Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei.

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.

Multidisciplinary cycles for protein engineering: site-directed mutagenesis and X-ray structural studies of aspartic proteinases.

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.