RESUMO
Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.
Assuntos
Crustáceos/enzimologia , Núcleosídeo-Difosfato Quinase/metabolismo , Nucleosídeos de Purina/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Animais , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Núcleosídeo-Difosfato Quinase/química , Conformação ProteicaRESUMO
Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca(2+) dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10(°) C and at least 50% more active than pig trypsin up to 50(°) C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca(2+) concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca(2+) dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.
Assuntos
Adaptação Fisiológica , Cálcio/química , Temperatura Baixa , Tripsina/química , Animais , Biotecnologia , Catálise , Clonagem Molecular , Crustáceos/enzimologia , Crustáceos/genética , Ativação Enzimática , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutação , Filogenia , Conformação Proteica , Engenharia de Proteínas , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Termodinâmica , Tripsina/biossíntese , Tripsina/genética , Tripsina/isolamento & purificaçãoRESUMO
Chitosanase production of Gongronella sp. JG cells immobilized in calcium alginate gel and polyurethane foam was compared with that of the free cells, there was a 60% increase in the enzyme yield (2429 U/L) compared to the highest yield obtained from free cells (1513 U/L). The optimal immobilization parameters (concentrations of sodium alginate, calcium chloride, bead inoculums, bead diameter, etc) for the enhanced production of chitosanase were determined as: sodium alginate 2% (w/v), 0.1 M calcium chloride, inoculum 10 mL beads to 100 mL production media and 2.7 mm bead diameter. Maximum chitosanase production was achieved with initial pH of 5.5 and temperature of 30 ºC. The alginate beads had well stability, retained 85% ability of enzyme production even after 7 cycles of repeated batch fermentation. These results showed the immobilization technique was a feasible and economical method for chitosansase production by Gongronella sp. JG.
Assuntos
Animais , Alginatos , Crustáceos/enzimologia , Crustáceos/microbiologia , Fermentação , Fungos Aquáticos/análise , Poliuretanos/análise , Quitosana/análise , Quitosana/isolamento & purificação , Sódio/análise , Atenção , Células Imobilizadas , Ativação Enzimática , Amostras de Alimentos , Métodos , Padrões de ReferênciaRESUMO
Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.
Assuntos
Proteínas Alimentares/metabolismo , Sistema Digestório/enzimologia , Invertebrados/enzimologia , Tripsina/metabolismo , Tripsinogênio/metabolismo , Adaptação Fisiológica , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Crustáceos/enzimologia , Sistema Endócrino/enzimologia , Ativação Enzimática , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Hidrólise , Hormônios de Inseto/metabolismo , Proteínas de Insetos/metabolismo , Insetos/enzimologia , Invertebrados/genética , Isoenzimas , Dados de Sequência Molecular , Conformação Proteica , Transcrição Gênica , Tripsina/química , Tripsina/genética , Tripsinogênio/química , Tripsinogênio/genéticaRESUMO
Linguatula serrata is a cosmopolitan parasite whose intermediate hosts are cattle, goats, sheep, and other ruminants. The adult form is found in the nasal airways, frontal sinuses, and tympanic cavity of canines and felines, and it produces hemorrhages and breathing difficulties. To elucidate if L. serrata produces enzymes that are capable of degrading tissues from the intermediate host, proteolytic activities in larval products were studied. Using the zymography technique, one major protease was detected in parasite in vitro-released products with an approximate molecular weight of 75 kDa. This enzyme was inhibited with phenylmethylsulfonyl fluoride, suggesting that it is a serine protease, which was also shown to degrade type I collagen. The serine protease exhibited maximal activity at alkaline pH and temperatures varying from 37 to 45 degrees C. To gather evidence about the physiological roles of the enzyme, further biochemical and functional studies are suggested.
Assuntos
Crustáceos/enzimologia , Doenças Parasitárias em Animais/parasitologia , Serina Endopeptidases/metabolismo , Animais , Colágeno Tipo I/metabolismo , Crustáceos/crescimento & desenvolvimento , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Larva/enzimologia , Peso Molecular , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/química , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/isolamento & purificação , TemperaturaRESUMO
In this paper, we review the current knowledge about the usage of carbohydrates, lipids and proteins as energy source by marine crustaceans during starvation. Crustaceans are a large and diverse group including some economically important species. The efforts to culture them for human consumption has prompted the interest to understand the preferences of energy sources to be applied for feed formulation and cost reduction. Important differences have been found among species and appear to be related not only to the biochemistry and physiology of nutrition, but also to the living environment of the crustaceans. Furthermore, crustaceans undergo morphological, physiological and behavioral changes due to their natural growing process that affect their feeding habits, an aspect that should be carefully considered. We discuss the current information on marine crustaceans about energy usage and describe areas of future research, where starvation studies render important insights.
Assuntos
Crustáceos/metabolismo , Inanição/metabolismo , Animais , Metabolismo dos Carboidratos , Crustáceos/enzimologia , Metabolismo dos Lipídeos , Modelos Biológicos , Proteínas/metabolismoRESUMO
Trypsin-like enzymes from two morphotypes (here called short and long) of the 'living fossil' Triops of Baja California Sur, Mexico were studied. Adults of both morphotypes were obtained from outdoor static cultures using dry soil from the natural habitats as a source of cysts and culture substrate. Individual and pooled extracts were made from dissected digestive tubes. The effect of pH and temperature on the trypsin activity was studied using N-alpha-benzoyl-DL-Arg-p-nitroanilide (BAPNA) as substrate. The highest proteolytic activity was found at the same pH with extracts of both morphotypes. At this pH, there was greater proteolytic activity at a lower temperature with the short morphotype extract than with the long morphotype extract. Substrate-SDS-PAGE zymograms showed bands of activity. Short morphotype extracts produced six bands; five of them were serine proteases of which three were trypsin-like enzymes. Long morphotype extracts revealed eight bands; six of them were serine proteases of which three were trypsin-like enzymes.