Recurrent mutation-selection to improve rennet production in Candida tsukubaensis

The yeast Candida tsukubaensis is of industrial importance for the production of microbial milk-clotting enzyme. Milk-clotting enzyme is an enzymatic complex capable of coagulating milk for cheese manufacturing. High clotting activity (CA) and low proteolytic activity (PA) are desirable qualities. To study the genetic nature of the CA and PA traits, we analyzed 179 colonies obtained after mutagenic treatment. Analysis of the data obtained for this populations showed that CA and PA are traits controlled by polygenes and that they are correlated (r = 0.3). The existence of a positive correlation indicates that selection for one trait without considering the other may alter one of the traits in an undesirable direction, since the objective of selection would be an increase in clotting activity and a decrease in proteolytic activity. Three cycles of recurrent mutation-selection were carried out to obtain improved strains. The ultraviolet light dose permitting a 5 per cent rate of cell survival was sufficient to generate genetic variability in the three selection cycles. At the end of the third cycle there was an increase of about 98 per cent in clotting activity and a decrease of about 20 per cent in proteolytic activity. Analysis of variance of the selective cycles showed that the linear effects were significant (P