RESUMO
The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.
Assuntos
Quimotripsina/biossíntese , Genes Fúngicos , Glicosídeo Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Trichoderma/genética , Animais , Bovinos , Celulose 1,4-beta-Celobiosidase , Quimotripsina/genética , Quimotripsina/metabolismo , Meios de Cultura/farmacologia , Indução Enzimática/efeitos dos fármacos , Vetores Genéticos , Glicosídeo Hidrolases/biossíntese , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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
Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 angstroms resolutions respectively. The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B. Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in Km with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3. Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98-112 of kappa-casein, the natural substrate, revealed an increase in the kcat compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.