RESUMEN
The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.
Asunto(s)
Quimosina/genética , Trichoderma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía por Intercambio Iónico , Quimosina/química , Quimosina/metabolismo , Clonación Molecular , Cristalización , ADN , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Trichoderma/enzimologíaRESUMEN
The specificity and pH profile of aspartic proteinases have evolved to include not only pepsin with a broad specificity and an optimal activity in acid media, but also renin, with high specificity for angiotensinogen and activity close to neutral pH. Comparisons of the structures and catalytic activities of aspartic proteinases provide helpful clues for engineering new activity profiles. We illustrate an approach that involves recombinant DNA techniques, biochemistry, structure determination and biocomputing. We use the 3-D structures of inhibitor complexes of several aspartic proteinases to define likely intermediates and specificity sub-sites. The multidisciplinary research is organised as cycles, in which each cycle tests a design hypothesis proposed in the previous cycle. We use one member of the aspartic proteinase family, chymosin, to illustrate these ideas in engineering enzymes with altered pH optima and specificities.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/genética , Secuencia de Bases , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Estructura Molecular , Difracción de Rayos XAsunto(s)
Quimosina/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Secuencia de Bases , Quimosina/biosíntesis , Gráficos por Computador , Cristalografía por Rayos X , Análisis de Fourier , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Mucor/enzimología , Pepsina A/química , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Rhizopus/química , Homología de Secuencia de Aminoácido , Programas Informáticos , Trichoderma/enzimologíaAsunto(s)
Quimosina/química , Quimosina/genética , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína , Aminoácidos , Animales , Cristalografía por Rayos X , Expresión Génica , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The loop exchange mutant chymosin 155-164 rhizopuspepsin was expressed in Trichoderma reesei and exported into the medium to yield a correctly folded and active product. The biochemical characterization and crystal structure determination at 2.5 A resolution confirm that the mutant enzyme adopts a native fold. However, the conformation of the mutated loop is unlike that in native rhizopuspepsin and involves the chelation of a water molecule in the loop. Kinetic analysis using two synthetic peptide substrates (six and 15 residues long) and the natural substrate, milk, revealed a reduction in the activity of the mutant enzyme with respect to the native when acting on both the long peptide substrate and milk. This may be a consequence of the different charge distribution of the mutated loop, its increased size and/or its different conformation.