Termoestabilización de proteínas de interés biotecnológico

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-12-2012Thermostability is a desired property for many biotechnological applications of enzymes because it allows an increase in their half-life at the working temperatures and produces a concomitant increase in the stability against organic solvents and detergents. Thermostable enzymes from thermophilic microorganisms are well adapted to such conditions, but not are always available or fulfill the role that the industry or specific applications require, leading to the development of methods to select thermostable variants from enzymes of mesophilic origin. Some of these methods rely on the deep knowledge of the structure-function relationships of the enzyme to design modifications that increase the thermostability. In other cases the methods of “directed evolution” mimics nature by selecting for a specific property (thermostability) among a high variety of random or semi-random mutants generated by in vitro or in vivo procedures. In directed evolution, the success of the selection depends on the capability to select over a huge amount of variants, and require either robotic system for the analysis of individual variants, or intermediate enrichment selection procedures to decrease the burden of selection. In this Thesis, we have applied the method of folding interference at high temperature in a thermophilic host (Thermus thermophilus) to select thermostable variants of two very different enzymes, a DNA polymerase form bacteriophage φ29, and the esterase I from Pseudomonas fluorescens (PFEI). The method is based on the expression in the thermophilic host of fusions between the target protein (N-terminal) and a thermostable kanamycin nucleotidyl transferase (C-terminal) as selectable reporter. In the first part of the experimental work, we isolated T. thermophilus deletion mutants defective in a major LonA-like protease, and checked their suitability as selection host, leading us to conclude that this protease is highly relevant to avoid toxicity of the fusions. In the second and third experimental sections, we isolated “folding-reinforced” mutants of the exonuclease and polymerase domains of the DNA polymerase of F29 and explained on structural ground their effects on stability. In the final experimental section, we went through the isolation of thermostable variants of the PFEI, and also were able to explain most of the observed effects on structural models. Some of the mutations obtained with both proteins are likely susceptible of patent protection. General conclusions about what can be expected for the general application of this method to isolate thermostable variants of any protein are discussed