form from levulinic acid as bifunctional adduct using convertible isocyanide in one-pot Ugi 4-center-3-component condensation reaction (U-4C-3CR). The product formed provides biologically interesting products in excellent yields in a short reaction time. The structures ofmore » the synthesized compounds were elucidated using spectroscopic data and elemental analysis.« less

Increased susceptibility to beta-lactam antibiotics and decreased porin content caused by envB mutations of Salmonella typhimurium.

PubMed Central

Oppezzo, O J; Avanzati, B; Antón, D N

1991-01-01

Isogenic derivatives carrying envB6, envB9, or envB+ alleles were obtained from a strain of Salmonella typhimurium that was partially resistant to mecillinam, a beta-lactam antibiotic specific for penicillin-binding protein 2 (PBP 2). Testing of the isogenic strains with several antibacterial agents demonstrated that envB mutations either increased resistance (mecillinam) or did not affect the response (imipemen) to beta-lactams that act primarily on PBP 2, while susceptibilities to beta-lactams that act on PBP 1B, PBP 3, or both were increased. Furthermore, the susceptibilities of envB strains to hydrophobic compounds such as rifampin, novobiocin, or chloramphenicol were not modified, even though their susceptibilities to deoxycholate and crystal violet were enhanced. Outer cell membranes of envB mutants presented a 50% reduction in protein content compared with that of the isogenic envB+ strains, and OmpF and OmpD porins were particularly affected by the reduction. No alteration in the amount or pattern of periplasmic proteins was noticed, and lipopolysaccharides from envB mutants appeared to be normal by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. By using derivatives that produced a plasmid-encoded beta-lactamase, it was demonstrated that envB cells are slightly less permeable to cephalothin than envB+ bacteria are. It is concluded that the high susceptibility of envB mutants to beta-lactams is due to the increased effectiveness of the antibiotics on PBP 1B, PBP 3, or both. Images PMID:1656857

Influence of thermal processing on hydrolysis and stability of folate poly-gamma-glutamates in broccoli (Brassica oleracea var. italica), carrot (Daucus carota) and tomato (Lycopersicon esculentum).

PubMed

Munyaka, Ann Wambui; Verlinde, Philippe; Mukisa, Ivan Muzira; Oey, Indrawati; Van Loey, Ann; Hendrickx, Marc

2010-04-14

The folate poly-gamma-glutamate profile, their concentrations, and hydrolysis by endogenous gamma-glutamyl hydrolase (GGH) were evaluated in broccoli, carrot and tomato. Further studies on the effect of time and temperature on folate poly-gamma-glutamate hydrolysis and stability were carried out in broccoli since this vegetable showed the highest long-chain and total folate poly-gamma-glutamate concentration. The evolution of l-ascorbic acid, total phenols and Trolox equivalent antioxidant capacity (TEAC) values was evaluated in parallel. Upon thermal inactivation of GGH prior to crushing, it was observed that broccoli, carrot and tomato contained poly-gamma-glutamates with one to seven glutamate residues but differed in the predominant poly-gamma-glutamates. Crushing of raw broccoli, carrot and tomato resulted in significant poly-gamma-glutamate profile changes in broccoli and carrot (indicating GGH-catalyzed hydrolysis) but not in tomato. In this study, the actual crushing of raw broccoli matrix had a greater effect on folate poly-gamma-glutamate hydrolysis than incubation conditions (0-30 min at 25-55 degrees C). During treatments at 25-140 degrees C, folate retention was higher at 80 and 100 degrees C than at the other temperatures. A similar trend in thermal stability was observed for folates, vitamin C, total phenols and TEAC value, an indication that conditions that result in endogenous antioxidants degradation might also result in folate degradation.

β-Lactam Antibiotics with a High Affinity for PBP2 Act Synergistically with the FtsZ-Targeting Agent TXA707 against Methicillin-Resistant Staphylococcus aureus

PubMed Central

Ferrer-González, Edgar; Kaul, Malvika; Parhi, Ajit K.; LaVoie, Edmond J.

2017-01-01

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. We have developed a promising new FtsZ-targeting agent (TXA707) with potent activity against MRSA isolates resistant to current standard-of-care antibiotics. We present here results that demonstrate differing extents of synergy between TXA707 and a broad range of β-lactam antibiotics (including six cephalosporins, two penicillins, and two carbapenems) against MRSA. To explore whether there is a correlation between the extent of synergy and the preferential antibacterial target of each β-lactam, we determined the binding affinities of the β-lactam antibiotics for each of the four native penicillin-binding proteins (PBPs) of S. aureus using a fluorescence anisotropy competition assay. A comparison of the resulting PBP binding affinities with our corresponding synergy results reveals that β-lactams with a high affinity for PBP2 afford the greatest degree of synergy with TXA707 against MRSA. In addition, we present fluorescence and electron microscopy studies that suggest a potential mechanism underlying the synergy between TXA707 and the β-lactam antibiotics. In this connection, our microscopy results show a disruption of septum formation in TXA707-treated MRSA cells, with a concomitant mislocalization of the PBPs from midcell to nonproductive peripheral sites. Viewed as a whole, our results indicate that PBP2-targeting β-lactam antibiotics are optimal synergistic partners with FtsZ-targeting agents for use in combination therapy of MRSA infections. PMID:28630190

Efficacy of β-lactam/β-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis

PubMed Central

Mishra, Saurabh; Shukla, Prashant; Bhaskar, Ashima; Anand, Kushi; Baloni, Priyanka; Jha, Rajiv Kumar; Mohan, Abhilash; Rajmani, Raju S; Nagaraja, Valakunja; Chandra, Nagasuma; Singh, Amit

2017-01-01

Mycobacterium tuberculosis (Mtb) expresses a broad-spectrum β-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, β-lactams. Nonetheless, β-lactams showed mycobactericidal activity in combination with β-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to β-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of β-lactam resistance (e.g. β-lactamase) and redox potential in Mtb. We also describe the role of WhiB4 in coordinating the activity of β-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb. Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions. DOI: http://dx.doi.org/10.7554/eLife.25624.001 PMID:28548640

Simultaneous determination of nine β-lactam antibiotics in human plasma by an ultrafast hydrophilic-interaction chromatography-tandem mass spectrometry.

PubMed

Abdulla, Alan; Bahmany, Soma; Wijma, Rixt A; van der Nagel, Bart C H; Koch, Birgit C P

2017-08-15

Contemporary β-lactam antibiotic dosing is debatable in severely ill patients, since the occurrence of pathophysiological changes in critical illness can result in great inter-individual variability. Therapeutic drug monitoring (TDM) is a commonly used dosing strategy to optimize exposure and thereby minimize toxicity and maximize the efficacy. Currently, TDM of β-lactam antibiotics is rarely performed, due to poor availability in clinical practice. We describe an ultrafast Hydrophilic-Interaction Chromatography (HILIC) based UPLC-MS/MS method for the determination of amoxicillin, benzylpenicillin, cefotaxime, cefuroxime, ceftazidime, flucloxacillin, imipenem, meropenem and piperacillin in human plasma. This method involves simple sample preparation steps and was comprehensively validated according to standard FDA guidelines. For all analytes, mean accuracy and precision values were within the acceptance value. The lower and upper limits of quantification were found to be sufficient to cover the therapeutic range for all antibiotics. Finally, the method was successfully applied in a large pharmacokinetic study performed in the intensive care setting, and the feasibility of the analytical procedure was demonstrated in routine clinical practice. To the best of our knowledge, we report here the first HILIC-based UPLC-MS/MS assay for the determination of β-lactam antibiotics in human plasma. This simple, sensitive and ultrafast assay requires small-volume samples and can easily be implemented in clinical laboratories to promote the TDM of β-lactam antibiotics. Copyright © 2017 Elsevier B.V. All rights reserved.

β-Lactam Combinations with Vancomycin Show Synergistic Activity against Vancomycin-Susceptible Staphylococcus aureus, Vancomycin-Intermediate S. aureus (VISA), and Heterogeneous VISA.

PubMed

Tran, Kieu-Nhi; Rybak, Michael J

2018-06-01

Increasing utilization of vancomycin due to the high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections has led to the emergence of vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) strains. In vitro data suggest the potential for potent synergy between several beta-lactams and vancomycin. The objective of this study is to evaluate the synergy between beta-lactams and vancomycin against MRSA that is vancomycin susceptible, vancomycin-susceptible Staphylococcus aureus (VSSA), hVISA, and VISA. Fifty randomly selected clinical MRSA strains with various susceptibility levels to vancomycin were evaluated for vancomycin alone and vancomycin in combination with various concentrations of cefazolin (CFZ), cefepime (FEP), ceftaroline (CPT), and nafcillin (NAF). The potential for synergy was assessed by 24-h time-kill studies. Beta-lactams reduced vancomycin MIC values against all strains (4- to 16-fold reduction). In time-kill studies against MRSA, CFZ, FEP, CPT, and NAF all demonstrated similar degrees of killing at 24 h, and all showed synergistic activity with vancomycin against VSSA, hVISA, and VISA. Each of these combinations was also superior to any single agent against isolates of all three phenotypes, and each was bactericidal ( P < 0.001 for all comparisons). All single-agent exposures demonstrated no activity at 24 h. The combination of vancomycin and beta-lactams significantly improved antibacterial activity against VSSA, hVISA, and VISA strains compared to the activity of any agent alone, supporting the potential use of vancomycin-beta-lactam combination therapy in infections caused by MRSA. Further clinical research is warranted to investigate the synergy of vancomycin against these Staphylococcus strains. Copyright © 2018 American Society for Microbiology.

Hydrolysis Batteries: Generating Electrical Energy during Hydrogen Absorption.

PubMed

Xiao, Rui; Chen, Jun; Fu, Kai; Zheng, Xinyao; Wang, Teng; Zheng, Jie; Li, Xingguo

2018-02-19

The hydrolysis reaction of aluminum can be decoupled into a battery by pairing an Al foil with a Pd-capped yttrium dihydride (YH 2 -Pd) electrode. This hydrolysis battery generates a voltage around 0.45 V and leads to hydrogen absorption into the YH 2 layer. This represents a new hydrogen absorption mechanism featuring electrical energy generation during hydrogen absorption. The hydrolysis battery converts 8-15 % of the thermal energy of the hydrolysis reaction into usable electrical energy, leading to much higher energy efficiency compared to that of direct hydrolysis. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

•OH and e-aq are yet good candidates for demolishing the β-lactam system of a penicillin eliminating the antimicrobial activity

NASA Astrophysics Data System (ADS)

Szabó, László; Tóth, Tünde; Rácz, Gergely; Takács, Erzsébet; Wojnárovits, László

2016-07-01

Tracking the pharmacophore of a drug subjected to advanced oxidation is essential for evaluating the efficiency of the process in terms of wastewater treatment. From this standpoint, the •OH and eaq- induced deactivation mechanism of amoxicillin, a penicillin derivative was investigated in dilute aqueous solution using pulse- and gamma-radiolysis techniques. Based on IR measurements, •OH and eaq- destroys the β-lactam system of amoxicillin with ~55% and ~84% efficiency, respectively. In aerated solution the elimination of the pharmacophore was slightly impaired since the reaction pathway of the ring-opening was disturbed owing to the reactivity of O2 and O2• - toward the intermediates of sulfur oxidation. The high potency of eaq- for β-lactam deactivation is attributed to the enhanced electron deficiency of the carbonyl carbon inside the lactam ring.

Electrospray ionization mass spectrometry for the hydrolysis complexes of cisplatin: implications for the hydrolysis process of platinum complexes.

PubMed

Feifan, Xie; Pieter, Colin; Jan, Van Bocxlaer

2017-07-01

Non-enzyme-dependent hydrolysis of the drug cisplatin is important for its mode of action and toxicity. However, up until today, the hydrolysis process of cisplatin is still not completely understood. In the present study, the hydrolysis of cisplatin in an aqueous solution was systematically investigated by using electrospray ionization mass spectrometry coupled to liquid chromatography. A variety of previously unreported hydrolysis complexes corresponding to monomeric, dimeric and trimeric species were detected and identified. The characteristics of the Pt-containing complexes were investigated by using collision-induced dissociation (CID). The hydrolysis complexes demonstrate distinctive and correlative CID characteristics, which provides tools for an informative identification. The most frequently observed dissociation mechanism was sequential loss of NH 3 , H 2 O and HCl. Loss of the Pt atom was observed as the final step during the CID process. The formation mechanisms of the observed complexes were explored and experimentally examined. The strongly bound dimeric species, which existed in solution, are assumed to be formed from the clustering of the parent compound and its monohydrated or dihydrated complexes. The role of the electrospray process in the formation of some of the observed ions was also evaluated, and the electrospray ionization-related cold clusters were identified. The previously reported hydrolysis equilibria were tested and subsequently refined via a hydrolysis study resulting in a renewed mechanistic equilibrium system of cisplatin as proposed from our results. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Infusional β-lactam antibiotics in febrile neutropenia: has the time come?

PubMed

Abbott, Iain J; Roberts, Jason A

2012-12-01

Febrile neutropenia presents a clinical challenge in which timely and appropriate antibiotic exposure is crucial. In the context of altered pharmacokinetics and rising bacterial resistance, standard antibiotic doses are unlikely to be sufficient. This review explores the potential utility of altered dosing approaches of β-lactam antibiotics to optimize treatment in febrile neutropenia. There is a dynamic relationship between the antibiotic, the infecting pathogen, and the host. Great advancements have been made in the understanding of the pharmacokinetic changes in critical illness and the pharmacodynamic relationships of antibiotics in these settings. Antibiotic treatment in febrile neutropenia is becoming increasingly difficult. Patients are of higher acuity, receive more intensive chemotherapy regimens leading to prolonged neutropenia, and are often exposed to multiple antibiotic courses. These patients display significant variability in antibiotic clearances and increases in volume of distribution compared with standard ward-based patients. Rising antibiotic resistance and a lack of new antibiotics in production have prompted alternative dosing strategies based on pharmacokinetic/pharmacodynamic data, such as extended or continuous infusions of β-lactam antibiotics, to maximize the likelihood of treatment success. A definitive study that describes a mortality benefit of such dosing regimens remains elusive and the theoretical advantages require testing in well designed clinical trials.

Direct oral provocation tests in non-immediate mild cutaneous reactions related to beta-lactam antibiotics.

PubMed

Vezir, Emine; Dibek Misirlioglu, Emine; Civelek, Ersoy; Capanoglu, Murat; Guvenir, Hakan; Ginis, Tayfur; Toyran, Muge; Kocabas, Can N

2016-02-01

Skin testing has a limited role in the diagnosis of non-immediate beta-lactam hypersensitivity in children. The aim of this study was to report the results of oral provocation tests performed without skin tests in children with non-immediate mild cutaneous reactions without systemic symptoms caused by beta-lactam antibiotics. Oral provocation tests with suspected antibiotics were performed to patients with non-immediate mild cutaneous reactions without systemic symptoms caused by beta-lactam antibiotics. Skin tests were not performed before provocation tests. A total of five doses were administered with half-an-hour intervals in increasing doses. Provocation was continued for 5 days. A total of 119 patients with a median age of 4.3 (IQR: 2-7.5) years, of whom 58% were males, were included in the study. Amoxicillin-clavulanic acid was the most frequently responsible agent in 87 (73.1%) patients, and most common type of rash was maculopapular in 74 (62.2%) patients. Four patients (3.4%) had an urticarial reaction during the provocation test. We did not experience any severe reactions during oral provocation test without previous skin tests performed to children with non-immediate mild cutaneous reactions without systemic symptoms. Omitting skin tests before oral provocation test in this group of children can help decreasing the burden of allergy clinics and alleviating the discomfort of children. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Surface-promoted hydrolysis of 2,4,6-trinitrotoluene and 2,4-dinitroanisole on pyrogenic carbonaceous matter.

PubMed

Ding, Kai; Byrnes, Cory; Bridge, Jarrod; Grannas, Amanda; Xu, Wenqing

2018-04-01

This study investigates the fate of sorbed nitroaromatics on the surface of pyrogenic carbonaceous matter (PCM) to assess the feasibility of a PCM-promoted hydrolysis. The degradation of two nitroaromatic compounds, 2,4,6-trinitrotoluene (TNT) and 2,4-dinitroanisole, was observed at pH 7 in the presence of graphite powder, a model PCM. By contrast, no decay occurred without graphite. Using TNT as a model compound, our results suggest that TNT decay demonstrated a strong pH dependence, with no reaction at pH 3-5 but rapid degradation at pH 6-10. Moreover, by fitting TNT decay at different pH conditions along with its sorption kinetics to the Langmuir Kinetic Model, our results suggest that the base-catalyzed hydrolysis was important. The activation energy for TNT decay was obtained by measuring reaction rates at different temperatures with or without graphite and no significant difference was observed. However, the addition of tetramethylammonium cation was able to promote TNT decay possibly due to its ability to attract more OH - from the aqueous solution, leading to an increase in the sorbed OH - concentrations. Nitrite and a Meisenheimer complex were identified as degradation products for TNT. Other PCM, such as biochar, also demonstrated a comparable ability in promoting TNT decay at pH 7. Furthermore, a rapid degradation of TNT at pH 7 was observed when biochar was used as a soil amendment (4% by weight). Our results suggest that PCM can facilitate TNT and 2,4-dinitroanisole decay via a surface-promoted hydrolysis at neutral pH conditions, suggesting a promising alternative for in situ soil remediation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of a new family of conformationally restricted peptides as potent nucleators of beta-turns. Design, synthesis, structure, and biological evaluation of a beta-lactam peptide analogue of melanostatin.

PubMed

Palomo, Claudio; Aizpurua, Jesus M; Benito, Ana; Miranda, José Ignacio; Fratila, Raluca M; Matute, Carlos; Domercq, Maria; Gago, Federico; Martin-Santamaria, Sonsoles; Linden, Anthony

2003-12-31

Novel enantiopure (i)-(beta-lactam)-(Gly)-(i+3) peptide models, defined by the presence of a central alpha-alkyl-alpha-amino-beta-lactam ring placed as the (i+1) residue, have been synthesized in a totally stereocontrolled way by alpha-alkylation of suitable N-[bis(trimethylsilyl)methyl]-beta-lactams. The structural properties of these beta-lactam pseudopeptides have been studied by X-ray crystallography, Molecular Dynamics simulation, and NOESY-restrained NMR simulated annealing techniques, showing a strong tendency to form stable type II or type II' beta-turns either in the solid state or in highly coordinating DMSO solutions. Tetrapeptide models containing syn- or anti-alpha,beta-dialkyl-alpha-amino-beta-lactam rings have also been synthesized and their conformations analyzed, revealing that alpha-alkyl substitution is essential for beta-turn stabilization. A beta-lactam analogue of melanostatin (PLG amide) has also been prepared, characterized as a type-II beta-turn in DMSO-d6 solution, and tested by competitive binding assay as a dopaminergic D2 modulator in rat neuron cultured cells, displaying moderate agonist activity in the micromolar concentration range. On the basis of these results, a novel peptidomimetic design concept, based on the separation of constraint and recognition elements, is proposed.

Effects of beta-lactamases and omp mutation on susceptibility to beta-lactam antibiotics in Escherichia coli.

PubMed Central

Hiraoka, M; Okamoto, R; Inoue, M; Mitsuhashi, S

1989-01-01

Four types of beta-lactamases consisting of a penicillinase type I (TEM-1), a penicillinase type II (OXA-1), a cephalosporinase of Citrobacter freundii, and a cephalosporinase of Proteus vulgaris were introduced into Escherichia coli MC4100 and its omp mutants, MH1160 (MC4100 ompR1) and MH760 (MC4100 ompR2), by transformation. Effects of the combination of the omp mutations and these beta-lactamases on the susceptibility of E. coli strains were studied with 15 beta-lactam antibiotics including cephalosporins, cephamycins, penicillins, imipenem, and aztreonam. The ompR1 mutant, MH1160, lacks OmpF and OmpC, and it showed reduced susceptibility to 11 of the 15 beta-lactam agents. The reduction in susceptibility to cefoxitin, moxalactam, and flomoxef was much greater than reduction in susceptibility to the other agents. When the ompR1 mutant produced the cephalosporinase of C. freundii, the susceptibility of the mutant to 12 of the 15 beta-lactam antibiotics decreased. The reduction in susceptibility of MH1160 to 10 of the 12 agents affected by the enzyme was two- to fourfold greater than that observed in MC4100. Such a synergistic effect was also observed with the cephalosporinase of P. vulgaris and ompR1 mutation against six cephalosporins, moxalactam, and aztreonam. Images PMID:2658786

Discovery of wall teichoic acid inhibitors as potential anti-MRSA β-lactam combination agents.

PubMed

Wang, Hao; Gill, Charles J; Lee, Sang H; Mann, Paul; Zuck, Paul; Meredith, Timothy C; Murgolo, Nicholas; She, Xinwei; Kales, Susan; Liang, Lianzhu; Liu, Jenny; Wu, Jin; Santa Maria, John; Su, Jing; Pan, Jianping; Hailey, Judy; Mcguinness, Debra; Tan, Christopher M; Flattery, Amy; Walker, Suzanne; Black, Todd; Roemer, Terry

2013-02-21

Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore β-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA β-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of novel amide amino-β-lactam derivatives as cholesterol absorption inhibitors.

PubMed

Dražić, Tonko; Sachdev, Vinay; Leopold, Christina; Patankar, Jay V; Malnar, Martina; Hećimović, Silva; Levak-Frank, Sanja; Habuš, Ivan; Kratky, Dagmar

2015-05-15

The β-lactam cholesterol absorption inhibitor ezetimibe is so far the only representative of this class of compounds on the market today. The goal of this work was to synthesize new amide ezetimibe analogs from trans-3-amino-(3R,4R)-β-lactam and to test their cytotoxicity and activity as cholesterol absorption inhibitors. We synthesized six new amide ezetimibe analogs. All new compounds exhibited low toxicity in MDCKIIwt, hNPC1L1/MDCKII and HepG2 cell lines and showed significant inhibition of cholesterol uptake in hNPC1L1/MDCKII cells. In addition, we determined the activity of the three compounds to inhibit cholesterol absorption in vivo. Our results demonstrate that these compounds considerably reduce cholesterol concentrations in liver and small intestine of mice. Thus, our newly synthesized amide ezetimibe analogs are cholesterol absorption inhibitors in vitro and in vivo. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mechanism of Polyphosphates Hydrolysis by Purified Polyphosphatases from the Dorsal Muscle of Silver Carp (Hypophthalmichthys Molitrix) as Detected by ³¹P NMR.

PubMed

Liu, Wei; Xu, Meng; Zhang, Yawei; Wang, Fulong; Hui, Teng; Cui, Baowei; Guo, Xiuyun; Peng, Zengqi

2015-11-01

The dynamic hydrolysis of tetrasodium pyrophosphate (TSPP), sodium tripolyphosphate (STPP) and polyphosphate compound, which was catalyzed by purified pyrophosphatase (PPase) and myosin- tripolyphosphatase (TPPase) from the silver carp dorsal muscle, was studied using (31) P NMR spectroscopy. In the PPase + TSPP system, the pyrophosphate (PP) was hydrolyzed quickly and completely within 8 h and the hydrolysis rate of PP was 12.51%/h. In the TPPase + STPP system, the first-order hydrolysis of tripolyphosphate (TPP) was not yet complete after 48 h, and the derived PP accumulated progressively. Given the coexistence of PPase and TPPase, only 1.20% of TPP in STPP alone remained after 48 h. However, the generation rate of Pi in the polyphosphate compound (TSPP: STPP: sodium hexametaphosphate = 1: 8: 1) was 0.76%/h, which was less than 0.88%/h in STPP alone. In the presence of polyphosphatases, the decrease of PP or TPP content in the polyphosphate compound was not as rapid as that in TSPP or STPP alone due to the inhibitory effect of PP on TPPase and the effect of low system pH on PPase. The understanding of polyphosphates hydrolysis mechanism was capable of developing the advanced polyphosphate mixture in order to reduce the phosphate residue in fish products. Processors appreciate the proven value of phosphates to increase the yield and functionality of the fish meat products. Our studies showed that the hydrolysis rate of PP or TPP in the blend was slower than that of polyphosphate alone. Thus, it is likely that the addition of PP and TPP in a polyphosphate blend had a prolonged interaction with proteins in fish meat processing and the effectiveness of polyphosphates was enhanced. © 2015 Institute of Food Technologists®

Redeploying β-Lactam Antibiotics as a Novel Antivirulence Strategy for the Treatment of Methicillin-Resistant Staphylococcus aureus Infections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Elaine M.; Rudkin, Justine K.; Coughlan, Simone

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. Here, the beta-lactam antibiotic oxacillin was shown to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Oxacillin-mediated repression of the Agr quorum-sensing system and altered cell wall architecture, was associated with reduced cytolytic activity and increased susceptibility to host killing. These findings support the inclusion of -lactam antibiotics as an adjunctive anti-virulence therapy in the treatment of MRSA infections, with the potential to significantly improve patient outcomes in a safe, cost effective manner.

Crystal Structures of Covalent Complexes of [beta]-Lactam Antibiotics with Escherichia coli Penicillin-Binding Protein 5: Toward an Understanding of Antibiotic Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicola, George; Tomberg, Joshua; Pratt, R.F.

Penicillin-binding proteins (PBPs) are the molecular targets for the widely used {beta}-lactam class of antibiotics, but how these compounds act at the molecular level is not fully understood. We have determined crystal structures of Escherichia coli PBP 5 as covalent complexes with imipenem, cloxacillin, and cefoxitin. These antibiotics exhibit very different second-order rates of acylation for the enzyme. In all three structures, there is excellent electron density for the central portion of the {beta}-lactam, but weak or absent density for the R1 or R2 side chains. Areas of contact between the antibiotics and PBP 5 do not correlate with themore » rates of acylation. The same is true for conformational changes, because although a shift of a loop leading to an electrostatic interaction between Arg248 and the {beta}-lactam carboxylate, which occurs completely with cefoxitin and partially with imipenem and is absent with cloxacillin, is consistent with the different rates of acylation, mutagenesis of Arg248 decreased the level of cefoxitin acylation only 2-fold. Together, these data suggest that structures of postcovalent complexes of PBP 5 are unlikely to be useful vehicles for the design of new covalent inhibitors of PBPs. Finally, superimposition of the imipenem-acylated complex with PBP 5 in complex with a boronic acid peptidomimetic shows that the position corresponding to the hydrolytic water molecule is occluded by the ring nitrogen of the {beta}-lactam. Because the ring nitrogen occupies a similar position in all three complexes, this supports the hypothesis that deacylation is blocked by the continued presence of the leaving group after opening of the {beta}-lactam ring.« less

Trihalomethane hydrolysis in drinking water at elevated temperatures.

PubMed

Zhang, Xiao-Lu; Yang, Hong-Wei; Wang, Xiao-Mao; Karanfil, Tanju; Xie, Yuefeng F

2015-07-01

Hydrolysis could contribute to the loss of trihalomethanes (THMs) in the drinking water at elevated temperatures. This study was aimed at investigating THM hydrolysis pertaining to the storage of hot boiled water in enclosed containers. The water pH value was in the range of 6.1-8.2 and the water temperature was varied from 65 to 95 °C. The effects of halide ions, natural organic matter, and drinking water matrix were investigated. Results showed that the hydrolysis rates declined in the order following CHBrCl2 > CHBr2Cl > CHBr3 > CHCl3. THM hydrolysis was primarily through the alkaline pathway, except for CHCl3 in water at relatively low pH value. The activation energies for the alkaline hydrolysis of CHCl3, CHBrCl2, CHBr2Cl and CHBr3 were 109, 113, 115 and 116 kJ/mol, respectively. No hydrolysis intermediates could accumulate in the water. The natural organic matter, and probably other constituents, in drinking water could substantially decrease THM hydrolysis rates by more than 50%. When a drinking water was at 90 °C or above, the first order rate constants for THM hydrolysis were in the magnitude of 10(-2)‒10(-1) 1/h. When the boiled real tap water was stored in an enclosed container, THMs continued increasing during the first few hours and then kept decreasing later on due to the competition between hydrolysis and further formation. The removal of THMs, especially brominated THMs, by hydrolysis would greatly reduce one's exposure to disinfection by-products by consuming the boiled water stored in enclosed containers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Kinetics of PTEN-mediated PI(3,4,5)P3 hydrolysis on solid supported membranes

PubMed Central

Liu, Chun; Deb, Sanghamitra; Ferreira, Vinicius S.; Xu, Eric; Baumgart, Tobias

2018-01-01

Phosphatidylinositides play important roles in cellular signaling and migration. Phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) is an important phosphatidylinositide because it acts as a secondary messenger to trigger cell movement and proliferation. A high level of PI(3,4,5)P3 at the plasma membrane is known to contribute to tumorigenesis. One key enzyme that regulates PI(3,4,5)P3 levels at the plasma membrane is phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which dephosphorylates PI(3,4,5)P3 through hydrolysis to form phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). It has been reported that PI(4,5)P2 is involved in positive feedback in the PI(3,4,5)P3 hydrolysis by PTEN. However, how PI(3,4,5)P3 dephosphorylation by PTEN is regulated, is still under debate. How other PI(3,4,5)P3-binding proteins affect the dephosphorylation kinetics catalyzed by PTEN also remains unclear. Here, we develop a fluorescent-protein biosensor approach to study how PI(3,4,5)P3 dephosphorylation is regulated by PTEN as well as its membrane-mediated feedback mechanisms. Our observation of sigmoidal kinetics of the PI(3,4,5)P3 hydrolysis reaction supports the notion of autocatalysis in PTEN function. We developed a kinetic model to describe the observed reaction kinetics, which allowed us to i) distinguish between membrane-recruitment and allosteric activation of PTEN by PI(4,5)P2, ii) account for the influence of the biosensor on the observed reaction kinetics, and iii) demonstrate that all of these mechanisms contribute to the kinetics of PTEN-mediated catalysis. PMID:29447222

Kinetics of PTEN-mediated PI(3,4,5)P3 hydrolysis on solid supported membranes.

PubMed

Liu, Chun; Deb, Sanghamitra; Ferreira, Vinicius S; Xu, Eric; Baumgart, Tobias

2018-01-01

Phosphatidylinositides play important roles in cellular signaling and migration. Phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) is an important phosphatidylinositide because it acts as a secondary messenger to trigger cell movement and proliferation. A high level of PI(3,4,5)P3 at the plasma membrane is known to contribute to tumorigenesis. One key enzyme that regulates PI(3,4,5)P3 levels at the plasma membrane is phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which dephosphorylates PI(3,4,5)P3 through hydrolysis to form phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). It has been reported that PI(4,5)P2 is involved in positive feedback in the PI(3,4,5)P3 hydrolysis by PTEN. However, how PI(3,4,5)P3 dephosphorylation by PTEN is regulated, is still under debate. How other PI(3,4,5)P3-binding proteins affect the dephosphorylation kinetics catalyzed by PTEN also remains unclear. Here, we develop a fluorescent-protein biosensor approach to study how PI(3,4,5)P3 dephosphorylation is regulated by PTEN as well as its membrane-mediated feedback mechanisms. Our observation of sigmoidal kinetics of the PI(3,4,5)P3 hydrolysis reaction supports the notion of autocatalysis in PTEN function. We developed a kinetic model to describe the observed reaction kinetics, which allowed us to i) distinguish between membrane-recruitment and allosteric activation of PTEN by PI(4,5)P2, ii) account for the influence of the biosensor on the observed reaction kinetics, and iii) demonstrate that all of these mechanisms contribute to the kinetics of PTEN-mediated catalysis.

Preparation of Rh/Ag bimetallic nanoparticles as effective catalyst for hydrogen generation from hydrolysis of KBH4

NASA Astrophysics Data System (ADS)

Huang, Liang; Jiao, Chengpeng; Wang, Liqiong; Huang, Zili; Liang, Feng; Liu, Simin; Wang, Yuhua; Zhang, Haijun; Zhang, Shaowei

2018-01-01

ISOBAM-104 protected Rh/Ag bimetallic nanoparticles (NPs) with average diameter less than 3.0 nm were synthesized by a co-reduction method. Ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), high-resolution TEM and x-ray photoelectron spectroscopy (XPS) were employed to characterize the structure, particle size, and electronic structure of the prepared bimetallic NPs. The catalytic activities of prepared bimetallic NPs for hydrogen generation from hydrolysis of a basic KBH4 solution were evaluated in detail. The results indicated that as-prepared Rh/Ag bimetallic NPs showed a higher catalytic activity than corresponding monometallic NPs. Among all the monometallic NPs and bimetallic NPs, Rh80Ag20 bimetallic NPs exhibited the highest catalytic activity with a value of 6010 mol-H2·h-1·mol-catalyst-1 at pH = 12 and 303 K. The high catalytic activities of Rh/Ag bimetallic NPs could be attributed to presence of negatively charged Rh atoms and positively charged Ag atoms, which is supported by the results of XPS and density functional theory calculation. Based on the kinetic study, the apparent activation energy for the hydrolysis reaction of the basic KBH4 solution catalyzed by Rh80Ag20 bimetallic NPs was about 47.0 ± 3.9 kJ mol-1.

2-aminoimidazoles potentiate ß-lactam antimicrobial activity against Mycobacterium tuberculosis by reducing ß-lactamase secretion and increasing cell envelope permeability

PubMed Central

Obregón-Henao, Andrés; Ackart, David F.; Podell, Brendan K.; Belardinelli, Juan M.; Jackson, Mary; Nguyen, Tuan V.; Blackledge, Meghan S.; Melander, Roberta J.; Melander, Christian; Johnson, Benjamin K.; Abramovitch, Robert B.

2017-01-01

There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment. PMID:28749949

ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS - ALKALINE HYDROLYSIS

EPA Science Inventory

SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

Microwave Pretreatment For Hydrolysis Of Cellulose

NASA Technical Reports Server (NTRS)

Cullingford, Hatice S.; George, Clifford E.; Lightsey, George R.

1993-01-01

Microwave pretreatment enhances enzymatic hydrolysis of cellulosic wastes into soluble saccharides used as feedstocks for foods, fuels, and other products. Low consumption of energy, high yield, and low risk of proposed hydrolysis process incorporating microwave pretreatment makes process viable alternative to composting.

Hydrolysis kinetics of secoisolariciresinol diglucoside oligomers from flaxseed.

PubMed

Yuan, Jian-Ping; Li, Xin; Xu, Shi-Ping; Wang, Jiang-Hai; Liu, Xin

2008-11-12

Flaxseed is the richest dietary source of the lignan secoisolariciresinol diglucoside (SDG) and contains the largest amount of SDG oligomers, which are often hydrolyzed to break the ester linkages for the release of SDG and the glycosidic bonds for the release of secoisolariciresinol (SECO). The alkaline hydrolysis reaction kinetics of SDG oligomers from flaxseed and the acid hydrolysis process of SDG and other glucosides were investigated. For the kinetic modeling, a pseudo-first-order reaction was assumed. The results showed that the alkaline hydrolysis of SDG oligomers followed first-order reaction kinetics under mild alkaline hydrolytic conditions and that the concentration of sodium hydroxide had a strong influence on the activation energy of the alkaline hydrolysis of SDG oligomers. The results also indicated that the main acid hydrolysates of SDG included secoisolariciresinol monoglucoside (SMG), SECO, and anhydrosecoisolariciresinol (anhydro-SECO) and that the extent and the main hydrolysates of the acid hydrolysis reaction depended on the acid concentration, hydrolysis temperature, and time. In addition, the production and change of p-coumaric acid glucoside, ferulic acid glucoside and their methyl esters and p-coumaric acid, ferulic acid, and their methyl esters during the process of hydrolysis was also investigated.

Effect of hydrolysis on identifying prenatal cannabis exposure

PubMed Central

Gray, Teresa R.; Barnes, Allan J.

2011-01-01

Identification of prenatal cannabis exposure is important due to potential cognitive and behavioral consequences. A two-dimensional gas chromatography–mass spectrometry method for cannabinol, Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 8β,11-dihydroxy-THC, and 11-nor-9-carboxy-THC (THCCOOH) quantification in human meconium was developed and validated. Alkaline, enzymatic, and enzyme–alkaline tandem hydrolysis conditions were optimized with THC- and THCCOOH-glucuronide reference standards. Limits of quantification ranged from 10 to 15 ng/g, and calibration curves were linear to 500 ng/g. Bias and intra-day and inter-day imprecision were <12.3%. Hydrolysis efficiencies were analyte-dependent; THC-glucuronide was effectively cleaved by enzyme, but not base. Conversely, THCCOOH-glucuronide was most sensitive to alkaline hydrolysis. Enzyme–alkaline tandem hydrolysis maximized efficiency for both glucuronides. Identification of cannabinoid-positive meconium specimens nearly doubled following alkaline and enzyme–alkaline hydrolysis. Although no 11-OH-THC glucuronide standard is available, enzymatic hydrolysis improved 11-OH-THC detection in authentic specimens. Maximal identification of cannabis-exposed neonates and the widest range of cannabis biomarkers are achieved with enzyme–alkaline tandem hydrolysis. PMID:20517601

Hydrolysis rate constants at 10-25 °C can be more than doubled by a short anaerobic pre-hydrolysis at 35 °C.

PubMed

Zhang, L; Gao, R; Naka, A; Hendrickx, T L G; Rijnaarts, H H M; Zeeman, G

2016-11-01

Hydrolysis is the first step of the anaerobic digestion of complex wastewater and considered as the rate limiting step especially at low temperature. Low temperature (10-25 °C) hydrolysis was investigated with and without application of a short pre-hydrolysis at 35 °C. Batch experiments were executed using cellulose and tributyrin as model substrates for carbohydrates and lipids. The results showed that the low temperature anaerobic hydrolysis rate constants increased by a factor of 1.5-10, when the short anaerobic pre-hydrolysis at 35 °C was applied. After the pre-hydrolysis phase at 35 °C and decreasing the temperature, no lag phase was observed in any case. Without the pre-hydrolysis, the lag phase for cellulose hydrolysis at 35-10 °C was 4-30 days. Tributyrin hydrolysis showed no lag phase at any temperature. The hydrolysis efficiency of cellulose increased from 40 to 62%, and from 9.6 to 40% after 9.1 days at 15 and 10 °C, respectively, when the pre-hydrolysis at 35 °C was applied. The hydrolysis efficiency of tributyrin at low temperatures with the pre-hydrolysis at 35 °C was similar to those without the pre-hydrolysis. The hydrolytic activity of the supernatant collected from the digestate after batch digestion of cellulose and tributyrin at 35 °C was higher than that of the supernatants collected from the low temperature (≤25 °C) digestates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrolysis of alkaline pretreated banana peel

NASA Astrophysics Data System (ADS)

Fatmawati, A.; Gunawan, K. Y.; Hadiwijaya, F. A.

2017-11-01

Banana peel is one of food wastes that are rich in carbohydrate. This shows its potential as fermentation substrate including bio-ethanol. This paper presented banana peel alkaline pretreatment and enzymatic hydrolysis. The pretreatment was intended to prepare banana peel in order to increase hydrolysis performance. The alkaline pretreatment used 10, 20, and 30% w/v NaOH solution and was done at 60, 70 and 80°C for 1 hour. The hydrolysis reaction was conducted using two commercial cellulose enzymes. The reaction time was varied for 3, 5, and 7 days. The best condition for pretreatment process was one conducted using 30% NaOH solution and at 80°C. This condition resulted in cellulose content of 90.27% and acid insoluble lignin content of 2.88%. Seven-day hydrolysis time had exhibited the highest reducing sugar concentration, which was7.2869 g/L.

Acid hydrolysis of cellulose to yield glucose

DOEpatents

Tsao, George T.; Ladisch, Michael R.; Bose, Arindam

1979-01-01

A process to yield glucose from cellulose through acid hydrolysis. Cellulose is recovered from cellulosic materials, preferably by pretreating the cellulosic materials by dissolving the cellulosic materials in Cadoxen or a chelating metal caustic swelling solvent and then precipitating the cellulose therefrom. Hydrolysis is accomplished using an acid, preferably dilute sulfuric acid, and the glucose is yielded substantially without side products. Lignin may be removed either before or after hydrolysis.

Enzymatic hydrolysis of biomimetic bacterial cellulose-hemicellulose composites.

PubMed

Penttilä, Paavo A; Imai, Tomoya; Hemming, Jarl; Willför, Stefan; Sugiyama, Junji

2018-06-15

The production of biofuels and other chemicals from lignocellulosic biomass is limited by the inefficiency of enzymatic hydrolysis. Here a biomimetic composite material consisting of bacterial cellulose and wood-based hemicelluloses was used to study the effects of hemicelluloses on the enzymatic hydrolysis with a commercial cellulase mixture. Bacterial cellulose synthesized in the presence of hemicelluloses, especially xylan, was found to be more susceptible to enzymatic hydrolysis than hemicellulose-free bacterial cellulose. The reason for the easier hydrolysis could be related to the nanoscale structure of the substrate, particularly the packing of cellulose microfibrils into ribbons or bundles. In addition, small-angle X-ray scattering was used to show that the average nanoscale morphology of bacterial cellulose remained unchanged during the enzymatic hydrolysis. The reported easier enzymatic hydrolysis of bacterial cellulose produced in the presence of wood-based xylan offers new insights to overcome biomass recalcitrance through genetic engineering. Copyright © 2018 Elsevier Ltd. All rights reserved.

Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters.

PubMed

Miyamoto, Naoki; Yoshimura, Miho; Okubo, Yuji; Suzuki-Nagata, Kayo; Tsumuraya, Takeshi; Ito, Nobutoshi; Fujii, Ikuo

2018-05-01

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (Asn H33 , Ser H95 , and Arg L96 ) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (Asn H33 Ala, Ser H95 Ala, and Arg L96 Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry. Copyright © 2017 Elsevier Ltd. All rights reserved.

Discovery of Novel Wall Teichoic Acid Inhibitors as Effective anti-MRSA β-lactam Combination Agents

PubMed Central

Wang, Hao; Gill, Charles J.; Lee, Sang H.; Mann, Paul; Zuck, Paul; Meredith, Timothy C.; Murgolo, Nicholas; She, Xinwei; Kales, Susan; Liang, Lianzhu; Liu, Jenny; Wu, Jin; Maria, John Santa; Su, Jing; Pan, Jianping; Hailey, Judy; Mcguinness, Debra; Tan, Christopher M.; Flattery, Amy; Walker, Suzanne; Black, Todd; Roemer, Terry

2013-01-01

Summary Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore β-lactam efficacy against MRSA. Performing a whole cell pathway-based screen we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole genome sequencing of WTAI resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA β-lactam combination agents. This work also highlights the emerging role of whole genome sequencing in antibiotic mode-of-action and resistance studies. PMID:23438756

Polymeric amylase nanoparticles as a new semi-synthetic enzyme system for hydrolysis of starch.

PubMed

Say, R; Şenay, R Hilal; Biçen, Özlem; Ersöz, Arzu; Şişman Yılmaz, Filiz; Akgöl, Sinan; Denizli, Adil

2013-05-01

α-Amylase (EC 3.2.1.1; α-D-1,4,glucan glucanohydrolase) catalyzes the hydrolysis of α-D-(1,4)-glucosidic linkages in starch, glycogen, and various malto-oligosaccharides, by releasing α-anomeric products. In this study, a novel method has been developed to prepare nanoprotein particles that carry α-amylase as a monomer by using a photosensitive microemulsion polymerization process. The nanostructured α-amylase with photosensitive features have been characterized by fluorescence spectroscopy, transmission electron microscopy (TEM) and Zeta Sizer. The fluorescence intensity of amylase nanoparticles was determined to be 658 a.u. at 610 nm and the average particle size of nanoamylase was found to be about 71.8 nm. Both free α-amylase and nanoparticles were used in the hydrolysis of starch under varying reaction conditions such as pH and temperature that affect enzyme activity and the results were compared to each other. Km values were 0.26 and 0.87 mM and Vmax values were 0.36 IU mg(-1) and 22.32 IU mg(-1) for nanoenzyme and free enzyme, respectively. Then, thermal stability, storage stability and reusability were investigated and according to the results, activity was preserved 60% at 60 °C; 20% at 70-80 °C temperature values and 80% after 105 days storage. Finally after 10 cycles, the activity was preserved 90% and this novel enzymatic polymeric amylase nanoparticle has showed considerable potential as reusable catalyst. Copyright © 2012 Elsevier B.V. All rights reserved.

Bioethanol production: an integrated process of low substrate loading hydrolysis-high sugars liquid fermentation and solid state fermentation of enzymatic hydrolysis residue.

PubMed

Chu, Qiulu; Li, Xin; Ma, Bin; Xu, Yong; Ouyang, Jia; Zhu, Junjun; Yu, Shiyuan; Yong, Qiang

2012-11-01

An integrated process of enzymatic hydrolysis and fermentation was investigated for high ethanol production. The combination of enzymatic hydrolysis at low substrate loading, liquid fermentation of high sugars concentration and solid state fermentation of enzymatic hydrolysis residue was beneficial for conversion of steam explosion pretreated corn stover to ethanol. The results suggested that low substrate loading hydrolysis caused a high enzymatic hydrolysis yield; the liquid fermentation of about 200g/L glucose by Saccharomyces cerevisiae provided a high ethanol concentration which could significantly decrease cost of the subsequent ethanol distillation. A solid state fermentation of enzymatic hydrolysis residue was combined, which was available to enhance ethanol production and cellulose-to-ethanol conversion. The results of solid state fermentation demonstrated that the solid state fermentation process accompanied by simultaneous saccharification and fermentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Two new cassane diterpene lactams from the fruits of Caesalpinia mimosoides Lam.

PubMed

Bi, Dewen; Xia, Guanghui; Li, Yuanping; Liang, Xuesong; Zhang, Lanjun; Wang, Liqin

2018-04-01

Two new cassane ditepenoid lactams, caesmimotam A (1) and B (2), along with eight known compounds (3-10) were isolated from the fruits of Caesalpinia mimosoides Lam. Their structures were identified by 1D and 2D NMR spectral data. Compounds 1 and 2 were evaluated for their cytotoxicity on HL-60, SMMC-7721, A-549, MCF-7 and SW-480 human cancer cell lines, but they were inactive.

Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity*

PubMed Central

Calvez, Philippe; Breukink, Eefjan; Roper, David I.; Dib, Mélanie; Contreras-Martel, Carlos; Zapun, André

2017-01-01

Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism. PMID:28062575

Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor

PubMed Central

Danishuddin, Mohd; Khan, Asad U.

2013-01-01

The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of bla CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with bla CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC50 value (6 nM), high affinity (K i = 0.017 µM) and better acylation efficiency (k +2/K′ = 0.44 µM−1s−1). It forms an acyl-enzyme covalent complex, which is quite stable (k +3 = 0.0057 s−1). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient

A kinetic analysis of the inhibition of FOX-4 β-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic β-lactams and carbapenems

PubMed Central

Papp-Wallace, Krisztina M.; Mallo, Susana; Bethel, Christopher R.; Taracila, Magdalena A.; Hujer, Andrea M.; Fernández, Ana; Gatta, Julian A.; Smith, Kerri M.; Xu, Yan; Page, Malcolm G. P.; Desarbre, Eric; Bou, Germán; Bonomo, Robert A.

2014-01-01

Objectives Class C β-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available β-lactamase inhibitors. In order to find novel scaffolds to inhibit class C β-lactamases, the comparative efficacy of monocyclic β-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam β-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C β-lactamase (pmAmpC). Methods The FOX-4 β-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the β-lactam scaffolds described. Results The Ki values for the monocyclic β-lactams against FOX-4 β-lactamase were 0.04 ± 0.01 μM (aztreonam) and 0.66 ± 0.03 μM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 μM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 μM (ertapenem) to 2.3 ± 0.3 μM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic β-lactams and carbapenems were reacted with FOX-4 β-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. Conclusions Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases. PMID:24235094

Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in α-Amanitin Biosynthesis

DOE PAGES

Luo, Hong; Hong, Sung-Yong; Sgambelluri, R.  Michael; ...

2014-12-04

Amatoxins are ribosomally encoded and posttranslationally modified peptides that account for the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic octapeptide α-amanitin, formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases have been proposed to play a role in α-amanitin posttranslational processing, the exact mechanistic details are not known. In this paper, we show that a specific POP (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPBmore » catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25-mer from the 35-mer propeptide and transpeptidation at the second Pro to produce the cyclic octamer. Finally on the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP.« less

Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction.

PubMed

Sun, Jingjing; Chen, Yiling; Sheng, Jun; Sun, Mi

2015-01-01

To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition.

In vitro and in vivo efficacy of β-lactams against replicating and slowly growing/nonreplicating Mycobacterium tuberculosis.

PubMed

Solapure, Suresh; Dinesh, Neela; Shandil, Radha; Ramachandran, Vasanthi; Sharma, Sreevalli; Bhattacharjee, Deepa; Ganguly, Samit; Reddy, Jitendar; Ahuja, Vijaykamal; Panduga, Vijender; Parab, Manish; Vishwas, K G; Kumar, Naveen; Balganesh, Meenakshi; Balasubramanian, V

2013-06-01

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.

In Vitro and In Vivo Efficacy of β-Lactams against Replicating and Slowly Growing/Nonreplicating Mycobacterium tuberculosis

PubMed Central

Dinesh, Neela; Shandil, Radha; Ramachandran, Vasanthi; Sharma, Sreevalli; Bhattacharjee, Deepa; Ganguly, Samit; Reddy, Jitendar; Ahuja, Vijaykamal; Panduga, Vijender; Parab, Manish; Vishwas, K. G.; Kumar, Naveen; Balganesh, Meenakshi; Balasubramanian, V.

2013-01-01

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model. PMID:23507276

Extended infusion of beta-lactam antibiotics: optimizing therapy in critically-ill patients in the era of antimicrobial resistance.

PubMed

Rizk, Nesrine A; Kanafani, Zeina A; Tabaja, Hussam Z; Kanj, Souha S

2017-07-01

Beta-lactams are at the cornerstone of therapy in critical care settings, but their clinical efficacy is challenged by the rise in bacterial resistance. Infections with multi-drug resistant organisms are frequent in intensive care units, posing significant therapeutic challenges. The problem is compounded by a dearth in the development of new antibiotics. In addition, critically-ill patients have unique physiologic characteristics that alter the drugs pharmacokinetics and pharmacodynamics. Areas covered: The prolonged infusion of antibiotics (extended infusion [EI] and continuous infusion [CI]) has been the focus of research in the last decade. As beta-lactams have time-dependent killing characteristics that are altered in critically-ill patients, prolonged infusion is an attractive approach to maximize their drug delivery and efficacy. Several studies have compared traditional dosing to EI/CI of beta-lactams with regard to clinical efficacy. Clinical data are primarily composed of retrospective studies and some randomized controlled trials. Several reports show promising results. Expert commentary: Reviewing the currently available evidence, we conclude that EI/CI is probably beneficial in the treatment of critically-ill patients in whom an organism has been identified, particularly those with respiratory infections. Further studies are needed to evaluate the efficacy of EI/CI in the management of infections with resistant organisms.

Enzymatic hydrolysis of potato pulp.

PubMed

Lesiecki, Mariusz; Białas, Wojciech; Lewandowicz, Grażyna

2012-01-01

Potato pulp constitutes a complicated system of four types of polysaccharides: cellulose, hemicellulose, pectin and starch. Its composition makes it a potential and attractive raw material for the production of the second generation bioethanol. The aim of this research project was to assess the usefulness of commercial enzymatic preparations for the hydrolysis of potato pulp and to evaluate the effectiveness of hydrolysates obtained in this way as raw materials for ethanol fermentation. Sterilised potato pulp was subjected to hydrolysis with commercial enzymatic preparations. The effectiveness of the preparations declared as active towards only one fraction of potato pulp (separate amylase, pectinase and cellulase activity) and mixtures of these preparations was analysed. The monomers content in hydrolysates was determined using HPLC method. The application of amylolytic enzymes for potato pulp hydrolysis resulted in the release of only 18% of raw material with glucose as the dominant (77%) constituent of the formed product. In addition, 16% galactose was also determined in it. The hydrolysis of the cellulose fraction yielded up to 35% raw material and the main constituents of the obtained hydrolysate were glucose (46%) and arabinose (40%). Simultaneous application of amylolytic, cellulolytic and pectinolytic enzymes turned out to be the most effective way of carrying out the process as its efficiency in this case reached 90%. The obtained hydrolysate contained 63% glucose, 25% arabinose and 12% other simple substances. The application of commercial enzymatic preparations made it possible to perform potato pulp hydrolysis with 90% effectiveness. This was achieved by the application of a complex of amylolytic, cellulolytic and pectinolytic enzymes and the hydrolysate obtained in this way contained, primarily, glucose making it a viable substrate for ethanol fermentation.

Inhibition of cellulase-catalyzed lignocellulosic hydrolysis by iron and oxidative metal ions and complexes.

PubMed

Tejirian, Ani; Xu, Feng

2010-12-01

Enzymatic lignocellulose hydrolysis plays a key role in microbially driven carbon cycling and energy conversion and holds promise for bio-based energy and chemical industries. Cellulases (key lignocellulose-active enzymes) are prone to interference from various noncellulosic substances (e.g., metal ions). During natural cellulolysis, these substances may arise from other microbial activities or abiotic events, and during industrial cellulolysis, they may be derived from biomass feedstocks or upstream treatments. Knowledge about cellulolysis-inhibiting reactions is of importance for the microbiology of natural biomass degradation and the development of biomass conversion technology. Different metal ions, including those native to microbial activity or employed for biomass pretreatments, are often tested for enzymatic cellulolysis. Only a few metal ions act as inhibitors of cellulases, which include ferrous and ferric ions as well as cupric ion. In this study, we showed inhibition by ferrous/ferric ions as part of a more general effect from oxidative (or redox-active) metal ions and their complexes. The correlation between inhibition and oxidation potential indicated the oxidative nature of the inhibition, and the dependence on air established the catalytic role that iron ions played in mediating the dioxygen inhibition of cellulolysis. Individual cellulases showed different susceptibilities to inhibition. It is likely that the inhibition exerted its effect more on cellulose than on cellulase. Strong iron ion chelators and polyethylene glycols could mitigate the inhibition. Potential microbiological and industrial implications of the observed effect of redox-active metal ions on enzymatic cellulolysis, as well as the prevention and mitigation of this effect in industrial biomass conversion, are discussed.

β-Lactam Ring Opening: A Useful Entry to Amino Acids and Relevant Nitrogen-Containing Compounds

NASA Astrophysics Data System (ADS)

Palomo, C.; Oiarbide, M.

The main strategies for the ring opening of β-lactams by chemical means are described. The discovery of each approach is put into context, sometimes in connection to processes occurring in biological systems, and the synthetic opportunities each approach offers are shown. Thus, this β-lactam route affords a number of synthetically relevant building-blocks, including α-amino acids, β-amino acids, their derived peptides, and other nitrogen containing heterocycles and open chain molecules. The content, which encompases references to initial work, further major development, and the most relevant recent literature contributions, is categorized according to the ring bond cleavaged (N 1-C 2, C 2-C 3, C 3 -C 4 , N 1-C 4), to finish with ring opening strategies leading to large heterocyclic compounds. Within each category, distinction has been made according to the type of nucleophilic agent employed, principally O-, N-, and C-nucleophiles. Also, a variety of applications of the strategy to the synthesis of interesting target compounds are shown.

β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

PubMed Central

Gutierrez, A.; Laureti, L.; Crussard, S.; Abida, H.; Rodríguez-Rojas, A.; Blázquez, J.; Baharoglu, Z.; Mazel, D.; Darfeuille, F.; Vogel, J.; Matic, I.

2013-01-01

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies. PMID:23511474

Stochastic molecular model of enzymatic hydrolysis of cellulose for ethanol production

PubMed Central

2013-01-01

Background During cellulosic ethanol production, cellulose hydrolysis is achieved by synergistic action of cellulase enzyme complex consisting of multiple enzymes with different mode of actions. Enzymatic hydrolysis of cellulose is one of the bottlenecks in the commercialization of the process due to low hydrolysis rates and high cost of enzymes. A robust hydrolysis model that can predict hydrolysis profile under various scenarios can act as an important forecasting tool to improve the hydrolysis process. However, multiple factors affecting hydrolysis: cellulose structure and complex enzyme-substrate interactions during hydrolysis make it diffucult to develop mathematical kinetic models that can simulate hydrolysis in presence of multiple enzymes with high fidelity. In this study, a comprehensive hydrolysis model based on stochastic molecular modeling approch in which each hydrolysis event is translated into a discrete event is presented. The model captures the structural features of cellulose, enzyme properties (mode of actions, synergism, inhibition), and most importantly dynamic morphological changes in the substrate that directly affect the enzyme-substrate interactions during hydrolysis. Results Cellulose was modeled as a group of microfibrils consisting of elementary fibrils bundles, where each elementary fibril was represented as a three dimensional matrix of glucose molecules. Hydrolysis of cellulose was simulated based on Monte Carlo simulation technique. Cellulose hydrolysis results predicted by model simulations agree well with the experimental data from literature. Coefficients of determination for model predictions and experimental values were in the range of 0.75 to 0.96 for Avicel hydrolysis by CBH I action. Model was able to simulate the synergistic action of multiple enzymes during hydrolysis. The model simulations captured the important experimental observations: effect of structural properties, enzyme inhibition and enzyme loadings on the

Computational Studies on the Synthesis of β-Lactams via [2+2] Thermal Cycloadditions

NASA Astrophysics Data System (ADS)

Arrieta, Ana; Lecea, Begoña; Cossío, Fernando P.

The main computational studies on the formation of β-lactams through [2+2] cycloadditions published during 1992-2008 are reported with special emphasis on the mechanistic and selectivity aspects of these reactions. Disconnection of the N1-C2 and C3-C4 bonds of the azetidin-2-one ring leads to the reaction between ketenes and imines. Computational and experimental results point to a stepwise mechanism for this reaction. The first step consists of a nucleophilic attack of the iminic nitrogen on the sp-hybridized carbon atom of the ketene. The zwitterionic intermediate thus formed yields the corresponding β-lactam by means of a four-electron conrotatoty electrocyclization. The steroecontrol and the periselectivity of the reaction support this two-step mechanism. The [2+2] cycloaddition between isocyanates and alkenes takes place via a concerted (but asynchronous) mechanism that can be interpreted in terms of a [π2s + (π2s + π2s)] interaction between both reactants. Both the regio and the stereochemistry observed are compatible with this computational model. However, the combination of solvent and substituent effects can result in a stepwise mechanism.

Mechanism of action of NB2001 and NB2030, novel antibacterial agents activated by beta-lactamases.

PubMed

Stone, Geoffrey W; Zhang, Qin; Castillo, Rosario; Doppalapudi, V Ramana; Bueno, Analia R; Lee, Jean Y; Li, Qing; Sergeeva, Maria; Khambatta, Gody; Georgopapadakou, Nafsika H

2004-02-01

Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit beta-lactamases to release triclosan through hydrolysis of the beta-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A beta-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C beta-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k(cat)/K(m)). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the beta-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.

N-Sulfonyl-β-lactam hapten as an effective labeling reagent for aldolase mAb.

PubMed

Inokuma, Tsubasa; Fuller, Roberta P; Barbas, Carlos F

2015-04-15

Utilization of chemically programmed antibodies (cpAbs) is regarded to be one of the most efficient methods for the development of therapeutic systems. cpAbs can extend the half-life of programming reagents, activate immune systems via the Fc region of antibodies and achieve universal vaccination by attaching varieties of small, programmed molecules. In the current study, we aimed to develop a novel labeling reagent for the preparation of cpAbs and found that N-sulfonyl-β-lactams (NSBLs) were optimal. NSBL can be synthesized from readily available 4-(bromomethyl)benzenesulfonyl chloride via few simple manipulations and can label the aldolase monoclonal antibody (mAb) 84G3, which could not be labeled effectively by the conventional labeling reagent, N-acyl-β-lactam (NABL). We also demonstrated that the conjugate, which consists of mAb 84G3 and an NSBL bearing a biotin moiety, maintained strong binding activity to streptavidin. In addition, the stability assay of NSBL revealed that NSBLs can tolerate aqueous media without significant decomposition over 24h. Copyright © 2015 Elsevier Ltd. All rights reserved.

beta-Lactam resistance of motile Aeromonas isolates from clinical and environmental sources.

PubMed Central

Morita, K; Watanabe, N; Kurata, S; Kanamori, M

1994-01-01

The MICs of various beta-lactams for 182 isolates of Aeromonas species, i.e., A. hydrophila (n = 101), A. sobria (n = 69), and A. caviae (n = 12), from clinical and environmental sources were determined by an agar dilution technique. All strains were resistant to ampicillin and susceptible to aztreonam. A. sobria and A. caviae demonstrated lower resistance rates than A. hydrophila. Penicillin-hydrolyzing beta-lactamases were detected in all strains. PMID:8192463

ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. I. ALKALINE HYDROLYSIS

EPA Science Inventory

SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

Supramolecular Complexes Formed by the Self-assembly of Hydrophobic Bis(Zn(2+)-cyclen) Complexes, Copper, and Di- or Triimide Units for the Hydrolysis of Phosphate Mono- and Diesters in Two-Phase Solvent Systems (Cyclen=1,4,7,10-Tetraazacyclododecane).

PubMed

Hisamatsu, Yosuke; Miyazawa, Yuya; Yoneda, Kakeru; Miyauchi, Miki; Zulkefeli, Mohd; Aoki, Shin

2016-01-01

We previously reported on supramolecular complexes 4 and 5, formed by the 4 : 4 : 4 or 2 : 2 : 2 assembly of a dimeric zinc(II) complex (Zn2L(1)) having 2,2'-bipyridyl linker, dianion of cyanuric acid (CA) or 5,5-diethylbarbituric acid (Bar), and copper(II) ion (Cu(2+)) in an aqueous solution. The supermolecule 4 possesses Cu2(μ-OH)2 centers and catalyzes hydrolysis of phosphate monoester dianion, mono(4-nitrophenyl)phosphate (MNP), at neutral pH. In this manuscript, we report on design and synthesis of hydrophobic supermolecules 9 and 10 by 4 : 4 : 4 and 2 : 2 : 2 self-assembly of hydrophobic Zn2L(2) and Zn2L(3) containing long alkyl chains, CA or Bar, and Cu(2+) and their phosphatase activity for the hydrolysis of MNP and bis(4-nitrophenyl)phosphate (BNP) in two-phase solvent systems. We assumed that the Cu2(μ-OH)2 active sites of 9 and 10 would be more stable in organic solvent than in aqueous solution and that product inhibition of the supermolecules might be avoided by the release of HPO4(2-) into the aqueous layer. The findings indicate that 9 and 10 exhibit phosphatase activity in the two-phase solvent system, although catalytic turnover was not observed. Furthermore, the hydrolysis of BNP catalyzed by the hydrophobic 2 : 2 : 2 supermolecules in the two-phase solvent system is described.

Hydrolysis reactor for hydrogen production

DOEpatents

Davis, Thomas A.; Matthews, Michael A.

2012-12-04

In accordance with certain embodiments of the present disclosure, a method for hydrolysis of a chemical hydride is provided. The method includes adding a chemical hydride to a reaction chamber and exposing the chemical hydride in the reaction chamber to a temperature of at least about 100.degree. C. in the presence of water and in the absence of an acid or a heterogeneous catalyst, wherein the chemical hydride undergoes hydrolysis to form hydrogen gas and a byproduct material.

Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams.

PubMed Central

Vakulenko, S B; Tóth, M; Taibi, P; Mobashery, S; Lerner, S A

1995-01-01

To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 [numbering of Ambler et al. (Biochem J. 267:269-272, 1991)]) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme. All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime. In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam. One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime. PMID:7486939

Mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucoside by germinating and outgrowing spores of Bacillus species.

PubMed

Setlow, B; Cabrera-Martinez, R-M; Setlow, P

2004-01-01

during spore outgrowth in the presence of beta-MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99.7%. Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P. Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH. Generation of MU from beta-MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P. The major phosphoglucosidase catalyzing MU generation from beta-MUG-P in spores of both species is probably BglH. This work provides new insight into the mechanism of uptake and hydrolysis of beta-MUG by germinating and outgrowing spores of Bacillus species, in particular B. atrophaeus. The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

PubMed

Park, Jong-Min; Kim, Jo-Il; Song, Hyun-Woo; Noh, Joo-Yoon; Kang, Min-Jung; Pyun, Jae-Chul

2015-09-15

This work presented a highly sensitive bacterial antibiotic susceptibility test through β-lactamase assay using Parylene-matrix chip. β-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to β-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate β-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/z<500). The β-lactamase assay for the Escherichia coli antibiotic susceptibility test was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a β-lactamase assay with minimal interference of low molecular weight noise peaks. The β-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay. Copyright © 2015 Elsevier B.V. All rights reserved.

Poly(2-oxazoline)-Antibiotic Conjugates with Penicillins.

PubMed

Schmidt, Martin; Bast, Livia K; Lanfer, Franziska; Richter, Lena; Hennes, Elisabeth; Seymen, Rana; Krumm, Christian; Tiller, Joerg C

2017-09-20

The conjugation of antibiotics with polymers is rarely done, but it might be a promising alternative to low-molecular-weight derivatization. The two penicillins penicillin G (PenG) and penicillin V (PenV) were attached to the end groups of different water-soluble poly(2-oxazoline)s (POx) via their carboxylic acid function. This ester group was shown to be more stable against hydrolysis than the β-lactam ring of the penicillins. The conjugates are still antimicrobially active and up to 20 times more stable against penicillinase catalyzed hydrolysis. The antibiotic activity of the conjugates against Staphylococcus aureus in the presence of penicillinase is up to 350 times higher compared with the free antibiotics. Conjugates with a second antimicrobial function, a dodecyltrimethylammonium group (DDA-X), at the starting end of the PenG and PenV POx conjugates are more antimicrobially active than the conjugates without DDA-X and show high activity in the presence of penicillinase. For example, the conjugates DDA-X-PEtOx-PenG and DDA-X-PEtOx-PenV are 200 to 350 times more active against S. aureus in the presence of penicillinase and almost as effective as the penicillinase stable cloxacollin (Clox) under these conditions. These conjugates show even greater activity compared to cloxacollin without this enzyme present. Further, both conjugates kill Escherichia coli more effectively than PenG and Clox.

Self-resistance in Streptomyces, with Special Reference to β-Lactam Antibiotics.

PubMed

Ogawara, Hiroshi

2016-05-10

Antibiotic resistance is one of the most serious public health problems. Among bacterial resistance, β-lactam antibiotic resistance is the most prevailing and threatening area. Antibiotic resistance is thought to originate in antibiotic-producing bacteria such as Streptomyces. In this review, β-lactamases and penicillin-binding proteins (PBPs) in Streptomyces are explored mainly by phylogenetic analyses from the viewpoint of self-resistance. Although PBPs are more important than β-lactamases in self-resistance, phylogenetically diverse β-lactamases exist in Streptomyces. While class A β-lactamases are mostly detected in their enzyme activity, over two to five times more classes B and C β-lactamase genes are identified at the whole genomic level. These genes can subsequently be transferred to pathogenic bacteria. As for PBPs, two pairs of low affinity PBPs protect Streptomyces from the attack of self-producing and other environmental β-lactam antibiotics. PBPs with PASTA domains are detectable only in class A PBPs in Actinobacteria with the exception of Streptomyces. None of the Streptomyces has PBPs with PASTA domains. However, one of class B PBPs without PASTA domain and a serine/threonine protein kinase with four PASTA domains are located in adjacent positions in most Streptomyces. These class B type PBPs are involved in the spore wall synthesizing complex and probably in self-resistance. Lastly, this paper emphasizes that the resistance mechanisms in Streptomyces are very hard to deal with, despite great efforts in finding new antibiotics.

Nitrogen-containing ecdysteroid derivatives vs. multi-drug resistance in cancer: Preparation and antitumor activity of oximes, oxime ethers and a lactam.

PubMed

Vágvölgyi, Máté; Martins, Ana; Kulmány, Ágnes; Zupkó, István; Gáti, Tamás; Simon, András; Tóth, Gábor; Hunyadi, Attila

2018-01-20

Multidrug resistance is a widespread problem among various diseases and cancer is no exception. We had previously described the chemo-sensitizing activity of ecdysteroid derivatives with low polarity on drug susceptible and multi-drug resistant (MDR) cancer cells. We have also shown that these molecules have a marked selectivity towards the MDR cells. Recent studies on the oximation of various steroid derivatives indicated remarkable increase in their antitumor activity, but there is no related bioactivity data on ecdysteroid oximes. In our present study, 13 novel ecdysteroid derivatives (oximes, oxime ethers and a lactam) and one known compound were synthesized from 20-hydroxyecdysone 2,3;20,22-diacetonide and fully characterized by comprehensive NMR techniques revealing their complete 1 H and 13 C signal assignments. The compounds exerted moderate to strong in vitro antiproliferative activity on HeLa, SiHa, MCF-7 and MDA-MB-231 cell lines. Oxime and particularly oxime ether formation strongly increased their inhibitory activity on the efflux of rhodamine 123 by P-glycoprotein (P-gp), while the new ecdysteroid lactam did not interfere with the efflux function. All compounds exerted potent chemo-sensitizing activity towards doxorubicin on a mouse lymphoma cell line and on its MDR counterpart, and, on the latter, the lactam was found the most active. Because of its MDR-selective chemo-sensitizing activity with no functional effect on P-gp, this lactam is of high potential interest as a new lead for further antitumor studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Asymmetric synthesis of a potent, aminopiperidine-fused imidazopyridine dipeptidyl peptidase IV inhibitor.

PubMed

Xu, Feng; Corley, Edward; Zacuto, Michael; Conlon, David A; Pipik, Brenda; Humphrey, Guy; Murry, Jerry; Tschaen, David

2010-03-05

A practical asymmetric synthesis of a novel aminopiperidine-fused imidazopyridine dipeptidyl peptidase IV (DPP-4) inhibitor 1 has been developed. Application of a unique three-component cascade coupling with chiral nitro diester 7, which is easily accessed via a highly enantioselective Michael addition of dimethyl malonate to a nitrostyrene, allows for the assembly of the functionalized piperidinone skeleton in one pot. Through a base-catalyzed, dynamic crystallization-driven process, the cis-piperidionone 16a is epimerized to the desired trans isomer 16b, which is directly crystallized from the crude reaction stream in high yield and purity. Isomerization of the allylamide 16b in the presence of RhCl(3) is achieved without any epimerization of the acid/base labile stereogenic center adjacent to the nitro group on the piperidinone ring, while the undesired enamine intermediate is consumed to <0.5% by utilizing a trace amount of HCl generated from RhCl(3). The amino lactam 4, obtained through hydrogenation and hydrolysis, is isolated as its crystalline pTSA salt from the reaction solution directly, as such intramolecular transamidation has been dramatically suppressed via kinetic control. Finally, a Cu(I) catalyzed coupling-cyclization allows for the formation of the tricyclic structure of the potent DPP-4 inhibitor 1. The synthesis, which is suitable for large scale preparation, is accomplished in 23% overall yield.

Short-time ultrasonication treatment in enzymatic hydrolysis of biomass

Treesearch

Zengqian Shi; Zhiyong Cai; Siqun Wang; Qixin Zhong; Joseph J. Bozell

2013-01-01

To improve the conversion of enzymatic hydrolysis of biomass in an energy-efficient manner, two shorttime ultrasonication strategies were applied on six types of biomass with different structures and components. The strategies include pre-sonication before the hydrolysis and intermittent sonication during the ongoing hydrolysis. The microstructures of each type of...

Failure of daptomycin β-Lactam combination therapy to prevent resistance emergence in Enterococcus faecium.

PubMed

Menon, Vidthiya; Davis, Rebecca; Shackel, Nick; Espedido, Bjorn A; Beukers, Alicia G; Jensen, Slade O; van Hal, Sebastiaan J

2018-02-01

Daptomycin β-Lactam combination therapy offers "protection" against daptomycin non-susceptibility (DNS) development in Enterococcus faecium. We report failure of this strategy and the importance of source control. Mutations were detected in the LiaF and cls genes in DNS isolates. A single DNS isolate contained an unrecognized mutation, which requires confirmation. Copyright © 2017 Elsevier Inc. All rights reserved.

Conformational analysis of a condensed macrocyclic β-lactam by NMR and molecular dynamics calculations

NASA Astrophysics Data System (ADS)

Keserű, György M.; Vásárhelyi, Helga; Makara, Gergely

1994-09-01

The conformation of the new macrocyclic β-lactam ( 1) was investigated by NMR and molecular dynamics (MD) calculations. Restraints obtained from NOESY and ROESY experiments were introduced into MD simulations which led to well-defined conformations. The preference for the calculated minimum energy conformation was confirmed by the analysis of vicinal coupling constants. Experimental coupling constants agreed with computed values.

Lipase-catalyzed modification of the flavor profiles in recombined skim milk products by enriching the volatile components.

PubMed

Zhang, X M; Ai, N S; Wang, J; Tong, L J; Zheng, F P; Sun, B G

2016-11-01

The purpose of this study was to modify the amount and composition of volatile components in bovine milk products, in an attempt to create a recombined skim milk product with full-fat milk flavor but with only 0.5% fat. The experimental plan included lipase-catalyzed hydrolysis and esterification reactions using Palatase 20000L (Novozymes, Bagsværd, Denmark). The results, measured by the methods of volatile compositional analysis and sensory evaluation, showed that the flavor profiles of the optimal recombined milk products were effectively modified in this way, possessing intensified characteristic volatile flavor components with rather low level of fat contents, and the sensory characters were quite realistic to natural whole milk flavor. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Single hydration of the peptide bond: the case of the Vince lactam.

PubMed

Écija, Patricia; Basterretxea, Francisco J; Lesarri, Alberto; Millán, Judith; Castaño, Fernando; Cocinero, Emilio J

2012-10-18

2-Azabicyclo[2.2.1]hept-5-en-3-one (ABH or Vince lactam) and its monohydrated complex (ABH···H(2)O) have been observed in a supersonic jet by Fourier transform microwave spectroscopy. ABH is broadly used in the synthesis of therapeutic drugs, whereas the ABH···H(2)O system offers a simple model to explain the conformational preferences of water linked to a constrained peptidic bond. A single predominant form of the Vince lactam and its singly hydrated complex have been detected, determining the rotational constants, centrifugal distortion constants, and nuclear quadrupole coupling tensor. The monohydrated complex is stabilized by two hydrogen bonds (C═O···H-O and N-H···O) closing a six-membered ring. The complexation energy has been estimated to be ∼10 kJ mol(-1) from experimental results. In addition, the observed structure in the gas phase has been compared with solid-phase diffraction data. The structural parameters and binding energies of ABH···H(2)O have also been compared with similar molecules containing peptide bonds. Ab initio (MP2) and density functional (M06-2X and B3LYP) methods have supported the experimental work, describing the rotational parameters and conformational landscape of the title compound and its singly hydrated complex.

Hydrolysis of the quinone methide of butylated hydroxytoluene in aqueous solutions.

PubMed

Willcockson, Maren Gulsrud; Toteva, Maria M; Stella, Valentino J

2013-10-01

Butylated hydroxytoluene or BHT is an antioxidant commonly used in pharmaceutical formulations. BHT upon oxidation forms a quinone methide (QM). QM is a highly reactive electrophilic species that can undergo nucleophilic addition. Here, the kinetic reactivity of QM with water at various apparent pH values in a 50% (v/v) water-acetonitrile solution at constant ionic strength of I = 0.5 (NaCl)4 , was studied. The hydrolysis of QM in the presence of added acid, base, sodium chloride, and phosphate buffer resulted in the formation of only one product--the corresponding 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (BA). The rate of BA formation was catalyzed by the addition of acid and base, but not chloride and phosphate species. Nucleophilic excipients, used in the pharmaceutical formulation, or nucleophilic groups on active pharmaceutical ingredient molecule may form adducts with QM, the immediate oxidative product of BHT degradation, thus having implications for drug product impurity profiles. Because of these considerations, BHT should be used with caution in formulations containing drugs or excipients capable of acting as nucleophiles. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Kinetics and mechanism of imazosulfuron hydrolysis.

PubMed

Morrica, P; Barbato, F; Della Iacovo, R; Seccia, S; Ungaro, F

2001-08-01

Knowledge of the kinetics and pathways of hydrolytic degradation is crucial to the prediction of the fate and transport mechanism of chemicals. This work first describes the kinetics of the chemical hydrolysis of imazosulfuron, a new sulfonylurea herbicide, and evaluates the results to propose a degradation pathway. The hydrolysis of imazosulfuron has been studied in aqueous buffers both within the pH range 1.9-12.3 at ambient temperature (thermostated at 25 +/- 2 degrees C) and at pH 3.6 within the temperature range of 15-55 degrees C. The hydrolysis rate of imazosulfuron was characterized by a first-order kinetics, pH- and temperature-dependent, and accelerated by acidic conditions and higher temperatures. The calculated half-lives at pH 4.5 and 5.9 were 36.5 and 578 days, respectively. At pH 6.6, 7.4, 9.2, and 12.3 no significant change in imazosulfuron concentration was observed after 150 days. Half-lives were much lower at pH <4 (= imazosulfuron pK(a)), at which they ranged from 3.3 to 6.3 days. Moreover, a change in temperature from 15 to 25 degrees C in acidic conditions (pH 3.6) decreased the half-life of imazosulfuron by a factor of approximately 4.0; in any case, a 3-5-fold increase in the rate of hydrolysis was found for each 10 degrees C increase in temperature. In acidic conditions the only hydrolysis products were the two molecules resulting from the cleavage of the sulfonylurea bridge.

Thermodynamics of Enzyme-Catalyzed Reactions Database

National Institute of Standards and Technology Data Gateway

SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

Effects of Cranberry Juice on Pharmacokinetics of β-Lactam Antibiotics following Oral Administration▿

PubMed Central

Li, Meng; Andrew, Marilee A.; Wang, Joanne; Salinger, David H.; Vicini, Paolo; Grady, Richard W.; Phillips, Brian; Shen, Danny D.; Anderson, Gail D.

2009-01-01

Cranberry juice consumption is often recommended along with low-dose oral antibiotics for prophylaxis for recurrent urinary tract infection (UTI). Because multiple membrane transporters are involved in the intestinal absorption and renal excretion of β-lactam antibiotics, we evaluated the potential risk of pharmacokinetic interactions between cranberry juice and the β-lactams amoxicillin (amoxicilline) and cefaclor. The amoxicillin-cranberry juice interaction was investigated in 18 healthy women who received on four separate occasions a single oral test dose of amoxicillin at 500 mg and 2 g with or without cranberry juice cocktail (8 oz) according to a crossover design. A parallel cefaclor-cranberry juice interaction study was also conducted in which 500 mg cefaclor was administered with or without cranberry juice cocktail (12 oz). Data were analyzed by noncompartmental methods and nonlinear mixed-effects compartmental modeling. We conclude that the concurrent use of cranberry juice has no significant effect on the extent of oral absorption or the renal clearance of amoxicillin and cefaclor. However, delays in the absorption of amoxicillin and cefaclor were observed. These results suggest that the use of cranberry juice at usual quantities as prophylaxis for UTI is not likely to alter the pharmacokinetics of these two oral antibiotics. PMID:19398645

Comparison of Enzymatic Hydrolysis and Acid Hydrolysis of Sterol Glycosides from Foods Rich in Δ(7)-Sterols.

PubMed

Münger, Linda H; Jutzi, Sabrina; Lampi, Anna-Maija; Nyström, Laura

2015-08-01

In this study, we present the difference in sterol composition of extracted steryl glycosides (SG) hydrolyzed by either enzymatic or acid hydrolysis. SG were analyzed from foods belonging to the plant families Cucurbitaceae (melon and pumpkin seeds) and Amaranthaceae (amaranth and beetroot), both of which are dominated by Δ(7)-sterols. Released sterols were quantified by gas chromatography with a flame ionization detector (GC-FID) and identified using gas chromatography/mass spectrometry (GC-MS). All Δ(7)-sterols identified (Δ(7)-stigmastenyl, spinasteryl, Δ(7)-campesteryl, Δ(7)-avenasteryl, poriferasta-7,25-dienyl and poriferasta-7,22,25-trienyl glucoside) underwent isomerization under acidic conditions and high temperature. Sterols with an ethylidene or methylidene side chain were found to form multiple artifacts. The artifact sterols coeluted with residues of incompletely isomerized Δ(7)-sterols, or Δ(5)-sterols if present, and could be identified as Δ(8(14))-sterols on the basis of relative retention time, and their MS spectra as trimethylsilyl (TMS) and acetate derivatives. For instance, SG from melon were composed of 66% Δ(7)-stigmastenol when enzymatic hydrolysis was performed, whereas with acid hydrolysis only 8% of Δ(7)-stigmastenol was determined. The artifact of Δ(7)-stigmastenol coeluted with residual non-isomerized spinasterol, demonstrating the high risk of misinterpretation of compositional data obtained after acid hydrolysis. Therefore, the accurate composition of SG from foods containing sterols with a double bond at C-7 can only be obtained by enzymatic hydrolysis or by direct analysis of the intact SG.

Validation of a rapid lateral flow test for the simultaneous determination of β-lactam drugs and flunixin in raw milk.

PubMed

Douglas, David; Banaszewski, Katie; Juskelis, Rima; Al-Taher, Fadwa; Chen, Yang; Cappozzo, Jack; McRobbie, Lindsay; Salter, Robert S

2012-07-01

β-Lactam antibiotics are the most commonly used drugs on dairy farms. β-Lactam residues in milk are kept out of the human milk supply with good agricultural practices and mandatory truck screening performed by the dairy industry under Appendix N of the Pasteurized Milk Ordinance. Flunixin, a nonsteroidal and anti-inflammatory drug, appears in dairy cattle tissue residues with a frequency similar to the occurrence of penicillin G. This creates concern that flunixin residues could be in milk and would go undetected under current milk screening programs. A single test that combines mandatory β-lactam screening with voluntary flunixin screening is an economical approach for monitoring and controlling for potential flunixin or 5-hydroxyflunixin, the primary flunixin metabolite marker in milk. The objective of this study was to validate a β-lactam and flunixin rapid lateral flow test (LFT) and compare the results obtained with a liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of flunixin and 5-hydroxyflunixin in raw milk with a limit of detection of , 1 ppb, equivalent to 1 ng/ml. Using the LFT, three combined manufactured lots of test strips detected penicillin G at 2.0 ppb, ampicillin at 6.8 ppb, amoxicillin at 5.9 ppb, cephapirin at 13.4 ppb, ceftiofur (total metabolites) at 63 ppb, and 5-hydroxyflunixin at 1.9 ppb at least 90% of the time with 95% confidence. The LFT also detected incurred flunixin milk samples that were analyzed with the LC-MS/MS and diluted to tolerance in raw milk. The detection levels for the LFT are lower than the U.S. safe levels or tolerances and qualify the test to be used in compliance with U.S. milk screening programs.

Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

PubMed Central

Richards, Donald E.; Irvine, Robin F.; Dawson, Rex M. C.

1979-01-01

(1) The hydrolysis of 32P- or myo-[2-3H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca2+ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca2+ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[3H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed. PMID:508301

Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design.

PubMed

Xu, Li; Liu, Xiaohong; Yin, Zhenhao; Liu, Qian; Lu, Lili; Xiao, Min

2016-12-01

The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp 252 , Asp 257 , Asp 264 , Glu 530 , Arg 548 , His 553 , and Trp 555 ) and may form the hydrophobic pocket in stabilizing donor (Trp 261 , Tyr 302 , Tyr 316 , and Trp 369 ) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp 257 . Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

Immunological aspects of nonimmediate reactions to beta-lactam antibiotics.

PubMed

Rodilla, Esther Morena; González, Ignacio Dávila; Yges, Elena Laffond; Bellido, Francisco Javier Múñoz; Bara, María Teresa Gracia; Toledano, Félix Lorente

2010-09-01

beta-lactam antibiotics are the agents most frequently implied in immune drug adverse reactions. These can be classified as immediate or nonimmediate according to the time interval between the last drug administration and their onset. Mechanisms of immediate IgE-mediated reactions are widely studied and are therefore better understood. Nonimmediate reactions include a broad number of clinical entities like mild maculopapular exanthemas, the most common, and other less frequent but more severe reactions such as Stevens-Johnson syndrome, toxic epidermal necrolysis, acute exanthematic pustulosis or cytopenias. These nonimmediate reactions are mainly mediated by T cells but the precise underlying mechanisms are not well elucidated. This fact complicates the allergological evaluation of patients with this type of reaction and available tests have demonstrated poor sensitivity and specificity.

Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

PubMed Central

Wu, Siva; Li, Xiaojin; Gunawardana, Manjula; Maguire, Kathleen; Guerrero-Given, Debbie; Schaudinn, Christoph; Wang, Charles; Baum, Marc M.; Webster, Paul

2014-01-01

Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended. PMID:25007395

Extended versus Bolus Infusion of Broad Spectrum β-Lactams for Febrile Neutropenia: an Unblinded Randomized Trial.

PubMed

Ram, Ron; Halavy, Yael; Amit, Odelia; Paran, Yael; Katchman, Eugene; Yachini, Bruria; Kor, Svetlana; Avivi, Irit; Ben-Ami, Ronen

2018-03-28

Febrile neutropenia may be a sign of severe infection, and is associated with significant morbidity and mortality in high-risk patients with hematologic malignancies. Extended infusion of β-lactam antibiotics is associated with greater clinical response than is bolus infusion in non-neutropenic critically ill patients, but data are lacking for febrile neutropenic patients. We designed a single-center, non-blinded randomized trial comparing extended infusion (4 hours) and bolus infusion (30 minutes) of piperacillin-tazobactam or ceftazidime in high-risk patients with febrile neutropenia. The primary end-point was overall response on day 4, defined as the combination of resolution of fever, sterile blood cultures, resolution of clinical signs and symptoms, and no need for a change in the antibiotic regimen. Outcome was adjudicated by investigators blinded to treatment allocation. Of 123 enrolled patients, 105 had febrile neutropenia and were included in the intention-to-treat analysis: 47 in the extended infusion arm and 58 in the bolus infusion arm. Overall response occurred in 35 (74.4%) patients treated with extended infusion and 32 (55.1%) patients treated with bolus infusion (P=0.044). The superiority of extended infusion compared with bolus infusion was greatest for patients with clinically documented infections (overall response, 68.4% [13/19] versus 35.7% [10/28]; P=0.039), and specifically for those with pneumonia (80% [4/5] versus 0% [0/8]; P=0.007). Extended infusion of β-lactams is associated with superior treatment outcomes as compared with bolus infusion for high-risk patients with febrile neutropenia. The benefit of extended β-lactam infusion may be greatest for patients with pulmonary infections.

Stepwise hydrolysis to improve carbon releasing efficiency from sludge.

PubMed

Liu, Hongbo; Wang, Yuanyuan; Wang, Ling; Yu, Tiantian; Fu, Bo; Liu, He

2017-08-01

Based on thermal alkaline hydrolysis (TAH), a novel strategy of stepwise hydrolysis was developed to improve carbon releasing efficiency from waste activated sludge (WAS). By stepwise increasing hydrolysis intensity, conventional sludge hydrolysis (the control) was divided into four stages for separately recovering sludge carbon sources with different bonding strengths, namely stage 1 (60 °C, pH 6.0-8.0), stage 2 (80 °C, pH 6.0-8.0), stage 3 (80 °C, pH 10.0) and stage 4 (90 °C, pH 12.0). Results indicate stepwise hydrolysis could enhance the amount of released soluble chemical oxygen demand (SCOD) for almost 2 times, from 7200 to 14,693 mg/L, and the released carbon presented better biodegradability, with BOD/COD of 0.47 and volatile fatty acids (VFAs) yield of 0.37 g VFAs/g SCOD via anaerobic fermentation. Moreover, stepwise hydrolysis also improved the dewaterability of hydrolyzed sludge, capillary suction time (CST) reducing from 2500 to 1600 s. Economic assessment indicates stepwise hydrolysis shows less alkali demand and lower thermal energy consumption than those of the control. Furthermore, results of this study help support the concepts of improving carbon recovery in wastewater by manipulating WAS composition and the idea of classifiably recovering the nutrients in WAS. Copyright © 2017 Elsevier Ltd. All rights reserved.

Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

PubMed

Scollar, M P; Sigal, G; Klibanov, A M

1985-03-01

Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach*

PubMed Central

Kim, Young-Min; Kiso, Yoshiaki; Muraki, Tomoe; Kang, Min-Sun; Nakai, Hiroyuki; Saburi, Wataru; Lang, Weeranuch; Kang, Hee-Kwon; Okuyama, Masayuki; Mori, Haruhide; Suzuki, Ryuichiro; Funane, Kazumi; Suzuki, Nobuhiro; Momma, Mitsuru; Fujimoto, Zui; Oguma, Tetsuya; Kobayashi, Mikihiko; Kim, Doman; Kimura, Atsuo

2012-01-01

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr451–Val1082), a portion of which shares identity (35% at Ala39–Ser1304 of PsDex) with Pro32–Ala755 of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val837–Met932 for PsDex and Tyr404–Tyr492 for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala39–Ser1304) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp189, Asp340, Glu412, and Asp1254 of PsDex) of catalytic candidates. Their amide mutants decreased activity (11,500 to 140,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp340 and Glu412 as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN3. PMID:22461618

Evaluation of the Charm maximum residue limit β-lactam and tetracycline test for the detection of antibiotics in ewe and goat milk.

PubMed

Beltrán, M C; Romero, T; Althaus, R L; Molina, M P

2013-05-01

The Charm maximum residue limit β-lactam and tetracycline test (Charm MRL BLTET; Charm Sciences Inc., Lawrence, MA) is an immunoreceptor assay utilizing Rapid One-Step Assay lateral flow technology that detects β-lactam or tetracycline drugs in raw commingled cow milk at or below European Union maximum residue levels (EU-MRL). The Charm MRL BLTET test procedure was recently modified (dilution in buffer and longer incubation) by the manufacturers to be used with raw ewe and goat milk. To assess the Charm MRL BLTET test for the detection of β-lactams and tetracyclines in milk of small ruminants, an evaluation study was performed at Instituto de Ciencia y Tecnologia Animal of Universitat Politècnica de València (Spain). The test specificity and detection capability (CCβ) were studied following Commission Decision 2002/657/EC. Specificity results obtained in this study were optimal for individual milk free of antimicrobials from ewes (99.2% for β-lactams and 100% for tetracyclines) and goats (97.9% for β-lactams and 100% for tetracyclines) along the entire lactation period regardless of whether the results were visually or instrumentally interpreted. Moreover, no positive results were obtained when a relatively high concentration of different substances belonging to antimicrobial families other than β-lactams and tetracyclines were present in ewe and goat milk. For both types of milk, the CCβ calculated was lower or equal to EU-MRL for amoxicillin (4 µg/kg), ampicillin (4 µg/kg), benzylpenicillin (≤ 2 µg/kg), dicloxacillin (30 µg/kg), oxacillin (30 µg/kg), cefacetrile (≤ 63 µg/kg), cefalonium (≤ 10 µg/kg), cefapirin (≤ 30 µg/kg), desacetylcefapirin (≤ 30 µg/kg), cefazolin (≤ 25 µg/kg), cefoperazone (≤ 25 µg/kg), cefquinome (20 µg/kg), ceftiofur (≤ 50 µg/kg), desfuroylceftiofur (≤ 50µg/kg), and cephalexin (≤ 50 µg/kg). However, this test could neither detect cloxacillin nor nafcillin at or below EU-MRL (CCβ >30 µg/kg). The

A process for producing lignin and volatile compounds from hydrolysis liquor.

PubMed

Khazraie, Tooran; Zhang, Yiqian; Tarasov, Dmitry; Gao, Weijue; Price, Jacquelyn; DeMartini, Nikolai; Hupa, Leena; Fatehi, Pedram

2017-01-01

Hot water hydrolysis process is commercially applied for treating wood chips prior to pulping or wood pellet production, while it produces hydrolysis liquor as a by-product. Since the hydrolysis liquor is dilute, the production of value-added materials from it would be challenging. In this study, acidification was proposed as a viable method to extract (1) furfural and acetic acid from hot water hydrolysis liquor and (2) lignin compounds from the liquor. The thermal properties of the precipitates made from the acidification of hydrolysis liquor confirmed the volatile characteristics of precipitates. Membrane dialysis was effective in removing inorganic salts associated with lignin compounds. The purified lignin compounds had a glass transition temperature (Tg) of 180-190 °C, and were thermally stable. The results confirmed that lignin compounds present in hot water hydrolysis liquor had different characteristics. The acidification of hydrolysis liquor primarily removed the volatile compounds from hydrolysis liquor. Based on these results, a process for producing purified lignin and precipitates of volatile compounds was proposed.

High-dose continuous infusion beta-lactam antibiotics for the treatment of resistant Pseudomonas aeruginosa infections in immunocompromised patients.

PubMed

Moriyama, Brad; Henning, Stacey A; Childs, Richard; Holland, Steven M; Anderson, Victoria L; Morris, John C; Wilson, Wyndham H; Drusano, George L; Walsh, Thomas J

2010-05-01

To report a case series of high-dose continuous infusion beta-lactam antibiotics for the treatment of resistant Pseudomonas aeruginosa infections. Continuous infusion ceftazidime or aztreonam was administered to achieve target drug concentrations at or above the minimum inhibitory concentration, when possible, in 3 patients with P. aeruginosa infections. The maximal calculated target drug concentration was 100 mg/L. In the first patient, with primary immunodeficiency, neutropenia, and aggressive cutaneous T-cell lymphoma/leukemia, continuous infusion ceftazidime (6.5-9.6 g/day) was used to successfully treat multidrug-resistant P. aeruginosa bacteremia. In the second patient, with leukocyte adhesion deficiency type 1, continuous infusion aztreonam (8.4 g/day) was used to successfully treat multidrug-resistant P. aeruginosa wound infections. In the third patient, with severe aplastic anemia, continuous infusion ceftazidime (7-16.8 g/day) was used to treat P. aeruginosa pneumonia and bacteremia. In each patient, bacteremia cleared, infected wounds healed, and pneumonia improved in response to continuous infusion ceftazidime or aztreonam. Treatment strategies for multidrug-resistant P. aeruginosa infections are limited. A novel treatment strategy, when no other options are available, is the continuous infusion of existing beta-lactam antibiotics to maximize their pharmacodynamic activity. High-dose continuous infusion ceftazidime or aztreonam was used for the successful treatment of resistant systemic P. aeruginosa infections in 3 chronically immunocompromised patients. Continuous infusion beta-lactam antibiotics are a potentially useful treatment strategy for resistant P. aeruginosa infections in immunocompromised patients.

Aminoacyl-nucleotide reactions - Studies related to the origin of the genetic code and protein synthesis

NASA Technical Reports Server (NTRS)

Mullins, D. W., Jr.; Senaratne, N.; Lacey, J. C., Jr.

1984-01-01

In the present paper, a report is presented on the effect of pH and carbonate on the hydrolysis rate constants of N-blocked and free aminoacyl adenylate anhydrides. Whereas the hydrolysis of free aminoacyl adenylates seems principally catalyzed by OH(-), the hydrolysis of the N-blocked species is also catalyzed by H(+), giving this compound a U-shaped hydrolysis vs. pH curve. Furthermore, at pH's less than 8, carbonate has an extreme catalytic effect on the hydrolysis of free aminoacyl-AMP anhydride, but essentially no effect on the hydrolysis of N-blocked aminoacyl-AMP anhydride. Furthermore, the N-blocked aminoacyl-AMP anhydride is a very efficient generator of peptides using free glycine as acceptor. The possible significance of the observations to prebiological peptide synthesis is discussed.

Esculin hydrolysis by Vibrio vulnificus.

PubMed

Tison, D L

1986-01-01

A clinical isolate of Vibrio vulnificus was found to hydrolyze esculin when tested on bile-esculin-azide agar during the initial characterization of the strain. Reports in the literature of esculin hydrolysis by V. vulnificus are conflicting. We tested herein 52 strains of V. vulnificus from clinical and environmental sources for the ability to hydrolyze esculin. Seventy-eight percent of the strains hydrolyzed esculin on bile-esculin-azide agar, whereas all strains of V. vulnificus tested were positive for esculin hydrolysis in a noninhibitory medium, whereas some strains failed to hydrolyze esculin on media containing inhibitory compounds.

Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline-oxidative pretreatment of hybrid poplar.

PubMed

Bhalla, Aditya; Fasahati, Peyman; Particka, Chrislyn A; Assad, Aline E; Stoklosa, Ryan J; Bansal, Namita; Semaan, Rachel; Saffron, Christopher M; Hodge, David B; Hegg, Eric L

2018-01-01

When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H 2 O 2 ) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significant loss in sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified. After Cu-AHP pretreatment of 120 °C NaOH-H 2 O pre-extracted and 120 °C NaOH-EtOH pre-extracted biomass, approximately 1.4-fold more total lignin was solubilized (78% and 74%, respectively) compared to the 30 °C NaOH-H 2 O pre-extraction (55%) carried out in a previous study. Consequently, increasing the temperature of the alkaline pre-extraction step to 120 °C in both ethanol and water allowed us to decrease bipyridine and H 2 O 2 during Cu-AHP and enzymes during hydrolysis with only a small reduction in sugar yields compared to 30 °C alkaline pre-extraction. Technoeconomic analysis indicated that 120 °C NaOH-H 2 O pre-extraction has the lowest installed ($246 million) and raw material ($175 million) costs compared to the other process configurations. We found that by increasing the temperature of the alkaline pre-extraction step, we could successfully lower the inputs for pretreatment and enzymatic hydrolysis. Based on sugar yields as well as

Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline–oxidative pretreatment of hybrid poplar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhalla, Aditya; Fasahati, Peyman; Particka, Chrislyn A.

When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H 2O 2) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significantmore » loss in sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified. After Cu-AHP pretreatment of 120 °C NaOH-H 2O pre-extracted and 120 °C NaOH-EtOH pre-extracted biomass, approximately 1.4-fold more total lignin was solubilized (78% and 74%, respectively) compared to the 30 °C NaOH-H 2O pre-extraction (55%) carried out in a previous study. Consequently, increasing the temperature of the alkaline pre-extraction step to 120 °C in both ethanol and water allowed us to decrease bipyridine and H 2O 2 during Cu-AHP and enzymes during hydrolysis with only a small reduction in sugar yields compared to 30 °C alkaline pre-extraction. Technoeconomic analysis indicated that 120 °C NaOH-H 2O pre-extraction has the lowest installed ($246 million) and raw material (175 million) costs compared to the other process configurations. We found that by increasing the temperature of the alkaline pre-extraction step, we could successfully lower the inputs for pretreatment and enzymatic hydrolysis. Based on sugar yields as well as

Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline–oxidative pretreatment of hybrid poplar

DOE PAGES

Bhalla, Aditya; Fasahati, Peyman; Particka, Chrislyn A.; ...

2018-05-17

When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H 2O 2) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significantmore » loss in sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified. After Cu-AHP pretreatment of 120 °C NaOH-H 2O pre-extracted and 120 °C NaOH-EtOH pre-extracted biomass, approximately 1.4-fold more total lignin was solubilized (78% and 74%, respectively) compared to the 30 °C NaOH-H 2O pre-extraction (55%) carried out in a previous study. Consequently, increasing the temperature of the alkaline pre-extraction step to 120 °C in both ethanol and water allowed us to decrease bipyridine and H 2O 2 during Cu-AHP and enzymes during hydrolysis with only a small reduction in sugar yields compared to 30 °C alkaline pre-extraction. Technoeconomic analysis indicated that 120 °C NaOH-H 2O pre-extraction has the lowest installed ($246 million) and raw material (175 million) costs compared to the other process configurations. We found that by increasing the temperature of the alkaline pre-extraction step, we could successfully lower the inputs for pretreatment and enzymatic hydrolysis. Based on sugar yields as well as

[Impact of liquid volume of recycled methanogenic effluent on anaerobic hydrolysis].

PubMed

Hao, Li-ping; Lü, Fan; He, Pin-jing; Shao, Li-ming

2008-09-01

Methanogenic effluent was recycled to regulate hydrolysis during two-phase anaerobic digestion of organic solid wastes. In order to study the impact of recycled effluent's volume on hydrolysis, four hydrolysis reactors filled with vegetable and flower wastes were constructed, with different liquid volumes of recycled methanogenic effluent, i.e., 0.1, 0.5, 1.0, 2.0 m3/(m3 x d), respectively. The parameters related to hydrolytic environment (pH, alkalinity, ORP, concentrations of ammonia and reducing sugar), microbial biomass and hydrolysis efficiency (accumulated SCOD, accumulated reducing sugar, and hydrolysis rate constants) were monitored. This research shows that recycling methanogenic effluent into the hydrolysis reactor can enhance its buffer capability and operation stability; higher recycled volume is favorable for microbial anabolism and further promotes hydrolysis. After 9 days of reaction, the accumulated SCOD in the hydrolytic effluent reach 334, 407, 413, 581 mg/g at recycled volumes of 0.1, 0.5, 1.0, 2.0 m3/(m3 x d) and their first-order hydrolysis rate kinetic constants are 0.065, 0.083, 0.089, 0.105 d(-1), respectively.

Optimization of hydrolysis conditions for bovine plasma protein using response surface methodology.

PubMed

Seo, Hyun-Woo; Jung, Eun-Young; Go, Gwang-Woong; Kim, Gap-Don; Joo, Seon-Tea; Yang, Han-Sul

2015-10-15

The purpose of this study was to establish optimal conditions for the hydrolysis of bovine plasma protein. Response surface methodology was used to model and optimize responses [degree of hydrolysis (DH), 2,2-diphenyl-1-picrydrazyl (DPPH) radical-scavenging activity and Fe(2+)-chelating activity]. Hydrolysis conditions, such as hydrolysis temperature (46.6-63.4 °C), hydrolysis time (98-502 min), and hydrolysis pH (6.32-9.68) were selected as the main processing conditions in the hydrolysis of bovine plasma protein. Optimal conditions for maximum DH (%), DPPH radical-scavenging activity (%) and Fe(2+)-chelating activity (%) of the hydrolyzed bovine plasma protein, were respectively established. We discovered the following three conditions for optimal hydrolysis of bovine plasma: pH of 7.82-8.32, temperature of 54.1 °C, and time of 338.4-398.4 min. We consequently succeeded in hydrolyzing bovine plasma protein under these conditions and confirmed the various desirable properties of optimal hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polysiloxanes derived from the controlled hydrolysis of tetraethoxysilane as precursors to silica for use in ceramic processing

NASA Technical Reports Server (NTRS)

Philipp, Warren H.

1990-01-01

Synthesis, properties, and potential applications in ceramic processing for two polysiloxane silica precursors derived from the controlled hydrolysis of tetraethoxysilane (TEOS) are presented. The higher molecular weight TEOS-A is a thick adhesive liquid of viscosity 8000 to 12,000 c.p. having a SiO2 char yield of about 55 percent. The lower molecular weight TEOS-B is a more fluid liquid of viscosity 150 to 200 c.p. having a SiO2 char yield of about 52 percent. The acid catalyzed hydrolysis of TEOS to hydrated silica gel goes through a series of polysiloxane intermediates. The rate of this transition increases with the quantity of water added to the TEOS; thus, for ease of polymer isolation, the amount of water added must be carefully determined so as to produce the desired polymer in a reasonable time. The water to TEOS mole ratio falls in the narrow range of 1.05 for TEOS-A and 0.99 for TEOS-B. Further polymerization or gelation is prevented by storing at -5 C in a freezer. Both polysiloxanes thermoset to a glassy solid at 115 C. The liquid polymers are organic in nature in that they are miscible with toluene and ethanol, slightly souble in heptane, but immiscible with water. For both polymers, results on viscosity versus time are given at several temperatures and water additions. Based on these results, some examples of practical utilization of the precursors for ceramic fabrication are given.

Optimization of NaOH-catalyzed steam pretreatment of empty fruit bunch.

PubMed

Choi, Won-Il; Park, Ji-Yeon; Lee, Joon-Pyo; Oh, You-Kwan; Park, Yong Chul; Kim, Jun Seok; Park, Jang Min; Kim, Chul Ho; Lee, Jin-Suk

2013-11-29

Empty fruit bunch (EFB) has many advantages, including its abundance, the fact that it does not require collection, and its year-round availability as a feedstock for bioethanol production. But before the significant costs incurred in ethanol production from lignocellulosic biomass can be reduced, an efficient sugar fractionation technology has to be developed. To that end, in the present study, an NaOH-catalyzed steam pretreatment process was applied in order to produce ethanol from EFB more efficiently. The EFB pretreatment conditions were optimized by application of certain pretreatment variables such as, the NaOH concentrations in the soaking step and, in the steam step, the temperature and time. The optimal conditions were determined by response surface methodology (RSM) to be 3% NaOH for soaking and 160°C, 11 min 20 sec for steam pretreatment. Under these conditions, the overall glucan recovery and enzymatic digestibility were both high: the glucan and xylan yields were 93% and 78%, respectively, and the enzymatic digestibility was 88.8% for 72 h using 40 FPU/g glucan. After simultaneous saccharification and fermentation (SSF), the maximum ethanol yield and concentration were 0.88 and 29.4 g/l respectively. Delignification (>85%) of EFB was an important factor in enzymatic hydrolysis using CTec2. NaOH-catalyzed steam pretreatment, which can remove lignin efficiently and requires only a short reaction time, was proven to be an effective pretreatment technology for EFB. The ethanol yield obtained by SSF, the key parameter determining the economics of ethanol, was 18% (w/w), equivalent to 88% of the theoretical maximum yield, which is a better result than have been reported in the relevant previous studies.

Optimization of NaOH-catalyzed steam pretreatment of empty fruit bunch

PubMed Central

2013-01-01

Background Empty fruit bunch (EFB) has many advantages, including its abundance, the fact that it does not require collection, and its year-round availability as a feedstock for bioethanol production. But before the significant costs incurred in ethanol production from lignocellulosic biomass can be reduced, an efficient sugar fractionation technology has to be developed. To that end, in the present study, an NaOH-catalyzed steam pretreatment process was applied in order to produce ethanol from EFB more efficiently. Results The EFB pretreatment conditions were optimized by application of certain pretreatment variables such as, the NaOH concentrations in the soaking step and, in the steam step, the temperature and time. The optimal conditions were determined by response surface methodology (RSM) to be 3% NaOH for soaking and 160°C, 11 min 20 sec for steam pretreatment. Under these conditions, the overall glucan recovery and enzymatic digestibility were both high: the glucan and xylan yields were 93% and 78%, respectively, and the enzymatic digestibility was 88.8% for 72 h using 40 FPU/g glucan. After simultaneous saccharification and fermentation (SSF), the maximum ethanol yield and concentration were 0.88 and 29.4 g/l respectively. Conclusions Delignification (>85%) of EFB was an important factor in enzymatic hydrolysis using CTec2. NaOH-catalyzed steam pretreatment, which can remove lignin efficiently and requires only a short reaction time, was proven to be an effective pretreatment technology for EFB. The ethanol yield obtained by SSF, the key parameter determining the economics of ethanol, was 18% (w/w), equivalent to 88% of the theoretical maximum yield, which is a better result than have been reported in the relevant previous studies. PMID:24286374

Role of asparagine 152 in catalysis of beta-lactam hydrolysis by Escherichia coli AmpC beta-lactamase studied by site-directed mutagenesis.

PubMed

Dubus, A; Normark, S; Kania, M; Page, M G

1995-06-13

The role of asparagine 152 in the catalytic mechanism of Escherichia coli AmpC beta-lactamase has been investigated by site-directed mutagenesis. The residue has been replaced by aspartic acid, glutamic acid, histidine, and leucine. All the substitutions had similar effects on the activity toward substrates and inhibitors. The rate of substrate hydrolysis decreased by factors of 500-5000. The rates of both acylation (2-50-fold decrease) and deacylation (50-500-fold decrease) were affected, indicating a role for Asn152 in both processes. The wild-type AmpC beta-lactamase appears to exist as an equilibrium mixture of two forms, identified by their different kinetic properties. The Asn152 mutations affected the activity of the slow-reacting form much more than that of the fast-reacting form, but they did not appear to affect the interconversion of these two kinetic forms. Comparison of these observations with results obtained with mutation of the equivalent residues in other classes of penicillin-sensitive enzyme indicates that there are quite profound differences between the catalytic mechanisms of these enzymes despite a high degree of conservation of amino acids in the active center, and of the overall three-dimensional structure.

Simultaneous pharmacodynamic analysis of the lag and bactericidal phases exhibited by beta-lactams against Escherichia coli.

PubMed Central

Li, R C

1996-01-01

Antibiotic-bacterium interactions are complex in nature. In many cases, bacterial killing does not commence immediately after the addition of an antibiotic, and a lag period is observed. Antibiotic permeation and/or the intermediate steps that exist between antibiotic-receptor binding and expression of cell death are two major possible causes for such lag period. This study was primarily designed to determine the relationship, if any, between antibiotic concentrations and the lag periods by a modeling approach. Short-term time-kill studies were conducted for amoxicillin, ampicillin, penicillin-G, oxacillin, and dicloxacillin against Escherichia coli. In conjunction with the use of a saturable rate model to describe the concentration-dependent killing process, a first-order induction (initiation) rate constant was used to characterize the delay in bacterial killing during the lag period. For all of the beta-lactams tested, parameters describing the bactericidal effect suggest that amoxicillin and ampicillin were much more potent than oxacillin and dicloxacillin. The induction rate constant estimates for both ampicillin and amoxicillin were found to relate linearly to concentrations. Nevertheless, these induction rate constant estimates were lower for penicillin-G, oxacillin, and dicloxacillin and increased nonlinearly with concentrations until an apparent plateau was observed. These findings support the hypothesis that the permeation process is potentially a rate-limiting step for the rapid bactericidal beta-lactams such as ampicillin and amoxicillin. However, as suggested by previous observations of the various morphological changes induced by beta-lactams, the contribution of the steps following antibiotic-receptor complex formation to the lag period might be significant for the less bactericidal antibiotics such as oxacillin and dicloxacillin. Findings from the present modeling approach can potentially be used to guide future bench experimentation. PMID:8891135

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

PubMed

Rival, S; Besson, C; Saulnier, J; Wallach, J

1999-02-01

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.

Structural Insight into the Mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tchigvintsev, A.; Xu, X.; Singer, A.

2010-08-01

Cyclic diguanylate (or bis-(3'-5') cyclic dimeric guanosine monophosphate; c-di-GMP) is a ubiquitous second messenger that regulates diverse cellular functions, including motility, biofilm formation, cell cycle progression, and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TBD1265 EAL domain was determined in free state (1.8 {angstrom}) and in complex with c-di-GMP (2.35 {angstrom}), andmore » unveiled the role of conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.« less

The enzymic hydrolysis of amygdalin

PubMed Central

Haisman, D. R.; Knight, D. J.

1967-01-01

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond β-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the β-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components. PMID:4291788

Theoretical Proposal for the Whole Phosphate Diester Hydrolysis Mechanism Promoted by a Catalytic Promiscuous Dinuclear Copper(II) Complex.

PubMed

Esteves, Lucas F; Rey, Nicolás A; Dos Santos, Hélio F; Costa, Luiz Antônio S

2016-03-21

The catalytic mechanism that involves the cleavage of the phosphate diester model BDNPP (bis(2,4-dinitrophenyl) phosphate) catalyzed through a dinuclear copper complex is investigated in the current study. The metal complex was originally designed to catalyze catechol oxidation, and it showed an interesting catalytic promiscuity case in biomimetic systems. The current study investigates two different reaction mechanisms through quantum mechanics calculations in the gas phase, and it also includes the solvent effect through PCM (polarizable continuum model) single-point calculations using water as solvent. Two mechanisms are presented in order to fully describe the phosphate diester hydrolysis. Mechanism 1 is of the S(N)2 type, which involves the direct attack of the μ-OH bridge between the two copper(II) ions toward the phosphorus center, whereas mechanism 2 is the process in which hydrolysis takes place through proton transfer between the oxygen atom in the bridging hydroxo ligand and the other oxygen atom in the phosphate model. Actually, the present theoretical study shows two possible reaction paths in mechanism 1. Its first reaction path (p1) involves a proton transfer that occurs immediately after the hydrolytic cleavage, so that the proton transfer is the rate-determining step, which is followed by the entry of two water molecules. Its second reaction path (p2) consists of the entry of two water molecules right after the hydrolytic cleavage, but with no proton transfer; thus, hydrolytic cleavage is the rate-limiting step. The most likely catalytic path occurs in mechanism 1, following the second reaction path (p2), since it involves the lowest free energy activation barrier (ΔG(⧧) = 23.7 kcal mol(-1), in aqueous solution). A kinetic analysis showed that the experimental k(obs) value of 1.7 × 10(-5) s(-1) agrees with the calculated value k1 = 2.6 × 10(-5) s(-1); the concerted mechanism is kinetically favorable. The KIE (kinetic isotope effect) analysis

High-Dose Continuous Infusion Beta-lactam Antibiotics for the Treatment of Resistant Pseudomonas aeruginosa Infections in Immunocompromised Patients

PubMed Central

Moriyama, Brad; Henning, Stacey A.; Childs, Richard; Holland, Steven M.; Anderson, Victoria L.; Morris, John C.; Wilson, Wyndham H.; Drusano, George L.; Walsh, Thomas J.

2011-01-01

OBJECTIVE To report a case series of high-dose continuous infusion beta-lactam antibiotics for the treatment of resistant Pseudomonas aeruginosa infections. CASE SUMMARY Continuous infusion ceftazidime or aztreonam was administered to achieve target drug levels at or above the MIC when possible in three patients with P. aeruginosa infections. The maximal calculated target drug level was 100 mg/L. In the first patient with primary immunodeficiency, neutropenia, and aggressive cutaneous T cell lymphoma/leukemia, continuous infusion ceftazidime (6.5 to 9.6 g/day) was used to successfully treat multidrug-resistant P. aeruginosa bacteremia. In the second patient with leukocyte adhesion deficiency type 1, continuous infusion aztreonam (8.4 g/day) was used to successfully treat multidrug-resistant P. aeruginosa wound infections. In the third patient with severe aplastic anemia, continuous infusion ceftazidime (7 to 16.8 g/day) was used to treat P. aeruginosa pneumonia and bacteremia. In each patient, the bacteremia cleared, infected wounds healed, and pneumonia improved in response to continuous infusion ceftazidime or aztreonam. DISCUSSION Treatment strategies for multidrug-resistant P. aeruginosa infections are limited. A novel treatment strategy when no other options are available is the administration of existing beta-lactam antibiotics by continuous infusion in order to maximize their pharmacodynamic activity. High-dose continuous infusion ceftazidime or aztreonam was used for the successful treatment of resistant systemic P. aeruginosa infections in three chronically immunocompromised patients. CONCLUSION Continuous infusion beta-lactam antibiotics are a potentially useful treatment strategy for resistant P. aeruginosa infections in immunocompromised patients. PMID:20371747

Enhanced hydrolysis of cellulose hydrogels by morphological modification.

PubMed

Alfassi, Gilad; Rein, Dmitry M; Cohen, Yachin

2017-11-01

Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to the high costs of conversion. Hydrogel particles of regenerated cellulose constitute a useful substrate for enzymatic hydrolysis, due to their porous and amorphous structure. This article describes the influence of several structural aspects of the cellulose hydrogel on its hydrolysis. The hydrogel density was shown to be directly proportional to the cellulose concentration in the initial solution, thus affecting its hydrolysis rate. Using high-resolution scanning electron microscopy, we show that the hydrogel particles in aqueous suspension exhibit a dense external surface layer and a more porous internal network. Elimination of the external surface layer accelerated the hydrolysis rate by up to sixfold and rendered the process nearly independent of cellulose concentration. These findings may be of practical relevance to saccharification processing costs, by reducing required solvent quantities and enzyme load.

Energetic approach of biomass hydrolysis in supercritical water.

PubMed

Cantero, Danilo A; Vaquerizo, Luis; Mato, Fidel; Bermejo, M Dolores; Cocero, M José

2015-03-01

Cellulose hydrolysis can be performed in supercritical water with a high selectivity of soluble sugars. The process produces high-pressure steam that can be integrated, from an energy point of view, with the whole biomass treating process. This work investigates the integration of biomass hydrolysis reactors with commercial combined heat and power (CHP) schemes, with special attention to reactor outlet streams. The innovation developed in this work allows adequate energy integration possibilities for heating and compression by using high temperature of the flue gases and direct shaft work from the turbine. The integration of biomass hydrolysis with a CHP process allows the selective conversion of biomass into sugars with low heat requirements. Integrating these two processes, the CHP scheme yield is enhanced around 10% by injecting water in the gas turbine. Furthermore, the hydrolysis reactor can be held at 400°C and 23 MPa using only the gas turbine outlet streams. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors.

PubMed

Hassan, Hammad Ali; Rani, Sadaf; Fatima, Tabeer; Kiani, Farooq Ahmad; Fischer, Stefan

2017-11-01

Hydrolysis of phosphate groups is a crucial reaction in living cells. It involves the breaking of two strong bonds, i.e. the O a H bond of the attacking water molecule, and the PO l bond of the substrate (O a and O l stand for attacking and leaving oxygen atoms). Mechanism of the hydrolysis reaction can proceed either by a concurrent or a sequential mechanism. In the concurrent mechanism, the breaking of O a H and PO l bonds occurs simultaneously, whereas in the sequential mechanism, the O a H and PO l bonds break at different stages of the reaction. To understand how protonation affects the mechanism of hydrolysis of phosphate monoester, we have studied the mechanism of hydrolysis of protonated and deprotonated phosphate monoester at M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory (where ZPE stands for zero point energy). Our calculations show that in both protonated and deprotonated cases, the breaking of the water O a H bond occurs before the breaking of the PO l bond. Because the two events are not separated by a stable intermediate, the mechanism can be categorized as semi-concurrent. The overall energy barrier is 41kcalmol -1 in the unprotonated case. Most (5/6th) of this is due to the initial breaking of the water O a H bond. This component is lowered from 34 to 25kcalmol -1 by adding one proton to the phosphate. The rest of the overall energy barrier comes from the subsequent breaking of the PO l bond and is not sensitive to protonation. This is consistent with previous findings about the effect of triphosphate protonation on the hydrolysis, where the equivalent protonation (on the γ-phosphate) was seen to lower the barrier of breaking the water O a H bond and to have little effect on the PO l bond breaking. Hydrolysis pathways of phosphate monoester with initial breaking of the PO l bond could not be found here. This is because the leaving group in phosphate monoester cannot be protonated, unlike in triphosphate hydrolysis, where protonation of the �

High β-Lactamase Levels Change the Pharmacodynamics of β-Lactam Antibiotics in Pseudomonas aeruginosa Biofilms

PubMed Central

Ciofu, Oana; Yang, Liang; Wu, Hong; Song, Zhijun; Oliver, Antonio; Høiby, Niels

2013-01-01

Resistance to β-lactam antibiotics is a frequent problem in Pseudomonas aeruginosa lung infection of cystic fibrosis (CF) patients. This resistance is mainly due to the hyperproduction of chromosomally encoded β-lactamase and biofilm formation. The purpose of this study was to investigate the role of β-lactamase in the pharmacokinetics (PK) and pharmacodynamics (PD) of ceftazidime and imipenem on P. aeruginosa biofilms. P. aeruginosa PAO1 and its corresponding β-lactamase-overproducing mutant, PAΔDDh2Dh3, were used in this study. Biofilms of these two strains in flow chambers, microtiter plates, and on alginate beads were treated with different concentrations of ceftazidime and imipenem. The kinetics of antibiotics on the biofilms was investigated in vitro by time-kill methods. Time-dependent killing of ceftazidime was observed in PAO1 biofilms, but concentration-dependent killing activity of ceftazidime was observed for β-lactamase-overproducing biofilms of P. aeruginosa in all three models. Ceftazidime showed time-dependent killing on planktonic PAO1 and PAΔDDh2Dh3. This difference is probably due to the special distribution and accumulation in the biofilm matrix of β-lactamase, which can hydrolyze the β-lactam antibiotics. The PK/PD indices of the AUC/MBIC and Cmax/MBIC (AUC is the area under concentration-time curve, MBIC is the minimal biofilm-inhibitory concentration, and Cmax is the maximum concentration of drug in serum) are probably the best parameters to describe the effect of ceftazidime in β-lactamase-overproducing P. aeruginosa biofilms. Meanwhile, imipenem showed time-dependent killing on both PAO1 and PAΔDDh2Dh3 biofilms. An inoculum effect of β-lactams was found for both planktonic and biofilm P. aeruginosa cells. The inoculum effect of ceftazidime for the β-lactamase-overproducing mutant PAΔDDh2Dh3 biofilms was more obvious than for PAO1 biofilms, with a requirement of higher antibiotic concentration and a longer period of treatment

Reaction Kinetic Model of Dilute Acid-Catalyzed Hemicellulose Hydrolysis of Corn Stover under High-Solid Conditions

DOE PAGES

Shi, Suan; Guan, Wenjian; Kang, Li; ...

2017-09-13

High solid conditions are desirable in pretreatment of lignocellulosic biomass. An advanced dilute-acid pretreatment reactor has been developed at National Renewable Energy Laboratory (NREL). It is a continuous auger-driven reactor that can be operated with high-solid charge at high temperature and with short residence time resulting high productivity and high sugar concentration. Here, we investigated the kinetics of the reactions associated with dilute-acid pretreatment of corn stover, covering the reaction conditions of the NREL reactor operation: 155-185 C, 1-2 wt% sulfuric acid concentration, and 1:2 solid to liquid ratio. The experimental data were fitted to a first-order biphasic model whichmore » assumes that xylan is comprised of two different fragments: fast and slow reacting fractions. Due to the high solid loading condition, significant amount of xylose oligomers was observed during the pretreatment. We also included the oligomers as an intermediate entity in the kinetic model. The effect of acid concentration was incorporated into the pre-exponential factor of Arrhenius equation. The kinetic model with bestfit kinetic parameters has shown good agreement with experimental data. The kinetic parameter values of the proposed model were noticeably different from those previously reported. The activation energies of xylan hydrolysis are lower and the acid exponents are higher than the average of literature values. The proposed model can serve as a useful tool for design and operation of pretreatment system pertaining to corn stover.« less

Reaction Kinetic Model of Dilute Acid-Catalyzed Hemicellulose Hydrolysis of Corn Stover under High-Solid Conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Suan; Guan, Wenjian; Kang, Li

High solid conditions are desirable in pretreatment of lignocellulosic biomass. An advanced dilute-acid pretreatment reactor has been developed at National Renewable Energy Laboratory (NREL). It is a continuous auger-driven reactor that can be operated with high-solid charge at high temperature and with short residence time resulting high productivity and high sugar concentration. Here, we investigated the kinetics of the reactions associated with dilute-acid pretreatment of corn stover, covering the reaction conditions of the NREL reactor operation: 155-185 C, 1-2 wt% sulfuric acid concentration, and 1:2 solid to liquid ratio. The experimental data were fitted to a first-order biphasic model whichmore » assumes that xylan is comprised of two different fragments: fast and slow reacting fractions. Due to the high solid loading condition, significant amount of xylose oligomers was observed during the pretreatment. We also included the oligomers as an intermediate entity in the kinetic model. The effect of acid concentration was incorporated into the pre-exponential factor of Arrhenius equation. The kinetic model with bestfit kinetic parameters has shown good agreement with experimental data. The kinetic parameter values of the proposed model were noticeably different from those previously reported. The activation energies of xylan hydrolysis are lower and the acid exponents are higher than the average of literature values. The proposed model can serve as a useful tool for design and operation of pretreatment system pertaining to corn stover.« less

Kinetics of Maleic Acid and Aluminum Chloride Catalyzed Dehydration and Degradation of Glucose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Ximing; Hewetson, Barron B.; Mosier, Nathan S.

We report the positive effect of maleic acid, a dicarboxylic acid, on the selectivity of hexose dehydration to 5-hydroxymethyfurfural (HMF) and subsequent hydrolysis to levulinic and formic acids. We also describe the kinetic analysis of a Lewis acid (AlCl3) alone and in combination with HCl or maleic acid to catalyze the isomerization of glucose to fructose, dehydration of fructose to HMF, hydration of HMF to levulinic and formic acids, and degradation of these compounds to humins. The results show that AlCl3 significantly enhances the rate of glucose conversion to HMF and levulinic acid in the presence of both maleic acidmore » and HCl. In addition, the degradation of HMF to humins, rather than levulinic and formic acids, is reduced by 50% in the presence of maleic acid and AlCl3 compared to HCl combined with AlCl3. The results suggest different reaction mechanisms for the dehydration of glucose and rehydration of HMF between maleic acid and HCl.« less

Rapid spot test for the determination of esculin hydrolysis.

PubMed

Edberg, S C; Gam, K; Bottenbley, C J; Singer, J M

1976-08-01

Esculin hydrolysis is a useful test in the differentiation of both gram-positive and gram-negative bacteria covering a wide spectrum of aerobes, facultative anaerobes, and anaerobes. Commonly utilized methods require a minimum of 18 h of incubation in broth or agar medium and utilize the production of a brown-black compound, due to the combination of ferric ions with the hydrolysis product esculetin, as indicator. A procedure is presented that requires 15 to 30 min for completion and utilizes fluorescence loss as the indicator of hydrolysis. Esculin fluoresces at 366 nm, whereas the hydrolysis product esculetin does not. Over 1,400 strains of gram-positive and gram-negative bacteria were tested. There was 98.4% of correlation between the spot test and esculin broth and 97% correlation with the bile-esculin agar.

Substrate-induced inactivation of the OXA2 beta-lactamase.

PubMed Central

Ledent, P; Frère, J M

1993-01-01

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. 'Burst' kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two 'bursts', and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product. PMID:8240304

Hydrolysis of dilute acid-pretreated cellulose under mild hydrothermal conditions.

PubMed

Chimentão, R J; Lorente, E; Gispert-Guirado, F; Medina, F; López, F

2014-10-13

The hydrolysis of dilute acid-pretreated cellulose was investigated in a conventional oven and under microwave heating. Two acids--sulfuric and oxalic--were studied. For both hydrothermal conditions (oven and microwave) the resultant total organic carbon (TOC) values obtained by the hydrolysis of the cellulose pretreated with sulfuric acid were higher than those obtained by the hydrolysis of the cellulose pretreated with oxalic acid. However, the dicarboxylic acid exhibited higher hydrolytic efficiency towards glucose. The hydrolysis of cellulose was greatly promoted by microwave heating. The Rietveld method was applied to fit the X-ray patterns of the resultant cellulose after hydrolysis. Oxalic acid preferentially removed the amorphous region of the cellulose and left the crystalline region untouched. On the other hand, sulfuric acid treatment decreased the ordering of the cellulose by partially disrupting its crystalline structure. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lysophosphatidylcholine synthesis by lipase-catalyzed ethanolysis.

PubMed

Yang, Guolong; Yang, Ruoxi; Hu, Jingbo

2015-01-01

Lysophosphatidylcholine (LPC) is amphiphilic substance, and possesses excellent physiological functions. In this study, LPC was prepared through ethanolysis of phosphatidylcholine (PC) in n-hexane or solvent free media catalyzed by Novozym 435 (from Candida antarctica), Lipozyme TLIM (from Thermomcyces lanuginosus) and Lipozyme RMIM (from Rhizomucor miehei). The results showed that three immobilized lipases from Candida Antarctica, Thermomcyces lanuginosus and Rhizomucor miehei could catalyze ethanolysis of PC efficiently. In n-hexane, the LPC conversions of ethanolysis of PC catalyzed by Novozyme 435, Lipozyme TLIM and Lipozyme RMIM could reach to 98.5 ± 1.6%, 94.6 ± 1.4% and 93.7 ± 1.8%, respectively. In solvent free media, the highest LPC conversions of ethanolysis of PC catalyzed by Novozyme 435, Lipozyme TL IM and Lipozyme RM IM were 97.7 ± 1.7%, 93.5 ± 1.2% and 93.8 ± 1.9%, respectively. The catalytic efficiencies of the three lipases were in the order of Novozyme 435 > Lipozyme TLIM > Lipozyme RMIM. Furthermore, their catalytic efficiencies in n-hexane were better than those in solvent free media.

Alkaline thermal sludge hydrolysis.

PubMed

Neyens, E; Baeyens, J; Creemers, C

2003-02-28

The waste activated sludge (WAS) treatment of wastewater produces excess sludge which needs further treatment prior to disposal or incineration. A reduction in the amount of excess sludge produced, and the increased dewaterability of the sludge are, therefore, subject of renewed attention and research. A lot of research covers the nature of the sludge solids and associated water. An improved dewaterability requires the disruption of the sludge cell structure. Previous investigations are reviewed in the paper. Thermal hydrolysis is recognized as having the best potential to meet the objectives and acid thermal hydrolysis is most frequently used, despite its serious drawbacks (corrosion, required post-neutralization, solubilization of heavy metals and phosphates, etc.). Alkaline thermal hydrolysis has been studied to a lesser extent, and is the subject of the detailed laboratory-scale research reported in this paper. After assessing the effect of monovalent/divalent cations (respectively, K(+)/Na(+) and Ca(2+)/Mg(2+)) on the sludge dewaterability, only the use of Ca(2+) appears to offer the best solution. The lesser effects of K(+), Na(+) and Mg(2+) confirm previous experimental findings. As a result of the experimental investigations, it can be concluded that alkaline thermal hydrolysis using Ca(OH)(2) is efficient in reducing the residual sludge amounts and in improving the dewaterability. The objectives are fully met at a temperature of 100 degrees C; at a pH approximately 10 and for a 60-min reaction time, where all pathogens are moreover killed. Under these optimum conditions, the rate of mechanical dewatering increases (the capillary suction time (CST) value is decreased from approximately 34s for the initial untreated sample to approximately 22s for the hydrolyzed sludge sample) and the amount of DS to be dewatered is reduced to approximately 60% of the initial untreated amount. The DS-content of the dewatered cake will be increased from 28 (untreated) to 46

Estimation of hydrolysis rate constants for carbamates ...

EPA Pesticide Factsheets

Cheminformatics based tools, such as the Chemical Transformation Simulator under development in EPA’s Office of Research and Development, are being increasingly used to evaluate chemicals for their potential to degrade in the environment or be transformed through metabolism. Hydrolysis represents a major environmental degradation pathway; unfortunately, only a small fraction of hydrolysis rates for about 85,000 chemicals on the Toxic Substances Control Act (TSCA) inventory are in public domain, making it critical to develop in silico approaches to estimate hydrolysis rate constants. In this presentation, we compare three complementary approaches to estimate hydrolysis rates for carbamates, an important chemical class widely used in agriculture as pesticides, herbicides and fungicides. Fragment-based Quantitative Structure Activity Relationships (QSARs) using Hammett-Taft sigma constants are widely published and implemented for relatively simple functional groups such as carboxylic acid esters, phthalate esters, and organophosphate esters, and we extend these to carbamates. We also develop a pKa based model and a quantitative structure property relationship (QSPR) model, and evaluate them against measured rate constants using R square and root mean square (RMS) error. Our work shows that for our relatively small sample size of carbamates, a Hammett-Taft based fragment model performs best, followed by a pKa and a QSPR model. This presentation compares three comp

Gastric protein hydrolysis of raw and roasted almonds in the growing pig.

PubMed

Bornhorst, Gail M; Drechsler, Krista C; Montoya, Carlos A; Rutherfurd, Shane M; Moughan, Paul J; Singh, R Paul

2016-11-15

Gastric protein hydrolysis may influence gastric emptying rate and subsequent protein digestibility in the small intestine. This study examined the gastric hydrolysis of dietary protein from raw and roasted almonds in the growing pig as a model for the adult human. The gastric hydrolysis of almond proteins was quantified by performing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent image analysis. There was an interaction between digestion time, stomach region, and almond type for gastric protein hydrolysis (p<0.05). Gastric emptying rate of protein was a significant (p<0.05) covariate in the gastric protein hydrolysis. In general, greater gastric protein hydrolysis was observed in raw almonds (compared to roasted almonds), hypothesized to be related to structural changes in almond proteins during roasting. Greater gastric protein hydrolysis was observed in the distal stomach (compared to the proximal stomach), likely related to the lower pH in the distal stomach. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Method to Determine 18O Kinetic Isotope Effects in the Hydrolysis of Nucleotide Triphosphates

PubMed Central

Du, Xinlin; Ferguson, Kurt; Sprang, Stephen R.

2007-01-01

A method to determine 18O kinetic isotope effects (KIE) in the hydrolysis of GTP is described that is generally applicable to reactions involving other nucleotide triphosphates. Internal competition, wherein the substrate of the reaction is a mixture of 18O-labeled and unlabeled nucleotides, is employed and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18O at sites of mechanistic interest also contains 13C at all carbon positions, while the 16O-nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink™ interface (ThermoFinnigan). Carbon isotope ratios can be determined with accuracy and precision greater than 0.04%, and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1333-catalyzed hydrolysis of [β18O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (< 0.1%). A single KIE measurement can be conducted in 25 minutes with less than 5 μg nucleotide reaction product. PMID:17963711

Rapid spot test for the determination of esculin hydrolysis.

PubMed Central

Edberg, S C; Gam, K; Bottenbley, C J; Singer, J M

1976-01-01

Esculin hydrolysis is a useful test in the differentiation of both gram-positive and gram-negative bacteria covering a wide spectrum of aerobes, facultative anaerobes, and anaerobes. Commonly utilized methods require a minimum of 18 h of incubation in broth or agar medium and utilize the production of a brown-black compound, due to the combination of ferric ions with the hydrolysis product esculetin, as indicator. A procedure is presented that requires 15 to 30 min for completion and utilizes fluorescence loss as the indicator of hydrolysis. Esculin fluoresces at 366 nm, whereas the hydrolysis product esculetin does not. Over 1,400 strains of gram-positive and gram-negative bacteria were tested. There was 98.4% of correlation between the spot test and esculin broth and 97% correlation with the bile-esculin agar. Images PMID:787006

Comparative outcomes of β-lactam antibiotics in outpatient parenteral antibiotic therapy: treatment success, readmissions and antibiotic switches.

PubMed

Lee, Boeun; Tam, Idy; Weigel, Bernard; Breeze, Janis L; Paulus, Jessica K; Nelson, Jason; Allison, Genève M

2015-08-01

β-Lactam antibiotics are commonly used in outpatient parenteral antimicrobial therapy (OPAT), but data regarding outcomes of long-term therapy are limited. The purpose of this study was to compare treatment success, readmission and antibiotic switch rates in patients treated with β-lactam antibiotics as OPAT. We carried out a retrospective review of all patients, discharged from Tufts Medical Center with cefazolin, ceftriaxone, ertapenem or oxacillin, between January 2009 and June 2013. A competing risks analysis was used to compare the cumulative incidence of first occurrence of treatment success, antibiotic switch and 30 day readmission for each drug. Four hundred patients were identified (cefazolin n = 38, ceftriaxone n = 104, ertapenem n = 128 and oxacillin n = 130). Baseline demographics were similar. Treatment success rates were higher for ceftriaxone and ertapenem (cefazolin 61%, ceftriaxone 81%, ertapenem 73% and oxacillin 58%; P < 0.001). Thirty-day all-cause readmissions were similar (cefazolin 21%, ceftriaxone 14%, ertapenem 20% and oxacillin 15%; P = 0.46). In 400 OPAT courses, 37 out of 50 antibiotic switches were accomplished without readmission. Adverse drug events (ADEs) were the most common reason for outpatient antibiotic switches (31/37, 84%). The ADE rate was higher for the oxacillin group (cefazolin 2.0 versus ceftriaxone 1.5 versus ertapenem 2.9 versus oxacillin 8.4 per 1000 OPAT days; P < 0.001). OPAT with β-lactam antibiotics is effective, but antibiotic switches for adverse events were more frequent with oxacillin use. Clinicians should be cognizant of the risk of readmissions and ADEs in OPAT patients, as the value of OPAT lies in reducing patient morbidity and readmissions by managing ADEs and preventing clinical failures. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Removal of β-lactam antibiotics from pharmaceutical wastewaters using photo-Fenton process at near-neutral pH.

PubMed

Giraldo-Aguirre, Ana L; Serna-Galvis, Efraím A; Erazo-Erazo, Edgar D; Silva-Agredo, Javier; Giraldo-Ospina, Héctor; Flórez-Acosta, Oscar A; Torres-Palma, Ricardo A

2017-02-03

In this work, the photo-Fenton process at near-neutral pH was applied for the removal of the β-lactam antibiotic oxacillin (OXA) in water using artificial and sunlight. Initially, the main variables of the process (Fe(II), H 2 O 2 , and light power) were optimized by a statistical factorial design (2 3 with center points). The experimental design indicated that 90 μmol L -1 of Fe(II), 10 mmol L -1 of H 2 O 2 , and 30 W of power light were the favorable conditions for degradation of OXA at 203 μmol L -1 . In the photo-Fenton system, the H 2 O 2 alone, UV-light/H 2 O 2 , and Fe(II)/H 2 O 2 subsystems presented a significant participation on antibiotic removal. Moreover, based on the primary organic transformation products, a mechanism of OXA degradation was proposed. Under the favorable operational conditions, both the pollutant and the antimicrobial activity were eliminated after 50 min of process application. Although at 480 min of treatment, only 5% of mineralization was achieved, the level of biodegradability of the solutions increased from 0.08 to 0.98. Interestingly, the presence of pharmaceutical additives (glucose, isopropanol, and oxalic acid) had a moderate interference on the efficiency of the pollutant removal. Additionally, the treatment at pilot scale of the β-lactam antibiotic in a pharmaceutical complex matrix using solar radiation allowed the complete removal of the pollutant and its associated antimicrobial activity in a very short time period (5 min). These results evidenced the applicability of the photo-Fenton process to treat wastewaters from pharmaceutical industry loaded with β-lactam antibiotics at near neutral pH values efficiently.

Hydrolysis of diacylglycerols by lipoprotein lipase.

PubMed

Morley, N H; Kuksis, A; Buchnea, D; Myher, J J

1975-05-10

Enantiomeric diacylglycerols were emulsified, mole for mole, with lyso(1-acyl) lecithin and were hydrolyzed with lipoprotein lipase in NH4Cl-beef serum albumin buffer at pH 8.6 after a brief incubation with delipidated rat serum. The enzyme was prepared from lyophilized and dialyzed bovine skim milk in a 4 percent solution. The course of hydrolysis for each set of enantiomers was determined by gas-liquid chromatography of the masses of the diacylglycerols remaining or monoacylglycerols released in the medium between 0 and 15 min. The majority of sets of sn-1,2- and 2,3-diacylglycerols, including an isotope-labeled true enantiomeric set which was assessed by mass spectrometry, demonstrated preference by the enzyme for lipolysis at position 1 but with less specificity than previously was shown in sn-triacylglycerol hydrolysis. The results preclude the possibility that the predominance of sn-2,3-diacylglycerol intermediates during triacylglycerol hydrolysis is due solely to a preferential breakdown of the 1,2-isomers and reinforce the conclusion that lipoprotein lipase is specific for position 1.

Modeling of autocatalytic hydrolysis of adefovir dipivoxil in solid formulations.

PubMed

Dong, Ying; Zhang, Yan; Xiang, Bingren; Deng, Haishan; Wu, Jingfang

2011-04-01

The stability and hydrolysis kinetics of a phosphate prodrug, adefovir dipivoxil, in solid formulations were studied. The stability relationship between five solid formulations was explored. An autocatalytic mechanism for hydrolysis could be proposed according to the kinetic behavior which fits the Prout-Tompkins model well. For the classical kinetic models could hardly describe and predict the hydrolysis kinetics of adefovir dipivoxil in solid formulations accurately when the temperature is high, a feedforward multilayer perceptron (MLP) neural network was constructed to model the hydrolysis kinetics. The build-in approaches in Weka, such as lazy classifiers and rule-based learners (IBk, KStar, DecisionTable and M5Rules), were used to verify the performance of MLP. The predictability of the models was evaluated by 10-fold cross-validation and an external test set. It reveals that MLP should be of general applicability proposing an alternative efficient way to model and predict autocatalytic hydrolysis kinetics for phosphate prodrugs.

Hydrolysis mechanism of methyl parathion evidenced by Q-Exactive mass spectrometry.

PubMed

Liu, Yuan; Zhang, Caixiang; Liao, Xiaoping; Luo, Yinwen; Wu, Sisi; Wang, Jianwei

2015-12-01

Organophosphorus pesticides (OPPs), a kind of widely used pesticides, are currently attracting great attention due to their adverse effects on human central nervous systems, particularly in children. Although the hydrolysis behavior of OPPs has been studied well, its hydrolysis mechanism remained controversial, especially at various pH conditions, partly due to their relatively complex structures and abundant moieties that were prone to be attacked by nucleophiles. The Q-Exactive mass spectrometer, part of those hybrid high-resolution mass spectrometers (HRMS), was used to determine hydrolysis products of methyl parathion (MP), a kind of OPPs in situ buffer aqueous solution with pH ranging from 1 to 13 in this study. Most of the complex hydrolysis products of MP were identified due to the high sensitivity and accuracy of HRMS. The results demonstrated that the hydrolysis rate and pathway of MP were strong pH dependent. With the increase of pH, the hydrolysis rate of MP increased, and two different reaction mechanisms were identified: SN (2)@P pathway dominated the hydrolysis process at high pH (e.g., pH ≥ 11) while SN (2)@C was the main behavior at low pH (e.g., pH ≤ 9). This study helps understand the hydrolysis mechanism of OPPs at various pH and extends the use of Q-Exactive mass spectrometry in identifying organic pollutants and their degradation products in environmental matrices.

Study on the technology of compound enzymatic hydrolysis of whole passion fruit

NASA Astrophysics Data System (ADS)

Yang, Yu-xia; Duan, Zhen-hua; Kang, Chao; Zhu, Xiang-hao; Li, Ding-jin

2017-12-01

Fresh Whole Passion Fruit was used as raw material, The enzymatic hydrolysis technology of Passion Fruit by Complex enzyme were studied, The effects of enzyme dosage, Enzyme ratio(cellulose: pectinase), pH, temperature and time on the hydrolysis were investigated by single-tests and orthogonal tests, the hydrolysis indicators of single-factor tests and orthogonal tests were juice yield. The optimal hydrolysis conditions of Passion Fruit by Complex enzyme were enzyme dosage 0.12%, Enzyme ratio 5:1, hydrolysis temperature 50°C, pH4.0 and time 3.5 h. Under such conditions, juice yield of Passion Fruit was 92.91%.

Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors

USDA-ARS?s Scientific Manuscript database

Removal of inhibitory compounds by bioabatement, combined with xylan hydrolysis, enables effective cellulose hydrolysis of pretreated corn stover, for fermentation of the sugars to fuel ethanol or other products. The fungus Coniochaeta ligniaria NRRL30616 eliminates most enzyme and fermentation inhi...

Accelerated hydrolysis of substituted cellulose for potential biofuel production: kinetic study and modeling.

PubMed

Mu, Bingnan; Xu, Helan; Yang, Yiqi

2015-11-01

In this work, kinetics of substitution accelerated cellulose hydrolysis with multiple reaction stages was investigated to lay foundation for mechanism study and molecular design of substituting compounds. High-efficiency hydrolysis of cellulose is critical for cellulose-based bioethanol production. It is known that, substitution could substantially decrease activation energy and increase reaction rate of acidic hydrolysis of glycosidic bonds in cellulose. However, reaction kinetics and mechanism of the accelerated hydrolysis were not fully revealed. In this research, it was proved that substitution therefore accelerated hydrolysis only occurred in amorphous regions of cellulose fibers, and was a process with multiple reaction stages. With molar ratio of substitution less than 1%, the overall hydrolysis rate could be increased for around 10 times. We also quantified the relationship between the hydrolysis rate of individual reaction stage and its major influences, including molar ratio of substitution, activation energy of acidic hydrolysis, pH and temperature. Copyright © 2015 Elsevier Ltd. All rights reserved.

In-silico modeling of a novel OXA-51 from β-lactam-resistant Acinetobacter baumannii and its interaction with various antibiotics.

PubMed

Tiwari, Vishvanath; Nagpal, Isha; Subbarao, Naidu; Moganty, Rajeswari R

2012-07-01

Acinetobacter baumannii, one of the major Gram negative bacteria, causes nosocomial infections such as pneumonia, urinary tract infection, meningitis, etc. β-lactam-based antibiotics like penicillin are used conventionally to treat infections of A. baumannii; however, they are becoming progressively less effective as the bacterium produces diverse types of β-lactamases to inactivate the antibiotics. We have recently identified a novel β-lactamase, OXA-51 from clinical strains of A. baumannii from our hospital. In the present study, we generated the structure of OXA-51 using MODELLER9v7 and studied the interaction of OXA-51 with a number of β-lactams (penicillin, oxacillin, ceftazidime, aztreonam and imipenem) using two independent programs: GLIDE and GOLD. Based on the results of different binding parameters and number of hydrogen bonds, interaction of OXA-51 was found to be maximum with ceftazidime and lowest with imipenem. Further, molecular dynamics simulation results also support this fact. The lowest binding affinity of imipenem to OXA-51 indicates clearly that it is not efficiently cleaved by OXA-51, thus explaining its high potency against resistant A. baumannii. This finding is supported by experimental results from minimum inhibitory concentration analysis and transmission electron microscopy. It can be concluded that carbapenems (imipenem) are presently effective β-lactam antibiotics against resistant strains of A. baumannii harbouring OXA-51. The results presented here could be useful in designing more effective derivatives of carbapenem.

Genetic characterization and plasmid replicon typing of ß-lactam resistant Escherichia coli from humans and companion animals in Egypt

USDA-ARS?s Scientific Manuscript database

Limited therapeutic options due to antimicrobial resistance (AR) is a major threat to human and animal health worldwide. There is a paucity of information on ß-lactam resistant Esherichia coli isolated from companion animals in developing countries; therefore their zoonotic impact is unknown. This s...

Sub-Equimolar Hydrolysis and Condensation of Organophosphates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alam, Todd M.; Kinnan, Mark K.; Wilson, Brendan W.

We characterized the in-situ hydrolysis and subsequent condensation reaction of the chemical agent simulant diethyl chlorophosphate (DECP) by high-resolution 31P NMR spectroscopy following the addition of water in sub-equimolar concentrations. Moreover, the identification and quantification of the multiple pyrophosphate and larger polyphosphate chemical species formed through a series of self-condensation reactions are reported. Finally, the DECP hydrolysis kinetics and distribution of breakdown species was strongly influenced by the water concentration and reaction temperature.

Sub-Equimolar Hydrolysis and Condensation of Organophosphates

DOE PAGES

Alam, Todd M.; Kinnan, Mark K.; Wilson, Brendan W.; ...

2016-07-16

We characterized the in-situ hydrolysis and subsequent condensation reaction of the chemical agent simulant diethyl chlorophosphate (DECP) by high-resolution 31P NMR spectroscopy following the addition of water in sub-equimolar concentrations. Moreover, the identification and quantification of the multiple pyrophosphate and larger polyphosphate chemical species formed through a series of self-condensation reactions are reported. Finally, the DECP hydrolysis kinetics and distribution of breakdown species was strongly influenced by the water concentration and reaction temperature.

Hydrolysis of the amorphous cellulose in cotton-based paper.

PubMed

Stephens, Catherine H; Whitmore, Paul M; Morris, Hannah R; Bier, Mark E

2008-04-01

Hydrolysis of cellulose in Whatman no. 42 cotton-based paper was studied using gel permeation chromatography (GPC), electrospray ionization-mass spectrometry (ESI-MS), and uniaxial tensile testing to understand the course and kinetics of the reaction. GPC results suggested that scission reactions passed through three stages. Additionally, the evolution of soluble oligomers in the ESI-MS data and the steady course of strength loss showed that the hydrolysis reaction occurred at a constant rate. These findings are explained with a more detailed description of the cellulose hydrolysis, which includes multiple chain scissions on amorphous segments. The breaks occur with increasing frequency near the ends of amorphous segments, where chains protrude from crystalline domains. Oligomers unattached to crystalline domains are eventually created. Late-stage reactions near the ends of amorphous segments produce a kinetic behavior that falsely suggests that hydrolysis had ceased. Monte Carlo simulations of cellulose degradation corroborated the experimental findings.

Evaluation of hydrolysis-esterification biodiesel production from wet microalgae.

PubMed

Song, Chunfeng; Liu, Qingling; Ji, Na; Deng, Shuai; Zhao, Jun; Li, Shuhong; Kitamura, Yutaka

2016-08-01

Wet microalgae hydrolysis-esterification route has the advantage to avoid the energy-intensive units (e.g. drying and lipid extraction) in the biodiesel production process. In this study, techno-economic evaluation of hydrolysis-esterification biodiesel production process was carried out and compared with conventional (usually including drying, lipid extraction, esterification and transesterification) biodiesel production process. Energy and material balance of the conventional and hydrolysis-esterification processes was evaluated by Aspen Plus. The simulation results indicated that drying (2.36MJ/L biodiesel) and triolein transesterification (1.89MJ/L biodiesel) are the dominant energy-intensive stages in the conventional route (5.42MJ/L biodiesel). By contrast, the total energy consumption of hydrolysis-esterification route can be reduced to 1.81MJ/L biodiesel, and approximately 3.61MJ can be saved to produce per liter biodiesel. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rapid enzymatic hydrolysis using a novel recombinant β-glucuronidase in benzodiazepine urinalysis.

PubMed

Morris, Ayodele A; Chester, Scot A; Strickland, Erin C; McIntire, Gregory L

2014-10-01

Only trace amounts of parent benzodiazepines are present in urine following extensive metabolism and conjugation. Thus, hydrolysis of glucuronides is necessary for improved detection. Enzyme hydrolysis is preferred to retain identification specificity, but can be costly and time-consuming. The assessment of a novel recombinant β-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant β-glucuronidase. Hydrolysis efficiency was assessed at 55°C and at room temperature (RT) using the recommended optimum pH. Hydrolysis efficiency for four other benzodiazepines was evaluated solely with positive patient samples. Maximum hydrolysis of glucuronide controls at 5 min at RT (mean analyte recovery ≥ 94% for oxazepam and lorazepam and ≥ 80% for temazepam) was observed. This was considerably faster than the optimized 30 min incubation time for the abalone β-glucuronidase at 65°C. Mean analyte recovery increased at longer incubation times at 55°C for temazepam only. Total analyte in patient samples compared well to targets from abalone hydrolysis after recombinant β-glucuronidase hydrolysis at RT with no incubation. Some matrix effect, differential reactivity, conjugation variability and transformation impacting total analyte recovery were indicated. The unique potential of the IMCSzyme™ recombinant β-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fuzzy logic feedback control for fed-batch enzymatic hydrolysis of lignocellulosic biomass.

PubMed

Tai, Chao; Voltan, Diego S; Keshwani, Deepak R; Meyer, George E; Kuhar, Pankaj S

2016-06-01

A fuzzy logic feedback control system was developed for process monitoring and feeding control in fed-batch enzymatic hydrolysis of a lignocellulosic biomass, dilute acid-pretreated corn stover. Digested glucose from hydrolysis reaction was assigned as input while doser feeding time and speed of pretreated biomass were responses from fuzzy logic control system. Membership functions for these three variables and rule-base were created based on batch hydrolysis data. The system response was first tested in LabVIEW environment then the performance was evaluated through real-time hydrolysis reaction. The feeding operations were determined timely by fuzzy logic control system and efficient responses were shown to plateau phases during hydrolysis. Feeding of proper amount of cellulose and maintaining solids content was well balanced. Fuzzy logic proved to be a robust and effective online feeding control tool for fed-batch enzymatic hydrolysis.

Mechanistic kinetic models of enzymatic cellulose hydrolysis-A review.

PubMed

Jeoh, Tina; Cardona, Maria J; Karuna, Nardrapee; Mudinoor, Akshata R; Nill, Jennifer

2017-07-01

Bioconversion of lignocellulose forms the basis for renewable, advanced biofuels, and bioproducts. Mechanisms of hydrolysis of cellulose by cellulases have been actively studied for nearly 70 years with significant gains in understanding of the cellulolytic enzymes. Yet, a full mechanistic understanding of the hydrolysis reaction has been elusive. We present a review to highlight new insights gained since the most recent comprehensive review of cellulose hydrolysis kinetic models by Bansal et al. (2009) Biotechnol Adv 27:833-848. Recent models have taken a two-pronged approach to tackle the challenge of modeling the complex heterogeneous reaction-an enzyme-centric modeling approach centered on the molecularity of the cellulase-cellulose interactions to examine rate limiting elementary steps and a substrate-centric modeling approach aimed at capturing the limiting property of the insoluble cellulose substrate. Collectively, modeling results suggest that at the molecular-scale, how rapidly cellulases can bind productively (complexation) and release from cellulose (decomplexation) is limiting, while the overall hydrolysis rate is largely insensitive to the catalytic rate constant. The surface area of the insoluble substrate and the degrees of polymerization of the cellulose molecules in the reaction both limit initial hydrolysis rates only. Neither enzyme-centric models nor substrate-centric models can consistently capture hydrolysis time course at extended reaction times. Thus, questions of the true reaction limiting factors at extended reaction times and the role of complexation and decomplexation in rate limitation remain unresolved. Biotechnol. Bioeng. 2017;114: 1369-1385. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Enzymatic hydrolysis of lignocellulosic biomass by Kitasatospora sp. to produce xylo-oligosaccharides (XOS)

NASA Astrophysics Data System (ADS)

Rahmani, Nanik; Jannah, Alifah Mafatikhul; Lisdiyanti, Puspita; Prasetya, Bambang; Yopi

2017-11-01

The optimizations of enzymatic hydrolysis to produce of xylo-oligosaccharides (XOs) from three different lignocellulosic biomasses were investigated. Sugarcane bagasse, oil palm empty fruit bunch, and rice straw contain rich hemicelluloses especially hetero-xylan which can be hydrolyzes by endo-xylanase enzyme. Enzymatic hydrolysis of sugarcane bagasse by endo-xylanase from Kitasatospora sp. was optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 4 % substrate concentrations, while oil palm empty fruit bunchwas optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 5 % substrate concentrations, and rice straw was optimum at 40 °C temperature hydrolysis using 16 U of enzyme concentrations and 4 % substrate concentrations. The hydrolysis products were analyzed by TLC and HPLC. The main product hydrolysis for sugarcane bagasse, oil palm empty fruit bunch and rice straw are xylobiose.

Internal Hydrolysis Indicator for Sample Specific Monitoring of β-Glucuronidase Activity.

PubMed

Taylor, Lacy L; Flint, Noah A; Ma, Vinh; Hill, Brandy M; Clark, Chantry J; Strathmann, Frederick G

2017-06-01

Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing β-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the β-Glucuronidase enzyme. In this study resorufin β-D-glucuronide, a substrate of the β-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin β-D-glucuronide. The β-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin β-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The β-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Theoretical studies of the ATP hydrolysis mechanism of myosin.

PubMed

Okimoto, N; Yamanaka, K; Ueno, J; Hata, M; Hoshino, T; Tsuda, M

2001-11-01

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis.

Process development of starch hydrolysis using mixing characteristics of Taylor vortices.

PubMed

Masuda, Hayato; Horie, Takafumi; Hubacz, Robert; Ohmura, Naoto; Shimoyamada, Makoto

2017-04-01

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor-Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.

Acid-Catalyzed Isomerization of Carvone to Carvacrol

ERIC Educational Resources Information Center

Kjonaas, Richard A.; Mattingly, Shawn P.

2005-01-01

The acid-catalyzed isomerization of carvone to carvacrol, first reported by Ritter and Ginsburg, is especially well suited with a permanent-magnet FT instrument. The acid-catalyzed isomerization of carvone to carvacrol produced a 61% yield after a three hour reflux with 30% aqueous sulfuric acid.

Histidine-functionalized water-soluble nanoparticles for biomimetic nucleophilic/general-base catalysis under acidic conditions.

PubMed

Chadha, Geetika; Zhao, Yan

2013-10-21

Cross-linking the micelles of 4-dodecyloxybenzyltripropargylammonium bromide by 1,4-diazidobutane-2,3-diol in the presence of azide-functionalized imidazole derivatives yielded surface-cross-linked micelles (SCMs) with imidazole groups on the surface. The resulting water-soluble nanoparticles were found, by fluorescence spectroscopy, to contain hydrophobic binding sites. The imidazole groups promoted the photo-deprotonation of 2-naphthol at pH 6 and catalyzed the hydrolysis of p-nitrophenylacetate (PNPA) in aqueous solution at pH ≥ 4. Although the overall hydrolysis rate slowed down with decreasing solution pH, the catalytic effect of the imidazole became stronger because the reactions catalyzed by unfunctionalized SCMs slowed down much more. The unusual ability of the imidazole–SCMs to catalyze the hydrolysis of PNPA under acidic conditions was attributed to the local hydrophobicity and the positive nature of the SCMs.

[Isolation of the ergot (Claviceps purpurea (Fr.) Tul., strain VKM-F-366D), producing the lactamic alkaloid ergocornam].

PubMed

Komarova, E L; Shain, S S; Sheĭchenko, V I

2002-01-01

A new ergot strain VKM-F-3662D producing lactamic alkaloid ergocornam with concomitant alkaloids valinamide and ergometrine was isolated during selective works with sclerotium MS-462, which was obtained from ergocryptine ergot strain VKM-F-2642D. The structure of these alkaloids was determined by 1H and 13C NMR.

Validation of lignocellulosic biomass carbohydrates determination via acid hydrolysis.

PubMed

Zhou, Shengfei; Runge, Troy M

2014-11-04

This work studied the two-step acid hydrolysis for determining carbohydrates in lignocellulosic biomass. Estimation of sugar loss based on acid hydrolyzed sugar standards or analysis of sugar derivatives was investigated. Four model substrates (starch, holocellulose, filter paper and cotton) and three levels of acid/material ratios (7.8, 10.3 and 15.4, v/w) were studied to demonstrate the range of test artifacts. The method for carbohydrates estimation based on acid hydrolyzed sugar standards having the most satisfactory carbohydrate recovery and relative standard deviation. Raw material and the acid/material ratio both had significant effect on carbohydrate hydrolysis, suggesting the acid to have impacts beyond a catalyst in the hydrolysis. Following optimal procedures, we were able to reach a carbohydrate recovery of 96% with a relative standard deviation less than 3%. The carbohydrates recovery lower than 100% was likely due to the incomplete hydrolysis of substrates, which was supported by scanning electron microscope (SEM) images. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of new drugs for an old target: the penicillin binding proteins.

PubMed

Zervosen, Astrid; Sauvage, Eric; Frère, Jean-Marie; Charlier, Paulette; Luxen, André

2012-10-24

The widespread use of β-lactam antibiotics has led to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to β-lactams by two main mechanisms: the production of β-lactamases, sometimes accompanied by a decrease of outer membrane permeability, and the production of low-affinity, drug resistant Penicillin Binding Proteins (PBPs). PBPs remain attractive targets for developing new antibiotic agents because they catalyse the last steps of the biosynthesis of peptidoglycan, which is unique to bacteria, and lies outside the cytoplasmic membrane. Here we summarize the “current state of the art” of non-β-lactam inhibitors of PBPs, which have being developed in an attempt to counter the emergence of β-lactam resistance. These molecules are not susceptible to hydrolysis by β-lactamases and thus present a real alternative to β-lactams. We present transition state analogs such as boronic acids, which can covalently bind to the active serine residue in the catalytic site. Molecules containing ring structures different from the β-lactam-ring like lactivicin are able to acylate the active serine residue. High throughput screening methods, in combination with virtual screening methods and structure based design, have allowed the development of new molecules. Some of these novel inhibitors are active against major pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and thus open avenues new for the discovery of novel antibiotics.

WCK 5107 (Zidebactam) and WCK 5153 Are Novel Inhibitors of PBP2 Showing Potent "β-Lactam Enhancer" Activity against Pseudomonas aeruginosa, Including Multidrug-Resistant Metallo-β-Lactamase-Producing High-Risk Clones.

PubMed

Moya, Bartolome; Barcelo, Isabel M; Bhagwat, Sachin; Patel, Mahesh; Bou, German; Papp-Wallace, Krisztina M; Bonomo, Robert A; Oliver, Antonio

2017-06-01

Zidebactam and WCK 5153 are novel β-lactam enhancers that are bicyclo-acyl hydrazides (BCH), derivatives of the diazabicyclooctane (DBO) scaffold, targeted for the treatment of serious infections caused by highly drug-resistant Gram-negative pathogens. In this study, we determined the penicillin-binding protein (PBP) inhibition profiles and the antimicrobial activities of zidebactam and WCK 5153 against Pseudomonas aeruginosa , including multidrug-resistant (MDR) metallo-β-lactamase (MBL)-producing high-risk clones. MIC determinations and time-kill assays were conducted for zidebactam, WCK 5153, and antipseudomonal β-lactams using wild-type PAO1, MexAB-OprM-hyperproducing ( mexR ), porin-deficient ( oprD ), and AmpC-hyperproducing ( dacB ) derivatives of PAO1, and MBL-expressing clinical strains ST175 ( bla VIM-2 ) and ST111 ( bla VIM-1 ). Furthermore, steady-state kinetics was used to assess the inhibitory potential of these compounds against the purified VIM-2 MBL. Zidebactam and WCK 5153 showed specific PBP2 inhibition and did not inhibit VIM-2 (apparent K i [ K i app ] > 100 μM). MICs for zidebactam and WCK 5153 ranged from 2 to 32 μg/ml (amdinocillin MICs > 32 μg/ml). Time-kill assays revealed bactericidal activity of zidebactam and WCK 5153. LIVE-DEAD staining further supported the bactericidal activity of both compounds, showing spheroplast formation. Fixed concentrations (4 or 8 μg/ml) of zidebactam and WCK 5153 restored susceptibility to all of the tested β-lactams for each of the P. aeruginosa mutant strains. Likewise, antipseudomonal β-lactams (CLSI breakpoints), in combination with 4 or 8 μg/ml of zidebactam or WCK 5153, resulted in enhanced killing. Certain combinations determined full bacterial eradication, even with MDR MBL-producing high-risk clones. β-Lactam-WCK enhancer combinations represent a promising β-lactam "enhancer-based" approach to treat MDR P. aeruginosa infections, bypassing the need for MBL inhibition. Copyright © 2017

111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging.

PubMed

Miao, Yubin; Gallazzi, Fabio; Guo, Haixun; Quinn, Thomas P

2008-02-01

The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.

Induction of rat liver microsomal epoxide hydrolase by thiazole and pyrazine: hydrolysis of 2-cyanoethylene oxide.

PubMed

Kim, S G; Kedderis, G L; Batra, R; Novak, R F

1993-08-01

mEH was the catalyst. The Vmax for the hydrolysis of CEO by hepatic microsomes from thiazole-treated rats (13.4 nmol/min/mg protein) was 1.5-fold greater than that with microsomes from pyrazine-treated rats, whereas similar Km values (approximately 1 mM) were observed for both microsomal preparations. These kinetic data correlate well with the increases in mEH mRNA observed after administration of thiazole or pyrazine to rats. These results provide evidence that administration of thiazole or pyrazine induces mEH with a large increase in mEH mRNA, and that the induced mEH catalyzes the hydrolysis of CEO.

Optimization of chip size and moisture content to obtain high, combined sugar recovery after sulfur dioxide-catalyzed steam pretreatment of softwood and enzymatic hydrolysis of the cellulosic component.

PubMed

Olsen, Colin; Arantes, Valdeir; Saddler, Jack

2015-01-01

The influence of chip size and moisture content on the combined sugar recovery after steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis of the cellulosic component were investigated using response surface methodology. Chip size had little influence on sugar recovery after both steam pretreatment and enzymatic hydrolysis. In contrast, the moisture of the chips greatly influenced the relative severity of steam pretreatment and, as a result, the combined sugar recovery from the hemicellulosic and cellulosic fractions. Irrespective of chip size and the pretreatment temperature, time, and SO2 loading that were used, the relative severity of pretreatment was highest at a moisture of 30-40w/w%. However, the predictive model indicated that an elevated moisture content of roughly 50w/w% (about the moisture content of a standard softwood mill chip) would result in the highest, combined sugar recovery (80%) over the widest range of steam pretreatment conditions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of beta-Lactamases and beta-Lactam-Related Proteins in Human Pathogenic Bacteria using a Computational Search Approach.

PubMed

Brambila-Tapia, Aniel Jessica Leticia; Perez-Rueda, Ernesto; Barrios, Humberto; Dávalos-Rodríguez, Nory Omayra; Dávalos-Rodríguez, Ingrid Patricia; Cardona-Muñoz, Ernesto Germán; Salazar-Páramo, Mario

2017-08-01

A systematic analysis of beta-lactamases based on comparative proteomics has not been performed thus far. In this report, we searched for the presence of beta-lactam-related proteins in 591 bacterial proteomes belonging to 52 species that are pathogenic to humans. The amino acid sequences for 19 different types of beta-lactamases (ACT, CARB, CifA, CMY, CTX, FOX, GES, GOB, IMP, IND, KPC, LEN, OKP, OXA, OXY, SHV, TEM, NDM, and VIM) were obtained from the ARG-ANNOT database and were used to construct 19 HMM profiles, which were used to identify potential beta-lactamases in the completely sequenced bacterial proteomes. A total of 2877 matches that included the word "beta-lactamase" and/or "penicillin" in the functional annotation and/or in any of its regions were obtained. These enzymes were mainly described as "penicillin-binding proteins," "beta-lactamases," and "metallo-beta-lactamases" and were observed in 47 of the 52 species studied. In addition, proteins classified as "beta-lactamases" were observed in 39 of the species included. A positive correlation between the number of beta-lactam-related proteins per species and the proteome size was observed (R 0.78, P < 0.00001). This correlation partially explains the high presence of beta-lactam-related proteins in large proteomes, such as Nocardia brasiliensis, Bacillus anthracis, and Mycobacterium tuberculosis, along with their absence in small proteomes, such as Chlamydia spp. and Mycoplasma spp. We detected only five types of beta-lactamases (TEM, SHV, CTX, IMP, and OXA) and other related proteins in particular species that corresponded with those reported in the literature. We additionally detected other potential species-specific beta-lactamases that have not yet been reported. In the future, better results will be achieved due to more accurate sequence annotations and a greater number of sequenced genomes.

Synthesis of 5/7-, 5/8- and 5/9-bicyclic lactam templates as constraints for external beta-turns.

PubMed

Duggan, Heather M E; Hitchcock, Peter B; Young, Douglas W

2005-06-21

The 5/7-, 5/8- and 5/9-bicyclic lactams 3, 17, 5 and 6 have been synthesised as single diastereoisomers by a route involving ring closing olefin metathesis. The X-ray crystal structure of the amino acid hydrochloride has been carried out and compared to that of the saturated external beta-turn constraint 18.

Formation of Acridones by Ethylene Extrusion in the Reaction of Arynes with β-Lactams and Dihydroquinolinones

PubMed Central

Fang, Yuesi; Rogness, Donald C.; Larock, Richard C.; Shi, Feng

2012-01-01

N-Unsubstituted β-lactams react with a molecule of aryne by insertion into the amide bond to form a 2,3-dihydroquinolin-4-one, which subsequently reacts with another molecule of aryne to form an acridone by extrusion of a molecule of ethylene. 2,3-Dihydroquinolin-4-ones react under the same reaction conditions to afford identical results. This is the first example of ethylene extrusion in aryne chemistry. PMID:22742883

Investigating Mass Transport Limitations on Xylan Hydrolysis During Dilute Acid Pretreatment of Poplar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mittal, Ashutosh; Pilath, Heid M.; Parent, Yves

2014-04-28

Mass transport limitations could be an impediment to achieving high sugar yields during biomass pretreatment and thus be a critical factor in the economics of biofuels production. The objective of this work was to study the mass transfer restrictions imposed by the structure of biomass on the hydrolysis of xylan during dilute acid pretreatment of biomass. Mass transfer effects were studied by pretreating poplar wood at particle sizes ranging from 10 micrometers to 10 mm. This work showed a significant reduction in the rate of xylan hydrolysis in poplar when compared to the intrinsic rate of hydrolysis for isolated xylanmore » that is possible in the absence of mass transfer. In poplar samples we observed no significant difference in the rates of xylan hydrolysis over more than two orders of magnitude in particle size. It appears that no additional mass transport restrictions are introduced by increasing particle size from 10 micrometers to 10 mm. This work suggests that the rates of xylan hydrolysis in biomass particles are limited primarily by the diffusion of hydrolysis products out of plant cell walls. A mathematical description is presented to describe the kinetics of xylan hydrolysis that includes transport of the hydrolysis products through biomass into the bulk solution. The modeling results show that the effective diffusion coefficient of the hydrolysis products in the cell wall is several orders of magnitude smaller than typical values in other applications signifying the role of plant cell walls in offering resistance to diffusion of the hydrolysis products.« less

Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae

PubMed Central

Ubukata, Kimiko; Shibasaki, Yumi; Yamamoto, Kentarou; Chiba, Naoko; Hasegawa, Keiko; Takeuchi, Yasuo; Sunakawa, Keisuke; Inoue, Matsuhisa; Konno, Masatoshi

2001-01-01

The affinity of [3H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of β-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae for which the AMP MIC was ≥1.0 μg/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the dacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-Thr-Gly (KTG) motif, was substituted for Arg-517 in group I strains (n = 9), and Lys-526 was substituted for Asn-526 in group II strains (n = 12). In group III strains (n = 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of β-lactams for H. influenzae transformants into which the ftsI gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for β-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in β-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to β-lactams in BLNAR strains. PMID:11353613

Surface-catalyzed air oxidation of hydrazines: Environmental chamber studies

NASA Technical Reports Server (NTRS)

Kilduff, Jan E.; Davis, Dennis D.; Koontz, Steven L.

1988-01-01

The surface-catalyzed air oxidation reactions of fuel hydrazines were studied in a 6500-liter fluorocarbon-film chamber at 80 to 100 ppm concentrations. First-order rate constants for the reactions catalyzed by aluminum, water-damaged aluminum (Al/Al2O3), stainless steel 304L, galvanized steel and titanium plates with surface areas of 2 to 24 sq m were determined. With 23.8 sq m of Al/Al2O3 the surface-catalyzed air oxidation of hydrazine had a half-life of 2 hours, diimide (N2H2) was observed as an intermediate and traces of ammonia were present in the final product mixture. The Al/Al2O3 catalyzed oxidation of monomethylhydrazine yielded methyldiazine (HN = NCH3) as an intermediate and traces of methanol. Unsymmetrical dimethylhydrazine gave no detectable products. The relative reactivities of hydrazine, MMH and UDMH were 130 : 7.3 : 1.0, respectively. The rate constants for Al/Al2O3-catalyzed oxidation of hydrazine and MMH were proportional to the square of the surface area of the plates. Mechanisms for the surface-catalyzed oxidation of hydrazine and diimide and the formation of ammonia are proposed.

Tryptophanol-derived oxazolopiperidone lactams: identification of a hit compound as NMDA receptor antagonist.

PubMed

Pereira, Nuno A L; Sureda, Francesc X; Esplugas, Roser; Pérez, Maria; Amat, Mercedes; Santos, Maria M M

2014-08-01

N-Methyl-D-aspartate receptors (NMDAR) exacerbated activation leads to neuron death through a phenomenon called excitotoxicity. These receptors are implicated in several neurological diseases (e.g., Alzheimer and Parkinson) and thus represent an important therapeutic target. We herein describe the study of enantiopure tryptophanol-derived oxazolopiperidone lactams as NMDA receptor antagonists. The most active hit exhibited an IC50 of 63.4 μM in cultured rat cerebellar granule neurons thus being 1.5 fold more active than clinically approved NMDA antagonist amantadine (IC50=92 μM). Copyright © 2014 Elsevier Ltd. All rights reserved.