ENDOPOLYGALACTURONASE SECRETION BY KLUYVEROMYCES-MARXIANUS AND OTHER COCOA PULP-DEGRADING YEASTS

Among 12 yeast strains isolated from cocoa fermentations, only four sh owed 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 methylest erases were produced. The pH and temperature optima for PG activity we re 5.0 and 40 degrees C, respectively. Purified PG compromised four pr oteins of M-r 47, 41, 35, and 33 kDa based on gel filtration and 45, 4 2, 39, and 36 according to SDS-PAGE, Activity-stained isoelectric focu sing gels showed three major bands (pls 5.9; 5.6, and 5.3) and up to s ix 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 visc osity of cocoa pulp. PG secretion started in early exponential phase a nd 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 i ntracellular PG located in low-density vesicles, vacuoles, and ER frac tions. In most mutants, there was a clear correlation between PG and i nulinase activity secreted from cells. The implications for hath cocoa and enzyme production are discussed. (C) 1997 Elsevier Science Inc.