RESUMO
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.
Assuntos
Quimosina/genética , Trichoderma/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica , Quimosina/química , Quimosina/metabolismo , Clonagem Molecular , Cristalização , DNA , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Trichoderma/enzimologiaRESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/química , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/genética , Sequência de Bases , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular , Difração de Raios XAssuntos
Quimosina/biossíntese , Genes Sintéticos , Proinsulina/biossíntese , Trichoderma/genética , Western Blotting , Quimosina/genética , Quimosina/isolamento & purificação , Clonagem Molecular , Vetores Genéticos , Plasmídeos , Proinsulina/genética , Proinsulina/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Mapeamento por RestriçãoAssuntos
Quimosina/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Quimosina/química , Leite/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Conformação ProteicaAssuntos
Quimosina/genética , Sequência de Aminoácidos , Animais , Quimosina/metabolismo , Clonagem Molecular , Escherichia coli/genética , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/metabolismo , Renina/metabolismo , Especificidade por SubstratoRESUMO
The aspartic proteinase chymosin exhibits a local network of hydrogen bonds involving the active site aspartates and surrounding residues which may have an influence on the rate and optimal pH of substrate cleavage. We have introduced into chymosin B the following substitutions: Asp304 to Ala (D304A), Thr218 to Ala (T218A) and Gly244 to Asp (G244D, chymosin A), using oligonucleotide-directed mutagenesis. Kinetic analysis of these active mutants shows shifts in their pH optima to 4.4 D304A, 4.2 T218A and 4.0 G244D compared with 3.8 for chymosin B using a synthetic octapeptide substrate. The upward shift of the D304A and T218A may be due to the loss of hydrogen bond interactions indirectly affecting the catalytic aspartates 32 and 215. The G244D mutation which is in a flexible loop on the surface of the enzyme may alter the conformation of the specificity pockets on the prime side of the scissile bond.
Assuntos
Quimosina/genética , Engenharia de Proteínas , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Escherichia coli/genética , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Conformação ProteicaRESUMO
Rapid advances in site-directed mutagenesis and total gene synthesis combined with new expression systems in prokaryotic and eukaryotic cells have provided the molecular biologist with tools for modification of existing proteins to improve catalytic activity, stability and selectivity, for construction of chimeric molecules and for synthesis of completely novel molecules that may be endowed with some useful activity. Such protein engineering can be seen as a cycle in which the structures of engineered molecules are studied by X-ray analysis and two-dimensional nuclear magnetic resonance. The results are used in the improvement of the design by using knowledge-based procedures that exploit facts, rules and observations about proteins of known three-dimensional structure.