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...
| Author: | |
|---|---|
| Format: | master thesis |
| Status: | Published version |
| Publication Date: | 2015 |
| Country: | México |
| Institution: | Universidad Autónoma Metropolitana |
| Repository: | Repositorio Institucional de la UAM Iztapalapa |
| Language: | Spanish |
| OAI Identifier: | oai:bindani.izt.uam.mx:qf85nb730 |
| Online Access: | https://doi.org/10.24275/uami.qf85nb730 |
| Access Level: | Open access |
| Keyword: | 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 |
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| dc.title.none.fl_str_mv |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii |
| title |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii |
| spellingShingle |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii JESUS ROJAS OSNAYA 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 |
| title_short |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii |
| title_full |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii |
| title_fullStr |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii |
| title_full_unstemmed |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii |
| title_sort |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecanii |
| dc.creator.none.fl_str_mv |
JESUS ROJAS OSNAYA |
| author |
JESUS ROJAS OSNAYA |
| author_facet |
JESUS ROJAS OSNAYA |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
CONCEPCION KEIKO SHIRAI MATSUMOTO ZAIZY ROCHA PINO |
| dc.subject.none.fl_str_mv |
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 |
| topic |
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 |
| description |
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. |
| publishDate |
2015 |
| dc.date.none.fl_str_mv |
2015-07-27 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/masterThesis info:eu-repo/semantics/publishedVersion |
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masterThesis |
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publishedVersion |
| dc.identifier.none.fl_str_mv |
https://doi.org/10.24275/uami.qf85nb730 |
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https://doi.org/10.24275/uami.qf85nb730 |
| dc.language.none.fl_str_mv |
spa |
| language |
spa |
| dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc/4.0/ |
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openAccess |
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https://creativecommons.org/licenses/by-nc/4.0/ |
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reponame:Repositorio Institucional de la UAM Iztapalapa instname:Universidad Autónoma Metropolitana instacron:UAM |
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Universidad Autónoma Metropolitana |
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UAM |
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UAM |
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Repositorio Institucional de la UAM Iztapalapa |
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Repositorio Institucional de la UAM Iztapalapa |
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| _version_ |
1858175557250842624 |
| spelling |
Estudio de la actividad de transglicosilación de las quitinasas producidas por Lecanicillium lecaniiJESUS ROJAS OSNAYAinfo:eu-repo/classification/LEM/Quitanasasinfo:eu-repo/classification/LEM/Chitinaseinfo:eu-repo/classification/LEM/Chitininfo:eu-repo/classification/LEM/Quitinainfo:eu-repo/classification/cti/6Chitinases 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.Las quitinasas son enzimas capaces de hidrolizar los enlaces β1-4 de la cadena de quitina, dependiendo de su secuencia, estructura y mecanismo de acción, éstas pueden pertenecer a una de las dos familias principales de las carbohidrato hidrolasas, a la familia 18 o 19. La familia 18 de las quitinasas son conocidas por catalizar no sólo la hidrólisis sino también una reacción de transglicosilación que resulta en quitin oligosacáridos con una longitud de cadena más larga que la del sustrato inicial. Estos derivados de la quitina son productos de alto valor agregado. Por lo anterior el objetivo del presente trabajo fue producir y purificar quitinasas de Lecanicillium lecanii y probar su actividad de transglicosilación. Se obtuvieron en cultivo sumergido, evaluando su producción utilizando como sustrato quitina coloidal con tres porcentajes de proteína diferentes. Los extractos enzimáticos se purificaron mediante precipitación con sulfato de amonio, cromatografía de exclusión molecular, seguido de cromatografía de intercambio aniónico. La reacción de transglicosilación se evaluó a dos pH y a dos concentraciones de enzima, tanto del precipitado con sulfato de amonio como de las enzimas purificadas. Se emplearon quitinas con grado de pureza diferente basada en su contenido de proteína residual, 6, 10 y 14%, las cuales fueron empleadas como sustrato en un biorreactor para producir las enzimas quitinolíticas. Cuando se utilizó quitina coloidal con 14% de proteína se obtuvieron actividades de 0.027 y 22.79 U/mg de proteína para Nacetilhexosaminidasa (Nhasa) y Endoquitinasa (Endo) respectivamente. La producción mayor de Nhasa se obtuvo al utilizar quitina coloidal con 10% de proteína residual obteniendo 0.115 U/mg de proteína, mientras que para la quitina coloidal con 6% de proteína no se detectó actividad quitinolítica. La presencia de actividad Nhasa se corroboró en las etapas de purificación mediante zimogramas. Una vez parcialmente purificada la Nhasa se obtuvieron 22 U/ mg de proteína, estas se sometieron a electroforesis mostrando dos bandas a 50 y 67 kDa. Además esta Nhasa logró llevar a cabo la reacción de transglicosilación utilizando 4-metil-N-acetil-D-glucosamina y 4-metil-N-acetilD-glucosamina formando oligómeros con grado de polimerización de 4 con diferentes grados de acetilación.CONCEPCION KEIKO SHIRAI MATSUMOTOZAIZY ROCHA PINO2015-07-27info:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/publishedVersionhttps://doi.org/10.24275/uami.qf85nb730reponame:Repositorio Institucional de la UAM Iztapalapainstname:Universidad Autónoma Metropolitanainstacron:UAMspainfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc/4.0/oai:bindani.izt.uam.mx:qf85nb7302025-11-26T19:18:06Z |
| score |
15,81155 |