Estudio de la producción y actividad de proteínas tipo hidrofobinas y quitinasas de Lecanicillium lecanii en cultivos en sustrato sólido y sumergido
Lecanicillium lecanii is an entomopathogenic fungus used commercially as a biopesticide in agriculture and horticulture. However, there is scarcity of information about how L. lecanii production of hydrophobins-like proteins (HfbLs) and chitinases are required for their development and enzymes relat...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2014 |
| País: | México |
| Institución: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:9g54xh76f |
| Acceso en línea: | https://doi.org/10.24275/uami.9g54xh76f |
| Access Level: | acceso abierto |
| Palabra clave: | info:eu-repo/classification/LEM/Lecanicillium lecanii info:eu-repo/classification/LEM/Entomopathogenic fungi info:eu-repo/classification/LEM/Ingeniería biomédica info:eu-repo/classification/LEM/Proteins -- Analysis info:eu-repo/classification/LEM/Hongos entomopatógenos info:eu-repo/classification/LEM/Proteínas -- Análisis info:eu-repo/classification/LEM/Biomedical engineering info:eu-repo/classification/cti/6 |
| Sumario: | Lecanicillium lecanii is an entomopathogenic fungus used commercially as a biopesticide in agriculture and horticulture. However, there is scarcity of information about how L. lecanii production of hydrophobins-like proteins (HfbLs) and chitinases are required for their development and enzymes related to pathogenic activity. Therefore, this study evaluated the effect of the hydrophobicity of the conditions culture, such as culture type, support type and carbon source type on the chitinolytic activity, and the production and surface activity of hydrophobins-like proteins (HfbLs) from L. lecanii. The hydrophobicity as result of extrinsic factors such as the culture type, for example, submerged culture, SmC (hydrophilic) and SSC (using PUF, hydrophobic), as well as the carbon source (for example, the chitin, insoluble in water and fructose, soluble in water) had significant effect on the chitinases and class I Hfb production of L. lecanii. SSC added with colloidal chitin as carbon source increased the β- N- acetyl hexosaminidases and Hfbs production ca. 3- and 10-folds, respectively, compared with the submerged culture. Interestingly, in this study showed that the chitin as carbon source acts as inductor of chitinases, as well as also to hydrophobins, it is due to the hydrophobins obtained from SSCchitin cultures showed surface activity to reduce the hydrophobicity of teflon (ca. 50 %), while hydrophobins from SSC added with fructose showed not surface activity on Teflon. Based on these results, the investigation moved forward on the research question of how the hydrophobicity of support type and chemical caracteristics of chitin used in SSC could affect the production and activity of hydrophobins of L. lecanii. This doctoral thesis showed that L. lecanii was able to produce class I and class II HfbLs in solid substrate culture (SSC) added with colloidal chitin as carbon source, the inert supports tested were perlite (P) and polyurethane (PUF). The purity and physicochemical properties as the degree of acetylation (DA) of the chitin, it had significant effect on the production of class I HfbLs of L. lecanii in SSC with PUF. Furthermore, it was observed that the hydrophilic character of the perlite and the hydrophobicity of the polyurethane were significant factors for the production and surface activity of the fungal HfbLs. The class I HfbLs were produced only in cultures with hydrophobic support, PUF, (302.1 ± 14.8 µg HfbL mL-1 ), and these showed surface activity to reduced ca. 50 % the hydrophobicity of teflon. The HfbLs class II were produced in cultures with either both supports, PUF or P, however, HfbLs produced in SSC with P were ca. 3-folds higher than in SSC with PUF. The class II HfbLs were able to reduce ca. 25 % the hydrophobicity of teflon and to reduce ca. 50 % of the surface tension of water. The hydrophobicity of volatile organic compounds (VOCs) used as carbon sources in submerged cultures of L. lecanii also showed significantly influence on the production of chitinases and HfbLs. L. lecanii showed be able to grow and consume n-hexane or toluene with or without addition of colloidal chitin as carbon source in submerged culture (SmC). L157 strain showed highest consumption of n- hexane (55.6 %) and toluene (52.9 %) as sole carbon source. In SmC cultures added with chitin and hydrocarbons (MCHT or MChH), the strain L157 showed ability to produce endochitinases and N- acetyl hexosaminidases, also, it increased ca. 10-folds their HfbLs class I production (548.6 ± 26.3 µg Hfb mL-1 protein) compared with cultures added with chitin as sole carbon source (57.4 ± 4.7 µg Hfb mL-1 protein). The hydrophobicity of VOCs tested. Also, it had significant effect on the surface activity of class I HfbLs. Class I HfbLs from MChT culture reduced ca. 48 % the hydrophobicity of teflon, in contrast to HfbLs from MChH (ca. 10 %). This study allowed us to observe the close relationship between the chitinases and hydrophobins production of L. lecanii. Noteworthy, the hydrophobicity of the culture, the support and the carbon source type were key elements to the production and surface activity of hydrophobins-like proteins, while the chitin was essential for the production of HfbLs with surface activity. The information obtained about the hydrofobicity effect on the chitinases and HfbLs production, allows us to deepen our knowledge about the development and pathogenesis of L. lecanii. Furthermore, it was possible to establish a method of transformation of L. lecanii using phosphinothricin (PPT) as a selective agent (bar gene), which could facilitate for studies about their development and interaction with the environment. Based on the above, this study could continue the purification, structural analysis and interaction of this fungus on different surfaces and with the study the involvement of HfbLs in the fungal development. |
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