Among 12 yeast strains isolated from cocoa fermentations, only four showed extracellular pectinase activity. Kluyveromyces marxianus was the most pectinolytic with 85% of total secreted protein consisting of a constitutive endopolygalacturonase (PG). No pectic lyases or methylesterases were produced. The pH and temperature optima for PG activity were 5.0 and 40 degrees C, respectively. Purified PG compromised four proteins of M-r 47, 41, 35, and 33 kDa based on gel filtration and 45, 42, 39, and 36 according to SDS-PAGE, Activity-stained isoelectric focusing gels showed three major bands (pls 5.9; 5.6, and 5.3) and up to six minor bands from pl 6.4-5.0. PG had a typical random mode of action, very high macerating activity on plant tissues, and reduced the viscosity of cocoa pulp. PG secretion started in early exponential phase and was completed after 24 h. Only five out of 138 mutants with altered PG levels produced after nitrosoguanidine mutagenesis showed modest (up to 25%) increase in PG production. Most mutants were underproducers of the full complement of PG isoforms including five which had high intracellular PG located in low-density vesicles, vacuoles, and ER fractions. In most mutants, there was a clear correlation between PG and inulinase activity secreted from cells. The implications for hath cocoa and enzyme production are discussed. (C) 1997 Elsevier Science Inc.
