Produção de enzimas por fungos filamentosos / Production of enzymes by filamental fungi

Estudos utilizando fungos endofíticos como produtores de metabólitos secundários de interesse biotecnológico vem sendo explorados. Assim, o presente trabalho objetivou avaliar a produção de enzimas por fungos filamentosos endofíticos, sendo escolhida a cepa 50 (C50), proveniente da coleção de cultura do Laboratório de Produtos Naturais e Biotecnologia (LPNBio) da UESB. A produção das enzimas amilase, celulase, invertase, lipase e poligalacturonase foi avaliada pelo o índice enzimático, atividade enzimática e verificado o tempo de fermentação de maior produtividade. Com exceção da invertase, a C50 apresentou atividade para as demais enzimas, destaque para lipase e poligalacturonase no tempo de 96 horas de fermentação. Estes resultados mostraram que a C50 tem potencial para ser explorada como produtora de enzimas.

Advances in enzyme bioelectrochemistry

ABSTRACT Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans) where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

Enzymatic reactions involving the heteroatoms from organic substrates

ABSTRACT Several enzymatic reactions of heteroatom-containing compounds have been explored as unnatural substrates. Considerable advances related to the search for efficient enzymatic systems able to support a broader substrate scope with high catalytic performance are described in the literature. These reports include mainly native and mutated enzymes and whole cells biocatalysis. Herein, we describe the historical background along with the progress of biocatalyzed reactions involving the heteroatom(S, Se, B, P and Si) from hetero-organic substrates.

Biotechnological applications of archaeal enzymes from extreme environments

To date, many industrial processes are performed using chemical compounds, which are harmful to nature. An alternative to overcome this problem is biocatalysis, which uses whole cells or enzymes to carry out chemical reactions in an environmentally friendly manner. Enzymes can be used as biocatalyst in food and feed, pharmaceutical, textile, detergent and beverage industries, among others. Since industrial processes require harsh reaction conditions to be performed, these enzymes must possess several characteristics that make them suitable for this purpose. Currently the best option is to use enzymes from extremophilic microorganisms, particularly archaea because of their special characteristics, such as stability to elevated temperatures, extremes of pH, organic solvents, and high ionic strength. Extremozymes, are being used in biotechnological industry and improved through modern technologies, such as protein engineering for best performance. Despite the wide distribution of archaea, exist only few reports about these microorganisms isolated from Antarctica and very little is known about thermophilic or hyperthermophilic archaeal enzymes particularly from Antarctica. This review summarizes current knowledge of archaeal enzymes with biotechnological applications, including two extremozymes from Antarctic archaea with potential industrial use, which are being studied in our laboratory. Both enzymes have been discovered through conventional screening and genome sequencing, respectively.

Use of enzymes in diets with different percentages of added fat for broilers / Uso de enzimas em dietas com diferentes porcentagens de adição de gordura para frangos de corte

We assessed the extent to which the removal of fat source, and consequently its compounds, such as linoleic acid, can affect the performance of broilers. We used 600 male Cobb 500 day old chicks. The birds were distributed in a completely randomized experimental design, with five treatments and six replicates of 20 birds each. The treatments were: (T1) diet - positive control (PC), which met the nutritional needs; (T2) diet - negative control (CN), a reduction of 100kcal/kg and low linoleic acid content; (T3): diet - negative control reformulated for low linoleic acid content and a set of Quantum phytase XT and Econase XT 25 (BAL + QFit-Eco), (T4): diet - negative control reformulated, with the percentage of linoleic acid adjusted to an intermediate value between the value of the diet and diet CP and CN to use a set of Quantum phytase XT and XT Econase 25 (IAL + QFit-Eco) and (T5): diet - negative control reformulated, with the percentage of linoleic acid adjusted to a value similar to that of the positive control diet and joint use of Quantum phytase XT and XT Econase 25 (AAL + QFit-Eco). The joint use of Quantum Phytase and Econase promoted improvement in the performance of broilers from 1 to 21 days. The greatest weight gain was obtained with diets containing percentages of total fat and linoleic acids. Dietary supplementation with enzymes resulted in higher levels of calcium in the tibia, whatever the percentage of linoleic studied.

Avaliou-se até que ponto a retirada de fonte de gordura e, consequentemente, de seus compostos, como o ácido linoleico, pode afetar o desempenho dos frangos de corte. Foram utilizados 600 pintos de um dia, machos da linhagem Cobb 500. As aves foram distribuídas num delineamento experimental inteiramente ao acaso, com cinco tratamentos e seis repetições de 20 aves cada. Os tratamentos foram: (T1) dieta-controle positivo (CP), que atendeu às necessidades nutricionais; (T2) dieta-controle negativo (CN), com redução de 100kcal de EM/kg e baixo teor de ácido linoleico; (T3): dieta- controle negativo reformulada, para baixo teor de ácido linoleico e com uso conjunto de Quantum fitase XT e Econase XT 25 (BAL+ Qfit-Eco); (T4): dieta-controle negativo reformulada, com porcentagem de ácido linoleico ajustada para um valor intermediário entre o valor da dieta CN e da dieta CP e com uso conjunto de Quantum fitase XT e Econase XT 25 (IAL + Qfit-Eco) e (T5): dieta-controle negativo reformulada, com porcentagem de ácido linoleico ajustada para um valor semelhante ao da dieta-controle positivo e com uso conjunto de Quantum fitase XT e Econase XT 25 (AAL+ Qfit-Eco ). O uso conjunto de Quantum Fitase e Econase promoveu melhora no desempenho dos frangos de corte de um a 21 dias. O maior ganho de peso foi obtido com dietas que continham porcentagens de gorduras totais e de ácido linoleico. A suplementação com as enzimas resultou em maior teor de cálcio nas tíbias, independentemente da porcentagem de ácido linoleico estudada.

Effect of inactivation and reactivation conditions on activity recovery of enzyme catalysts

Enzymes are labile catalysts with reduced half-life time that can be however improved by immobilization and, furthermore, already inactivated catalyst can be recovered totally or partially, therefore allowing the large scale application of enzymes as process catalysts. In recent years a few studies about reactivation of enzyme catalysts have been published as a strategy to prolong the catalyst lifetime. Reported results are very good, making this strategy an interesting tool to be applied to industrial process. These studies have been focused in the evaluation of different variables that may have a positive impact both in the rate and level of activity recovery, being then critical variables for conducting the reactivation process at productive scale. The present work summarizes the studies done about reactivation strategies considering different variables: type of immobilization, enzyme-support interaction, level of catalyst inactivation prior to reactivation, temperature and presence of modulators.

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

Enzymes have been long used in man-made biochemical processes, from brewing and fermentation to current industrial production of fine chemicals. The ever-growing demand for enzymes in increasingly specific applications requires tailoring naturally occurring enzymes to the non-natural conditions found in industrial processes. Relationships between enzyme sequence, structure and activity are far from understood, thus hindering the capacity to design tailored biocatalysts. In the field of protein engineering, directed enzyme evolution is a powerful algorithm to generate and identify novel and improved enzymes through iterative rounds of mutagenesis and screening applying a specific evolutive pressure. In practice, critical checkpoints in directed evolution are: selection of the starting point, generation of the mutant library, development of the screening assay and analysis of the output of the screening campaign. Each step in directed evolution can be performed using conceptually and technically different approaches, all having inherent advantages and challenges. In this article, we present and discuss in a general overview, challenges of designing and performing a directed enzyme evolution campaign, current advances in methods, as well as highlighting some examples of its applications in industrially relevant enzymes.

Digestive enzyme as benchmark for insecticide resistance development in culex pipiens larvae to chemical and bacteriologic insecticides

This work monitored changes in some digestive enzymes [trypsin and aminopeptidase] associated with the building up of resistance in Cx. pipiens larvae to two chemical insecticides [methomyl and/or malathion] and one biological insecticide [Bacillus thuringiensis-H14 or B.t HI4]. The LC[50] value of methomyl for both field and the 12[th] generation [F12] of the selected strain was 1.789 ppm and 8.925 ppm respectively. The LC[50] value of malathion for both field and the F12 of the selected strain was 0.082 ppm and 0.156 ppm respectively, and those of B.t H14 of field strain and the F12 was 2.550ppm and 2.395ppm respectively. The specific activity of trypsin enzyme in control susceptible colony was 20.806 +/- 0.452[micromol/min/mg protein; but at F4 and F8 for malathion and methomyl treated larvae were 10.810 +/- 0.860 and 15.616 +/- 0.408 [micromol/min/mg protein, respectively. Trypsin activity of F12 in treated larvae with B.t.H14 was 2.097 +/- 0.587micromol/min/mg protein. Aminopeptidase specific activity for susceptible control larvae was 173.05 +/- 1.3111micromol/min/mg protein. This activity decreased to 145.15 +/- 4.12, 152.497 +/- 6.775 and 102.04 +/- 3.58 [a]micromol/min/mg protein after larval [F12] treatment with methomyl, malathion and B.t H14 respectively

Invertase, glucose oxidase and catalase for converting sucrose to fructose and gluconic acid through batch and membrane-continuous reactors / Invertase de glicose oxidase e catalase para a conversão de sacarose para ácido glucónico e frutose através de lote e de membrana-contínuas reactores

Conversion of sucrose into fructose and gluconic acid using invertase, glucose oxidase and catalase was studied by discontinuous (sequential or simultaneous addition of the enzymes) and continuous (simultaneous addition of the enzymes in a 100 kDa-ultrafiltration membrane reactor) processes. The following parameters were varied: concentration of enzymes, initial concentration of substrates (sucrose and glucose), pH, temperature and feeding rate (for continuous process). The highest yield of conversion (100 percent) was attained through the discontinuous (batch) process carried out at pH 4.5 and 37 ºC by the sequential addition of invertase (14.3 U), glucose oxidase (10,000 U) and catalase (59,000 U).

Neste trabalho estudou-se a conversão da sacarose em frutose e ácido glicônico, usando as enzimas invertase, glicose oxidase e catalase, através do emprego de processo descontínuo (com adição sequencial ou simultânea das enzimas) e contínuo (adição simultânea das enzimas em reator com membrana acoplado à membrana de ultrafiltração de 100 kDa). Os parâmetros variados foram: a concentração das enzimas, a concentração inicial dos substratos (sacarose e glicose), o pH, a temperatura e a vazão específica de alimentação (processo contínuo). Obteve-se rendimento de 100 por cento, quando a conversão foi conduzida por processo descontínuo em pH 4,5 e a 37 ºC com adição seqüencial das enzimas invertase (14,3 U), glicose oxidase (10.000 U) e catalase (59.000 U).

Avaliação comparativa de características estruturais do amido e enzimas relacionadas à sua degradação em cultivares de banana como padrão distinto do modelo representado pela cultuvar Nanicão / Comparative evaluation of structural characteristics of starch and enzymes related to their degradation in banana cultivars as distinct from the standard model represented by cultuvar Nanicão

O acúmulo de açúcares solúveis observados em banana madura é resultado da ação de diversas enzimas que atuam sob o amido acumulado durante o seu desenvolvimento. Este processo pode ocorrer fora da planta e, em um período relativamente curto conhecido como amadurecimento. A rápida mobilização associado a escassez de trabalhos que enfoquem o processo de degradação de amido em Orgãos que armazenam amido temporariamente, como bananas, o objetivo desse estudo foi localizar cultivares que se comportem diferente do modelo Nanicão, que é objeto de estudo do laboratório ha muito tempo, sendo por isso, a cultivar mais conhecida e estudada. Bananas pertencentes a diferentes grupos genômicos foram selecionadas e analisadas, sendo elas: Nanicão (AAA), Terra (AAB), Mysore (AAB), Pacovan (AAB) e Figo (ABB). Quanto aos parâmetros de amadurecimento analisados, as cultivares Nanicão, Terra e Pacovan tiveram um comportamento climatérico típico. A sacarose foi o açúcar predominante em todas as cultivares seguida pela glicose e frutose que mantiveram uma proporção 1:1. A cultivar Nanicão teve maiores teores de açúcares solúveis seguida pela Figo, Pacovan, Terra e Mysore. Em extrato bruto de banana, os maiores valores de atividade da β-amilase foi verificada nos estágios finais do amadurecimento, enquanto que a da α-amilase foi, praticamente, constante durante o período analisado, com exceção da Figo. Para detectar a presença das amido-fosforilases foi realizado ensaio, em extrato bruto de banana, de eletroforese em condições não-desnaturantes (PAGE-nativo) e, duas bandas com atividade foram visualizadas nas cultivares, com exceção da Mysore. O inicio do processo de degradação de amido ocorreu anterior a produção autocatalitica de etileno e, as cultivares tiveram diferentes percentuais de degradagao de amido, sendo os maiores obtidos/na Pacovan seguida pela Mysore, Nanicão, Figo e Terra. Os grânulos de amido das cultivares Terra e Figo mostraram-se resistentes a...

The soluble sugars accumulation in mature banana is consequence of several enzymes action on accumulate starch obtained during its development process. The starch degradation can occur outside the plant in a relatively short time called ripening. Associate at the faster starch mobilization, few works are available focusing this process in organs that store starch temporarily like banana. The aim of this study was identified cultivars that show different pattern of starch degradation when compared with Nanicao model. Bananas of different genomic groups were selected and analyzed, Nanicao (AAA), Terra (AAB), Mysore (AAB), Pacovan (AAB) and Figo (ABB). The Nanicao, Terra and Pacovan cultivars had a typical climacteric behavior. Sucrose was the predominant sugar followed by glucose and fructose that maintained 1:1 ratio, in all cultivars. Nanicao had the higher levels of soluble sugars followed by Figo, Pacovan, Terra and Mysore. The onset of starch degradation seems to be independent of ethylene. The higher activities of β-amylase, in banana pulp, was obtained in the last ripening stages, while α-amylase activities was constant and low at Nanicao and Pacovan. Two bands with activities were visualized in native PAGE which corresponded to cytosolic and plastidial forms of starchphosphorylases, with exception of Mysore. All cultivars had different perceptual of starch degradation and the higher one was observed at Pacovan, followed by Mysore, Nanicao, Figo and Terra. Starches granules of Terra and Figo showed to be resistant to enzymatic hydrolysis. All cultivars had a and β-amylase activities associated to granule throughout the ripening process. Only Mysore showed αamylase activity associated to granule higher than β-amylase. No significant change along the ripening was observed in amylose content and amylopectin chain length distribution. The micrographs revealed that granule surface suffer changes along the ripening, exposing structures...

Polaron hopping in some biomolecular solids and their charge transfer complexes.

The solid state spectroscopy of charge transfer complexes of biomolecules such as fatty acids, tripalmitin, lysozyme. folic acid, beta-carotene, cytochrome c, valinomycin and gramicidin has been carried out. The absorption coefficient is related with electronic conductivity. A half-power beta density is found common among these macromolecular solids, indicating photon-induced polaron hopping or hopping of a charge carrier between two branches of a polariton. Band gap vs full width at half-maximum of the mid-IR peak also reveals a linear relation.

Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate.

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.

Studies on glucose-metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules.

Nitrogen is exported in the form of ureides or amides from the nodules in pulse crops. In order to understand the carbon metabolism in ureide and amide exporting nodules, activities of enzymes involved in glucose metabolism were compared in cytosolic and bacteroidal fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules during development. Activities of hexokinase, fructokinase, phosphoglucomutase, fructose-1,6-bisphosphatase, phosphohexose isomerase and UDP-glucose pyrophosphorylase were detected in cytosolic fraction of nodules of both the crops during development. Out of these enzymes, specific activity of phosphohexose isomerase was the highest in nodules of both the crops, in comparison with other enzymes. In comparison with mungbean, activities of various enzymes were less in cytosolic fraction of lentil. Activities of hexokinase, fructokinase, phosphoglucomutase were present only in cytosolic fraction of mungbean (Vigna radiata L.), however, low activity of these enzymes was also observed in lentil (Lens culinaris L.) bacteroids. Activities of phosphohexose isomerase and fructose-1,6-bisphosphatase were higher in bacteroids of lentil, as compared to mungbean during early nodule development, but this pattern was reversed with progress of crop development. Higher activities of phosphoglucomutase and fructose-1,6-phosphatase in mungbean cytosolic fraction could lead to increased flow of carbon towards pentose phosphate pathway.

Predicting enzyme class from protein structure using Bayesian classification

Predicting enzyme class from protein structure parameters is a challenging problem in protein analysis. We developed a method to predict enzyme class that combines the strengths of statistical and data-mining methods. This method has a strong mathematical foundation and is simple to implement, achieving an accuracy of 45%. A comparison with the methods found in the literature designed to predict enzyme class showed that our method outperforms the existing methods.

Reference Map of Soluble Proteins from Salmonella enterica Serovar Enteritidis by Two-Dimensional Electrophoresis

Protein identification by peptide mass fingerprinting using matrix-assisted laser desorption ionization time of fight (MALDI-TOF) mass spectrometry (MS) can analyze unambiguously identity of the spots from a 2-dimensional electrophoresis (2-DE) gel. This study developed a technique for 2-DE of Salmonella enterica serovar Enteritidis (S. enteritidis) by improving the dissolution conditions by 2-DE using a pH 4 - 7 immobilized pH gradient (IPG) strip. This report examines the protein components from the patterns of the S. enteritidis protein. The most abundant protein displayed a great number of clusters within the pH 4.5 - 7 range with a molecular mass ranging from 35-80 kDa. Some of these spots were identified as metabolic related enzymes. The protein fraction was also analyzed using an immobilized pH gradient strip. Different proteins were identified on the spot according to the elongation factors. In addition, this study showed that the 2-DE analysis of S. enteritidis provides useful information regarding the S. enteritidis proteome, and this approach might provide a strategy for identifying bacterial proteins using a proteome technology.

Transmembrane domains participate in functions of integral membrane proteins.

Integral membrane proteins have one or more transmembrane alpha-helical domains and carry out a variety of functions such as enzyme catalysis, transport across membranes, transducing signals as receptors of hormones and growth factors, and energy transfer in ATP synthesis. These transmembrane domains are not mere structural units anchoring the protein to the lipid bilayer but seem to-contribute in the overall activity. Recent findings in support of this are described using some typical examples-LDL receptor, growth factor receptor tyrosine kinase, HMG-CoA reductase, F0-ATPase and adrenergic receptors. The trends in research indicate that these transmembrane domains participate in a variety of ways such as a linker, a transducer or an exchanger in the overall functions of these proteins in transfer of materials, energy and signals.

Protein engineering as a powerful tool for the chemical modification of enzymes

This article discusses the techniques of site-specific mutagenesis and protein engineering and their application in the study of enzyme active sites and the mechanism of enzyme action. Particular emphasis is given to beta-lactamase

Microbial production of L-tyrosine: a review.

Microbial production of L-tyrosine by direct fermentation and by enzymatic methods has been reviewed. Achievements in this regard made through recombinant DNA techniques have also been included. The review also includes biosynthesis and regulation of tyrosine.