Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii
Chitinases are enzymes capable of hydrolyzing the β1-4 bond of the chitin chain, these belong to two main families of carbohydrate hydrolases, 18 and 19, and this classification depends on sequence, structure and mechanism of action. Some chitinases of family 18, in addition to their hydrolytic acti...
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| Tipo de recurso: | tesis de maestría |
| Estado: | Versión publicada |
| Fecha de publicación: | 2015 |
| País: | México |
| Institución: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:qf85nb730 |
| Acceso en línea: | https://doi.org/10.24275/uami.qf85nb730 |
| Access Level: | acceso abierto |
| Palabra clave: | info:eu-repo/classification/LEM/Quitanasas info:eu-repo/classification/LEM/Chitinase info:eu-repo/classification/LEM/Chitin info:eu-repo/classification/LEM/Quitina info:eu-repo/classification/cti/6 |
| Sumario: | Chitinases are enzymes capable of hydrolyzing the β1-4 bond of the chitin chain, these belong to two main families of carbohydrate hydrolases, 18 and 19, and this classification depends on sequence, structure and mechanism of action. Some chitinases of family 18, in addition to their hydrolytic activity possess transglycosylation activity, resulting in products derived from chitin (chitin oligosaccharides) which present a high added value with a chain length longer than the initial substrate. The aim of this work was to produce and purify chitinases from Lecanicillium lecanii and investigate if the enzymes were capable to transglycosylate. Chitinases were obtained in submerged culture using colloidal chitin as sole carbon source. The chitin employed presented three different percentages of residual protein, 6, 10, 14%. Crude enzyme was purified by precipitation with ammonium sulphate, size exclusion chromatography followed by anionic exchange chromatography. The transglycosylation reaction was evaluated with both salting out and purified enzyme at two pH and two concentrations of the enzyme. Enzymes produced with colloidal chitin with at 14% of protein presenting activities of 0.027 and 22.79 U/mg of N-acetylhexosaminidase (Nhasa) and Endochitinase (Endo), respectively. The highest production of Nhasa was obtained when colloidal chitin with 10% of protein was used, specifically 0.115 U/mg. Whereas, submerged culture with colloidal chitin with 6% of protein did not present chitinolytic activity. The presence of chitinolytic activity was determined for each step of purification by zymograms. Purified Nhasa displayed an activity of 3.085 U/mg, electrophoretic profile evidenced a protein band of 50 kDa. Tetramers were obtained as products of the transglycosylation reaction with different degree of acetylation. |
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