Analyzing Current Trends and Possible Strategies to Improve Sucrose Isomerases' Thermostability.

Due to their ability to produce isomaltulose, sucrose isomerases are enzymes that have caught the attention of researchers and entrepreneurs since the 1950s. However, their low activity and stability at temperatures above 40 °C have been a bottleneck for their industrial application. Specifically, the instability of these enzymes has been a challenge when it comes to their use for the synthesis and manufacturing of chemicals on a practical scale. This is because industrial processes often require biocatalysts that can withstand harsh reaction conditions, like high temperatures. Since the 1980s, there have been significant advancements in the thermal stabilization engineering of enzymes. Based on the literature from the past few decades and the latest achievements in protein engineering, this article systematically describes the strategies used to enhance the thermal stability of sucrose isomerases. Additionally, from a theoretical perspective, we discuss other potential mechanisms that could be used for this purpose.

Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk.

Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 µg.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

Herbivorous Caterpillars Can Utilize Three Mechanisms to Alter Green Leaf Volatile Emission.

Green plants emit green leaf volatiles (GLVs) as a general damage response. These compounds act as signals for the emitter plant, neighboring plants, and even for insects in the ecosystem. However, when oral secretions from certain caterpillars are applied to wounded leaves, GLV emissions are significantly decreased or modified. We examined four caterpillar species representing two lepidopteran families for their capacity to decrease GLV emissions from Zea mays leaf tissue. We also investigated the source of the GLV modifying components in the alimentary tract of the various caterpillars. In Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), we found three distinct mechanisms to modify GLV emission: a heat-stable compound in the gut, a heat-labile enzyme in salivary gland homogenate (previously described in Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), and an isomerase in the salivary gland homogenate, which catalyzes the conversion of (Z)-3-hexenal to (E)-2-hexenal (previously described in M. sexta). These mechanisms employed by caterpillars to suppress or modify GLV emission suggest a counteraction against the induced indirect volatile defenses of a plant and provides further insights into the ecological functions of GLVs.

High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma.

PURPOSE: In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma EXPERIMENTAL DESIGN: To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils are isolated from control and surgical trauma rats in this study. Extracted proteins are analyzed using nano liquid chromatography coupled with tandem mass spectrometry. RESULTS: A total of 2924 rat neutrophil proteins are identified in our analysis, of which 393 are found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma, we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations are then confirmed by in silico protein-protein interaction analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage.

Enzyme kinetics and transport in a system crowded by mobile macromolecules.

The dynamics of an elastic network model for the enzyme 4-oxalocrotonate tautomerase is studied in a system crowded by mobile macromolecules, also modeled by elastic networks. The system includes a large number of solvent molecules, as well as substrate and product molecules which undergo catalytic reactions with this hexameric protein. The time evolution of the entire system takes place through a hybrid dynamics that combines molecular dynamics for solute species and multiparticle collision dynamics for the solvent. It is shown that crowding leads to subdiffusive dynamics for the protein, in accord with many studies of diffusion in crowded environments, and increases orientational relaxation times. The enzyme reaction kinetics is also modified by crowding. The effective Michaelis constant decreases with crowding volume fraction, and this decrease is attributed to excluded volume effects, which dominate over effects due to reduced substrate diffusion that would cause the Michaelis constant to increase.

Health impact assessment of decreases in PM10 and ozone concentrations in the Mexico City Metropolitan Area: A basis for a new air quality management program / Evaluación de impacto en salud ante reducciones de PM10 y ozono en la Zona Metropolitana del Valle de México: Base para un nuevo programa de calidad del aire

Objective. To conduct a health impact assessment (HIA) to quantify health benefits for several PM and O3 air pollution reduction scenarios in the Mexico City Metropolitan Area (MCMA). Results from this HIA will contribute to the scientific support of the MCMA air quality management plan (PROAIRE) for the period 2011-2020. Materials and methods. The HIA methodology consisted of four steps: 1) selection of the air pollution reduction scenarios, 2) identification of the at-risk population and health outcomes for the 2005 baseline scenario, 3) selection of concentration-response functions and 4) estimation of health impacts. Results. Reductions of PM10 levels to 20 μg/m³ and O3 levels to 0.050ppm (98 µg/m³) would prevent 2300 and 400 annual deaths respectively. The greatest health impact was seen in the over-65 age group and in mortality due to cardiopulmonary and cardiovascular disease. Conclusion. Improved air quality in the MCMA could provide significant health benefits through focusing interventions by exposure zones.

Objetivo. Realizar una evaluación de impacto en salud (EIS) que documente los beneficios en salud ante diversos escenarios de reducción de PM10 y O3 en el aire de la Zona Metropolitana del Valle de México (ZMVM). Los resultados contribuyen al sustento científico del plan de gestión de calidad del aire (PROAIRE 2011-2020). Material y métodos. La metodología de EIS comprende cuatro pasos: 1) selección de los escenarios de reducción, 2) identificación de la población en riesgo y de los eventos en salud para el año basal 2005, 3) selección de las funciones de concentración-respuesta y 4) estimación del impacto en la salud. Resultados. Reducciones de PM10 a 20μg/m³ y de O3 a 0.050ppm (98 µg/m³) evitarían, respectivamente, cerca de 2 300 y 400 muertes por año. El mayor impacto se observa en el grupo de más de 65 años y en la mortalidad por causas cardiopulmonares y cardiovasculares. Conclusiones. Mejorar la calidad del aire en la ZMVM podría reflejar importantes beneficios para la salud focalizados por zonas o áreas de exposición.

Un modelo del mecanismo de entrada de los rotavirus a la célula hospedera / A model for the mechanism of entry of rotaviruses into the host cell

Antecedentes. La infección rotaviral es causa principal de gastroenteritis aguda severa en niños menores de cinco años. La capa protéica externa de la partícula viral está implicada en las interacciones iniciales virus-superficie celular. El mecanismo rotaviral de unión y entrada a la célula parece ser un proceso de múltiples pasos donde las proteínas rotavirales VP4 y VP7 interaccionan con diferentes moléculas de la superficie celular. Objetivo. Proponer un mecanismo de entrada de rotavirus a la célula que incorpore la actividad de la proteína disulfuro isomerasa (PDI). Material y métodos. Utilizando bases de datos electrónicas, se realizó una búsqueda de literatura original y de revisión publicada entre 1990 y 2009 sobre moléculas de la superficie rotaviral o celular participantes en el proceso de entrada del virus. El análisis de los resultados enfatizó las bases moleculares y celulares de las interacciones temporo-espaciales de las proteínas virales y las moléculas de unión/receptoras de la célula. Resultados. Se encontró fundamentos moleculares y celulares para incorporar la actividad de PDI a un mecanismo coherente de vías secuenciales o alternativas previas a la penetración viral. Se propone un mecanismo en que interaccionan las proteínas virales VP4, VP6 y VP7 con las moléculas de la superficie celular ácido siálico, integrinas, Hsc70 y PDI en un proceso endocítico caveola/“raft”-dependiente, caveolina/clatrina-independiente, dinamina-dependiente y sensible a depleción de colesterol. Conclusión. Se amplía el concepto de múltiples pasos en el proceso de entrada de rotavirus, donde la participación de PDI podría ser un blanco potencial de la acción de inhibidores de grupos tiol/disulfuro...

[Peroxisomal D-bifunctional enzyme deficiency. A case report]. / Deficencia enzimática D-bifuncional peroxlsomal. Caso clínico.

Newborn was referred with diagnosis of neonatal epilepsy. Medical team could suspect and confirm D-bifunctional peroxisomal enzymatic deficiency diagnosis. It was made by family antecedents, severe neonatal hypotonia, uncontrolled neonatal seizures, craniofacial dysmorphic features, psychomotor retardation, neuronal migration defect and a positive peroxisomal panel. The full study in skin fibroblasts involved enzyme analysis, complementation studies and DNA analysis. The accumulation of very long chain fatty acids, partial deficiency in phytanic acid oxidation, and abnormal morphology of peroxisomes was consistent with a defect in peroxisomal fatty acid oxidation, involving D-bifunctional protein. It is very important to make a diagnosis of this innate error of metabolism in order to give preconceptional genetic counseling, to identify recurrence risk and to perform mutation analysis for the D-bifunctional protein gene, and to offer the prenatal diagnosis.

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease.

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da via em resposta à relação NADP/NADPH, é codificada por um número de genes por genoma haplóide e é induzida até 46-vezes por peróxido de hidrogênio em trypomastigotas metacíclicos. Os genes que codificam 6-fosfogluconolactonase, 6-fosfogluconato desidrogenase, transaldolase e transcetolase estão presentes no clone CL Brener como cópia única por genoma haplóide. 6-fosfogluconato desidrogenase é muito instável, mas foi estabilizada introduzindo duas pontes salinas por mutagênese sítio-dirigida. A Ribose-5-fosfato isomerase pertence ao Tipo B; genes que codificam enzimas Tipo A, presentes em mamíferos estão ausentes. A Ribulose-5-fosfato epimerase é codificada por dois genes. As enzimas da via têm um componente citosólico principal, embora várias delas tenham uma localização glicosomal secundária e também, localizações em menor número em outras organelas.

Isolation and characterization of unsaturated fatty acid auxotrophs of Streptococcus pneumoniae and Streptococcus mutans.

Unsaturated fatty acid (UFA) biosynthesis is essential for the maintenance of membrane structure and function in many groups of anaerobic bacteria. Like Escherichia coli, the human pathogen Streptococcus pneumoniae produces straight-chain saturated fatty acids (SFA) and monounsaturated fatty acids. In E. coli UFA synthesis requires the action of two gene products, the essential isomerase/dehydratase encoded by fabA and an elongation condensing enzyme encoded by fabB. S. pneumoniae lacks both genes and instead employs a single enzyme with only an isomerase function encoded by the fabM gene. In this paper we report the construction and characterization of an S. pneumoniae 708 fabM mutant. This mutant failed to grow in complex medium, and the defect was overcome by addition of UFAs to the growth medium. S. pneumoniae fabM mutants did not produce detectable levels of monounsaturated fatty acids as determined by gas chromatography-mass spectrometry and thin-layer chromatography analysis of the radiolabeled phospholipids. We also demonstrate that a fabM null mutant of the cariogenic organism Streptococcus mutants is a UFA auxotroph, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci. Finally we report that the fabN gene of Enterococcus faecalis, coding for a dehydratase/isomerase, complements the growth of S. pneumoniae fabM mutants. Taken together, these results suggest that FabM is a potential target for chemotherapeutic agents against streptococci and that S. pneumoniae UFA auxotrophs could help identify novel genes encoding enzymes involved in UFA biosynthesis.

Degradação e isomerização da vitamina A em preparações para terapia nutricional enteral

O estudo recente mostrou severa degradação de vitamina A em preparações para terapia nutricional enteral, durante armazenamento. Por outro lado, informações sobre a degradação e isomerização deste fármaco durante infusão dessas preparações ao paciente não têm sido relatadas na literatura. O presente estudo teve por objetivo: a) propor e validar método analítico para sepração e determinação do retinol, seus isômeros geométricos e, simultaneamente, os carotenos totais em preparações enterais comerciais; b) Avaliar a degradação e isomerização da vitamina A nessas preparações, durante armazenamento e infusão simulada. Foram estudadas 17 preparações enterais comerciais, fabricadas por 5 laboratórios farmacêuticos que exportam seus produtos para o Brasil. Os resultados obtidos mostraram que o métodod proposto pode ser utilizado com segurança. Apesar da alta concentração de isômeros...

Enzimologia clínica / Clinical enzymology

A Enzimologia Clínica é uma das especialidades da clínica médica que mais tem se desenvolvido ultimamente. Hoje, devido aos enormes progressos alcançados pela bioquímica, biologia molecular, imunologia e biofísica, as avaliações das atividades totais das diversas enzimas estão sendo substituídas pelas determinações das suas atividades isoenzimáticas. Estas, por sua vez, são muito mais órgano-específicas e, portanto, permitem o diagnóstico de determinada doença de modo bem mais específico, seletivo e seguro. As mais importantes metas que nortearam este trabalho foram: 1. A inexistência de um livro dedicado à Enzimologia Clínica no Brasil ou mesmo na literatura médica da América Latina; 2. Contribuir para a difusão das inúmeras aplicações da enzimologia clínica como instrumento de diagnóstico e acompanhamento da evolução e ou terapêutica de grande número de doenças; 3. Como as técnicas mais utilizadas nas medidas das atividades enzimáticas são colorimétricas ou espectrofotométricas e, portanto, acessíveis mesmo aos laboratórios e hospitais mais modestos, este livro vem estimular maiores investimentos técnico-científicos nesta importante área do conhecimento médico; 4. Servir como obra de referência para atualizações e consultas técnicas

Di-fluoresceinthiocarbamyl-insulin: a fluorescent substrate for the assay of protein disulfide oxidoreductase activity.

We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate. The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity. In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml). Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase. Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.

Genetic analysis of Leishmania mexicana populations from Texas, Latin America, and the Caribbean.

A genetic analysis using enzyme data of 72 Leishmania mexicana isolated from hosts in Texas, Latin America, and the Caribbean is presented. All isolates from each country were combined and considered as local populations. Allomorph (allele determined by electrophoresis) frequencies for 20 enzyme (loci) were calculated and 7 populations (Texas, Mexico, Belize, Guatemala, Ecuador [EC], Venezuela, and the Dominican Republic [DR]) were compared pairwise in the statistic of genetic identity (I) (level of genetic similarity). All populations were found to be genetically similar with a mean I value for all comparisons of 0.890 +/- 0.067. When DR was included as one of the pair compared, I = 0.811 +/- 0.034. Among comparisons that include EC (excluding EC vs. DR), I = 0.875 +/- 0.026. The mean I for the other comparisons was less than 0.9. The data indicate that the DR population is divergent enough from the others that it can be considered at the subspecies/incipient species level of evolutionary divergence; the EC population is, to a lesser extent, distinct from the others, and the other 5 represent geographic populations of 1 widely distributed species. A diagrammatic representation of the allomorphs among the 72 isolates is included. There were some allomorph/geographical (or local) population relationships noted.

Contrasting effects of norepinephrine and 5-hydroxytryptamine on contractility of abdominal aorta of two kidney-two clip hypertensive rats. Effects of inhibitors of arachidonic acid metabolic enzymes.

This study intends to: 1) define reactivity in vessels of two kidney-two clip (2K2C) hypertensive rats (6-11 days after clipping); 2) determine the possible involvement of prostaglandins in modulating contractile vascular responses. Cumulative dose-response curves to norepinephrine (NE), 5-hydroxytryptamine (5-HT) and potassium chloride (KCl) were elicited on helical strips of abdominal aorta both in the absence and in the presence of prostacyclin synthetase (transylcypromine, TCP, 0.25mM) or cyclooxygenase (indomethacin, IND, 0.014 mM and acetylsalicylic acid, ASA, 0.20 mM) inhibitors Vessels of hypertensive animals developed significantly less tension to NE (n = 21) but higher tension and lower ED50 in response to 5-HT (n = 9) than sham control rat vessels. Force development to KCl (n = 9) was not statistically different between hypertensive and sham vessels. Vascular responses were decreased with the inhibitors, but the contrasting effects of NE and 5-HT on clip vessels were maintained. Threshold doses of PGE2 significantly reversed the effect of IND but not that of TCP on NE responses. Threshold doses of PGI2 had no significant effect on NE and 5-HT responses under TCP. The results would indicate: 1) different functional alterations for contractions to NE and 5-HT appear to have developed in vessels of 2K2C hypertensive rats; 2) PGE2 effectively contributes to modulation of NE responses in rat aorta strips; 3) these experiments suggest that prostaglandins do not play a significant role in the altered contractility of vessels in hypertensive rats.