Modificación química dirigida de lipasas en fase sólida
Author
Romero Ormazabal, ÓscarEntity
UAM. Departamento de Química Analítica y Análisis Instrumental; CSIC. Instituto de Catálisis y Petroleoquímica (ICP)Date
2014-02-25Subjects
Lipasa - Tesis doctorales; QuímicaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Analítica y Ánalisis Instrumental. Fecha de lectura: 25-02-2014Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Enzymes are versatile biocatalysts finding an increasing application in many industrial fields including fine
and organic chemistry, pharmacy, cosmetics or food industry. The enzymes unsurpassed chemo-, regioand
enantioselectivity characteristics are making them interesting as fine chemistry biocatalysts since the
preparation of pure intermediates is a key step for many industrially relevant processes. However, when
using non-natural substrates enzyme specificity may not be as high as required for industrial purposes
requesting the need of improvement of this enzyme property, possible through very different techniques,
like screening, controlled immobilization, enzyme engineering, directed evolution and rational design
approaches, chemical modification or combinations thereof.
Strategies for the selective and efficient chemical modification of proteins have been implemented
successfully for the study and modulation of enzyme specificity. However, despite the efforts made the
past years the site-directed incorporation of different moieties, like carbohydrates, peptides, polymers,
DNA, etc. specifically to the required position at the enzyme remains a challenging task. Applying
conventional methods of chemical modification on a protein attached covalently or reversible to a surface
or matrix can provide an important simplification of current protocols of modification.
The main objective of this Doctoral Thesis has been the development and implementation of new
strategies of site-directed chemical modifications of proteins on solid phase to improve their catalytic
properties. Joining genetic and chemical modification approaches 5 strategies were developed using
immobilized lipases as model enzymes:
- Chemical glycosylation of lipases on solid phase
- Promotion of the open conformation o lipase by two cysteine ligation
- Development of semisynthetic lipases conjugates
- Active center redesign with non-natural moieties
- Development of hybrid catalysts: lipase active-site anchoring of organometallics
These strategies were successfully applied, obtaining modified proteins with quantitative yields in almost
all the cases. These new modified lipases showed improved activity, regioselectivity, enantioselectivity in
several important biotransformations. The strategies developed during this thesis combine simplified and
easy handling of the enzyme during all modification steps with highly selective chemical modification
protocol. Methods are potentially extendable to other proteins and fields therefore offering general
strategies for obtaining improved enzyme variants applying solid-phase chemical modification strategies.
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