Desempenho de juvenis de tilápia-do-nilo alimentados com rações contendo complexo enzimático

Avaliou-se o efeito da inclusão de um complexo enzimático em dietas para tilápias-do-nilo (Oreochromis niloticus) sobre o desempenho, a composição química da carcaça e a qualidade da água. Foram utilizados 200 alevinos revertidos (4,57 ± 1,24 g), distribuídos em delineamento inteiramente casualizado em 20 tanques de 500 litros, com quatro tratamentos e cinco repetições, considerando a unidade experimental uma caixa com dez peixes. Os peixes foram alimentados com dietas contendo 0; 0,033; 0,066 ou 0,099% de complexo enzimático. As dietas foram processadas na forma peletizada e fornecidas quatro vezes ao dia, às 8, 11, 14 e 17 h. Os valores médios de pH, condutividade elétrica, oxigênio dissolvido, temperatura, fósforo total, amônia e nitrato da água de cultivo não foram influenciados pela dieta. A inclusão do complexo enzimático na dieta não afetou o ganho de peso, as taxas de sobrevivência e de crescimento específico, mas influenciou o consumo de ração e a conversão alimentar, cujos valores foram maiores nos peixes alimentados com a dieta com 0,066% de complexo enzimático. Não foram observadas diferenças nos teores de matéria seca, umidade, proteína bruta, matéria mineral, cálcio e fósforo na carcaça dos peixes, no entanto, o teor de extrato etéreo reduziu de forma linear com o aumento do nível de complexo enzimático. A utilização de complexo enzimático (amilase, protease, celulase, lipase, pectinase, xilanase, β-glucanase e fitase) no nível de 0,066% em dietas para juvenis de tilápia-do-nilo piora a conversão alimentar, mas não influencia o desempenho e a composição corporal dos peixes.

A complex zinc finger controls the enzymatic activities of nidovirus helicases.

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

Evaluation of the beta-glucanolytic enzyme complex of Trichoderma harzianum Rifai for the production of gluco-oligosaccharide fragments by enzymatic hydrolysis of 1,3;1,6-beta-D-glucans

Botryosphaeran, a new exopolysaccharide from the endophytic fungus Botryosphaeria rhodina MAMB-05, and algal laminarin were hydrolyzed by partially-fractionated enzymes of the beta-glucanolytic complex from Trichoderma harzianum Rifai. beta-Glucanase fractions (F-I and F-II) separated by gel permeation chromatography presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was hydrolyzed to the extent of 66% (F-I) and 98% (F-II) within 30 min, and its main hydrolysis products were gluco-oligosaccharides of DP >= 4, with lesser amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted in 15% conversion to glucose, while the percentage of saccharification was radically different (70% for F-I and 25% for F-II). The different product arrays within the polysaccharide hydrolysates can be explained by the difference in the enzymes' specificities within each enzyme fraction, and the molecular structures of the polysaccharides and their complexity.

Electropolymerization mechanism in aqueous solution of the oxo-manganese complex biomimicking of the enzymatic center present on the photosystem II

The [Mn4 IVO5(terpy)4(H 2O)2]6+ complex, show great potential for electrode modification by electropolymerization using cyclic voltammetry. The electropolymerization mechanism was based on the electronic transfer between dx2-y2 orbitals of the center metallic and pπ orbital of the ligand, which show great complexity of the system due to orbitals overlap present in octahedral complex of the metal-μ-oxo. The voltammetric behavior both in and after electropolymerization process were also discussed, where the best condition of electropolymerization was observed for low scan rate and 50 potential cycles. A study in ITO/glass electrode for better characterization of polymer was also performed. ©The Electrochemical Society.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5 L (15–25 FPU/g glucan) was in the following order: glycerol/HCl > HCl > H2SO4 > NaOH, with the glycerol system achieving ∼100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6–6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2–6 times that of acid substrates. However, glucan conversions (15–60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10–30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.

Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins

Viruses are submicroscopic, infectious agents that are obligate intracellular parasites. They adopt various types of strategies for their parasitic replication and proliferation in infected cells. The nucleic acid genome of a virus contains information that redirects molecular machinery of the cell to the replication and production of new virions. Viruses that replicate in the cytoplasm and are unable to use the nuclear transcription machinery of the host cell have developed their own transcription and capping systems. This thesis describes replication strategies of two distantly related viruses, hepatitis E virus (HEV) and Semliki Forest virus (SFV), which belong to the alphavirus-like superfamily of positive-strand RNA viruses. We have demonstrated that HEV and SFV share a unique cap formation pathway specific for alphavirus-like superfamily. The capping enzyme first acts as a methyltransferase, catalyzing the transfer of a methyl group from S-adenosylmethionine to GTP to yield m7GTP. It then transfers the methylated guanosine to the end of viral mRNA. Both reactions are virus-specific and differ from those described for the host cell. Therefore, these capping reactions offer attractive targets for the development of antiviral drugs. Additionally, it has been shown that replication of SFV and HEV takes place in association with cellular membranes. The origin of these membranes and the intracellular localization of the components of the replication complex were studied by modern microscopy techniques. It was demonstrated that SFV replicates in cytoplasmic membranes that are derived from endosomes and lysosomes. According to our studies, site for HEV replication seems to be the intermediate compartment which mediates the traffic between endoplasmic reticulum and the Golgi complex. As a result of this work, a unique mechanism of cap formation for hepatitis E virus replicase has been characterized. It represents a novel target for the development of specific inhibitors against viral replication.

Diastereomeric Drug Glucuronides: Enzymatic Glucuronidation and Analysis by Capillary Electrophoresis and Liquid Chromatography-Mass Spectrometry

Foreign compounds, such as drugs are metabolised in the body in numerous reactions. Metabolic reactions are divided into phase I (functionalisation) and phase II (conjugation) reactions. Uridine diphosphoglucuronosyltransferase enzymes (UGTs) are important catalysts of phase II metabolic system. They catalyse the transfer of glucuronic acid to small lipophilic molecules and convert them to hydrophilic and polar glucuronides that are readily excreted from the body. Liver is the main site of drug metabolism. Many drugs are racemic mixtures of two enantiomers. Glucuronidation of a racemic compound yields a pair of diastereomeric glucuronides. Stereoisomers are interesting substrates in glucuronidation studies since some UGTs display stereoselectivity. Diastereomeric glucuronides of O-desmethyltramadol (M1) and entacapone were selected as model compounds in this work. The investigations of the thesis deal with enzymatic glucuronidation and the development of analytical methods for drug metabolites, particularly diastereomeric glucuronides. The glucuronides were analysed from complex biological matrices, such as urine or from in vitro incubation matrices. Various pretreatment techniques were needed to purify, concentrate and isolate the analytes of interest. Analyses were carried out by liquid chromatography (LC) with ultraviolet (UV) or mass spectrometric (MS) detection or with capillary electromigration techniques. Commercial glucuronide standards were not available for the studies. Enzyme-assisted synthesis with rat liver microsomes was therefore used to produce M1 glucuronides as reference compounds. The glucuronides were isolated by LC/UV and ultra performance liquid chromatography (UPLC)/MS, while tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed in structural characterisation. The glucuronides were identified as phenolic O-glucuronides of M1. To identify the active UGT enzymes in (±)-M1 glucuronidation recombinant human UGTs and human tissue microsomes were incubated with (±)-M1. The study revealed that several UGTs can catalyse (±)-M1 glucuronidation. Glucuronidation in human liver microsomes like in rat liver microsomes is stereoselective. The results of the studies showed that UGT2B7, most probably, is the main UGT responsible for (±)-M1 glucuronidation in human liver. Large variation in stereoselectivity of UGTs toward (±)-M1 enantiomers was observed. Formation of M1 glucuronides was monitored with a fast and selective UPLC/MS method. Capillary electromigration techniques are known for their high resolution power. A method that relied on capillary electrophoresis (CE) with UV detection was developed for the separation of tramadol and its free and glucuronidated metabolites. The suitability of the method to identify tramadol metabolites in an authentic urine samples was tested. Unaltered tramadol and four of its main metabolites were detected in the electropherogram. A micellar electrokinetic chromatography (MEKC) /UV method was developed for the separation of the glucuronides of entacapone in human urine. The validated method was tested in the analysis of urine samples of patients. The glucuronides of entacapone could be quantified after oral entacapone dosing.

Modeling of angiogenin-3 '-NMP complex

Angiogenin belongs to the Ribonuclease superfamily and has a weak enzymatic activity that is crucial for its biological function of stimulating blood vessel growth. Structural studies on ligand bound Angiogenin will go a long way in understanding the mechanism of the protein as well as help in designing drugs against it. In this study we present the first available structure of nucleotide ligand bound Angiogenin obtained by computer modeling. The importance of this study in itself notwithstanding, is a precursor to modeling a full dinucleotide substrate onto Angiogenin. Bovine Angiogenin, the structure of which has been solved at a high resolution, was earlier subjected to Molecular Dynamics simulations for a nanosecond. The MD structures offer better starting points for docking as they offer lesser obstruction than the crystal structure to ligand binding. The MD structure with the least serious short contacts was modeled to obtain a steric free Angiogenin - 3' mononucleotide complex structure. The structures were energetically minimized and subjected to a brief spell of Molecular Dynamics. The results of the simulation show that all the li,ligand-Angiogenin interactions and hydrogen bonds are retained, redeeming the structure and docking procedure. Further, following ligand - protein interactions in the case of the ligands 3'-CMP and 3'-UMP we were able to speculate on how Angiogenin, a predominantly prymidine specific ribonuclease prefers Cytosine to Uracil in the first base position.

Non-enzymatic electronic detection of glucose using aminophenylboronic acid functionalized reduced graphene oxide

We report the non-enzymatic electronic detection of glucose using field effect transistor (FET) devices made of aminophenylboronic acid (APBA) functionalized reduced graphene oxide (RGO). Detection of glucose molecules was carried out over a wide dynamic range of concentration varying from 100 pM to 100 mM with a detection limit of similar to 2 nM using both covalently and non-covalently functionalized APBA-RGO complex. The normalized change in electrical conductance data shows that the FET devices made of non-covalently functionalized APBA-RGO complex (nc-APBA-RGO) exhibited a linear response to glucose aqueous solution of concentrations varying from 1 nM to 10 mM and showed 4 times enhanced sensitivity over the devices made of covalently functionalized APBA-RGO complex (c-APBA-RGO). Specificity of APBA-RGO complex to glucose is confirmed from the observation of negligible change in electrical conductance after exposure to 0.1 mM of lactose and other interfering factors. (C) 2015 Elsevier B.V. All rights reserved.

Mechanistic studies of a model reaction for enzymatic hydroxylation

A study has been made of the reaction mechanism of a model system for enzymatic hydroxylation. The results of a kinetic study of the hydroxylation of 2-hydroxyazobenzene derivatives by cupric ion and hydrogen peroxide are presented. An investigation of kinetic orders indicates that hydroxylation proceeds by way of a coordinated intermediate complex consisting of cupric ion and the mono anions of 2-hydroxyazobenzene and hydrogen peroxide. Studies with deuterated substrate showed the absence of a primary kinetic isotope effect and no evidence of an NIH shift. The effect of substituents on the formation of intermediate complexes and the overall rate of hydroxylation was studied quantitatively in aqueous solution. The combined results indicate that the hydroxylation step is only slightly influenced by ring substitution. The substituent effect is interpreted in terms of reaction by a radical path or a concerted mechanism in which the formation of ionic intermediates is avoided. The reaction mechanism is discussed as a model for enzymatic hydroxylation.

Heterometallic Complexes as Models of Enzymatic Active Sites

This dissertation describes studies on two multinucleating ligand architectures: the first scaffold was designed to support tricopper complexes, while the second platform was developed to support tri- and tetrametallic clusters.

In Chapter 2, the synthesis of yttrium (and lanthanide) complexes supported by a tripodal ligand framework designed to bind three copper centers in close proximity is described. Tricopper complexes were shown to react with dioxygen in a 1:1 [Cu₃]/O₂ stoichiometry to form intermediates in which the O–O bond was fully cleaved, as characterized via UV-Vis spectroscopy and determination of the reaction stoichiometry. Pre-arrangement of the three Cu centers was pivotal to cooperative O₂ activation, as mono-copper complexes reacted differently with dioxgyen. The reactivity of the observed intermediates was studied with various substrates (reductants, O-atom acceptors, H-atom donors, Brønsted acids) to determine their properties. In Chapter 3, the reactivity of the same yttrium-tricopper complex with nitric oxide was explored. Reductive coupling to form a trans-hyponitrite complex (characterized by X-ray crystallography) was observed via cooperative reactivity by an yttrium and a copper center on two distinct tetrametallic units. The hyponitrite complex was observed to release nitrous oxide upon treatment with a Brønsted acid, supporting its viability as an intermediate in nitric oxide reduction to nitrous oxide.

In Chapter 4, a different multinucleating ligand scaffold was employed to synthesize heterometallic triiron clusters containing one oxide and one hydroxide bridges. The effects of the redox-inactive, Lewis acidic heterometals on redox potential was studied by cyclic voltammetry, unveiling a linear correlation between redox potential and heterometal Lewis acidity. Further studies on these complexes showed that the Lewis acidity of the redox-inactive metals also affected the oxygen-atom transfer reactivity of these clusters. Comparisons of this reactivity with manganese systems, collaborative efforts to reassign the structures of related manganese oxo-hydroxo clusters, and synthetic attempts to access related dioxo clusters are also described.

In Appendix A, ongoing efforts to synthesize new clusters supported by the same multinucleating ligand platform are described. Studies of novel approaches towards ligand exchange in tetrametallic clusters and incorporation of new supporting and bridging ligand motifs in trinuclear complexes are presented.

Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Background: Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results: A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 x 10(5) and 1.7 x 10(5) per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion: The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the slippage model for initiation of reverse transcription, retropositional parasitism of SINEs on LINEs, the formation of the stem loop structure in 3'tail region of some SINEs and LINEs and the mechanism of template switching in generating new SINE family.

Kinetic Model Study on Enzymatic Hydrolysis of Cellulose Using Artificial Neural Networks

Enzymatic hydrolysis of cellulose was highly complex because of the unclear enzymatic mechanism and many factors that affect the heterogeneous system. Therefore, it is difficult to build a theoretical model to study cellulose hydrolysis by cellulase. Artificial neural network (ANN) was used to simulate and predict this enzymatic reaction and compared with the response surface model (RSM). The independent variables were cellulase amount X-1, substrate concentration X-2, and reaction time X-3, and the response variables were reducing sugar concentration Y-1 and transformation rate of the raw material Y-2. The experimental results showed that ANN was much more suitable for studying the kinetics of the enzymatic hydrolysis than RSM. During the simulation process, relative errors produced by the ANN model were apparently smaller than that by RSM except one and the central experimental points. During the prediction process, values produced by the ANN model were much closer to the experimental values than that produced by RSM. These showed that ANN is a persuasive tool that can be used for studying the kinetics of cellulose hydrolysis catalyzed by cellulase.

The complex kinetics of protein folding in wide temperature ranges

The complex protein folding kinetics in wide temperature ranges is studied through diffusive dynamics on the underlying energy landscape. The well-known kinetic chevron rollover behavior is recovered from the mean first passage time, with the U-shape dependence on temperature. The fastest folding temperature T-0 is found to be smaller than the folding transition temperature T-f. We found that the fluctuations of the kinetics through the distribution of first passage time show rather universal behavior, from high-temperature exponential Poissonian kinetics to the relatively low-temperature highly nonexponential kinetics. The transition temperature is at T-k and T-0, T-k, T-f. In certain low-temperature regimes, a power law behavior at long time emerges. At very low temperatures ( lower than trapping transition temperature T< T-0/(4&SIM;6)), the kinetics is an exponential Poissonian process again.

Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.