Identificación y caracterización de proteasas del pepino de mar
Holothurians, commonly known as sea cucumbers, belong to the Phylum echinoderms. These marine organisms contain a wide variety of active ingredients, such as carbohydrates, lipids, neurotoxins and enzymes. The knowledge of digestive enzymes in the sea cucumber could clarify its nutritional biochemis...
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| Tipo de recurso: | tesis de maestría |
| Estado: | Versión publicada |
| Fecha de publicación: | 2010 |
| País: | México |
| Institución: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:3484zh141 |
| Acceso en línea: | https://doi.org/10.24275/uami.3484zh141 |
| Access Level: | acceso abierto |
| Palabra clave: | info:eu-repo/classification/LEM/Pepino de mar info:eu-repo/classification/LEM/Proteolytic enzymes info:eu-repo/classification/LEM/Histología info:eu-repo/classification/LEM/Histology info:eu-repo/classification/LEM/Biotechnology info:eu-repo/classification/LEM/Biotecnología info:eu-repo/classification/LEM/Enzimas info:eu-repo/classification/LEM/Proteasas info:eu-repo/classification/LEM/Enzymes info:eu-repo/classification/LEM/Sea cucumbers info:eu-repo/classification/cti/6 |
| Sumario: | Holothurians, commonly known as sea cucumbers, belong to the Phylum echinoderms. These marine organisms contain a wide variety of active ingredients, such as carbohydrates, lipids, neurotoxins and enzymes. The knowledge of digestive enzymes in the sea cucumber could clarify its nutritional biochemistry and physiology. Determining the digestive capabilities of these animals would allow efficient selection of ingredients for diet formulation aimed for production. Moreover, the activity of extracted proteases from marine organisms is very high and have particular characteristics that can be efficiently exploited. In addition, sea cucumbers are illegally exploited in Mexico, the profit is generally for other countries. Therefore it is important to contribute to basic research of this resource. The objective of this study was to identify and characterize the proteases present in several organs of the sea cucumbers Holothuria impatiens and Isostichopus fuscus and to relate these active compounds to their histology. Specimens of sea cucumber (H. impatiens and I. fuscus) from the Sea of Cortés, off the coast of Sonora, collected by the Research Center for Food and Development (CIAD) were studied. They were measured, weighted and cut along the anteroposterior axis. The following regions were considered for analysis: podia or tentacles (covering the oral region), digestive tract, respiratory tract and different skin regions. For histology studies, samples of selected anatomical parts or organs were fixed with 70 % alcohol, dehydrated and embedded in paraffin; 7 to 12 μm thick sections were stained with hematoxylin-eosin, and observed under a light microscope. For enzyme characterization, we determined the endogenous proteolytic activity. Proteases were classified according to the pH of acidic and basic activity by the methods of Anson and Kunitz, respectively. Enzymatic extracts were obtained from each anatomical part or organ with a ratio of 1:2 (tissue: phosphate buffer) 20 mM pH 7, under non-denaturing conditions. The substrates, hemoglobin (pH 2-5) and casein (pH 6-10), were handled separately to 1 % and 4 ºC. The enzymatic reaction was carried out for 10 minutes at 37 °C by stopping with 50 % trichloroacetic acid, and finally, spectrophotometric readings were obtained at 280 nm. It was later determined the protein concentration by the biuret method. We estimated the molecular weights of the protease enzymatic extracts obtained by denaturing and nondenaturing electrophoresis according to the method of Laemmli (1970). We used a separation gel 12 % acrylamide gel and acrylamide concentration of 4 %. The results showed that the organs and anatomical parts of I. fuscus and H. impatiens have large amounts of tissue and cell aggregates with digestive activity, and a type of inclusions that suggest defense action of hemocytes against outside the body. Also, that may exist within these parasitic organisms and residual algae used as food. In particular, tissues that have an obvious number of cells with digestive activity in the histological study, it was the podiums or tentacles, internal tubules, breathing tube and the dorsal skin. The results obtained in enzymology studies complemented the histological study. They showed that, in general, the bodies of sea cucumbers with lower activity at acidic pH compared with the activity at basic pH. In analyzing the results of activity of different organs within species, we found that proteolytic activity was higher in the podiums were statistically significant at pH 6 to 10 (up to 81 + 3 UAP/mg for H. impatiens), suggesting the presence of serine proteases and pH 2 (up to 27 + 14 UAP/mg for H. impatiens) the presence of aspartic proteases. Also, it was observed that outer skin extracts (pH 2-4 and 10) (up to 68 + 36 UAP/mg) and the ventral skin (pH 10) (61 + 35 UAP/mg) of I. fuscus, and ventral skin extracts of H. impatiens (pH 4 and 7) have significantly higher activity than other organs, possibly by contact with the skin is the environment. It also suggests that the metabolic differences between type of family (Holothuriidae or Stichopodidae) of sea cucumbers influences the proteolytic activity. We found high molecular weight proteins, between 30 and 120 kDa, in most organs of the two species studied. In general, most of the organs of sea cucumber I. fuscus had estimated molecular weight proteins of 39 kDa, whereas in H. Impatiens proteins were between 45 and 66 kDa. The tentacles were identified in proteins of approximately 39, 45, 66 and 103 kDa, determined for I. fuscus a high concentration of protein higher than 5.5 mg/mL. In H. impatiens were identified in proteins of approximately 84.64 y 56.87 kDa. Therefore, relating the results of histology and Enzymology about the presence of certain inclusions with digestive activity, it is suggested that these hemocytes in conducting defense methods have proteolysis action acidic or basic, hence the presence of digestive activity in all anatomical parts or organs of sea cucumber. In addition to suggesting a alternate mechanism of digestion for these organisms. Thus, knowledge of digestive enzymes could clarify the nutritional biochemistry and physiology of sea cucumbers, helping to determine their digestive capacity. As evidence of the sui generis characteristics of proteases present in these species and their relation to certain tissues contribute to basic knowledge of the activity and function of these enzymes, hoping to lay the groundwork for future structural studies and potential biochemical applications. |
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