Yeast cell permeabilization by osmotic shock allows determination of enzymatic activities in situ.

Yeast cells were permeabilized by incubation in 0.8 M sorbitol followed by suspension in dilute buffer. A preincubation with 2-mercaptoethanol was also included for optimal permeabilization. More than 90% of the treated cells were stainable with methylene blue. Determinations of cell wall-synthesizing enzymes (beta(1 --> 3)glucan and chitin synthases) and cytosolic enzymes in permeabilized cells yielded similar or higher activities than those in cell extracts. With chitin synthase III, the activity obtained with cells was 4- to 6-fold higher than in membrane preparations. Little protein leaks from the cells during permeabilization; yet the cells appear to be readily permeable to substrates and even proteins. Thus, these preparations may be of wide use for the study of enzymes and of biological processes in situ.

Studies of pectic enzymes produced by Talaromyces flavus in submerged and solid substrate cultures.

Tons of peel and rag are generated each year by industries of citrus fruit juices. These by-products are used either for the elaboration of pectin or as substrate for enzyme production. Talaromyces flavus produces extracellular pectinesterase and polygalacturonase after 24 h in submerged culture supplemented with 0.5-0.8% citrus pectin preceded by preculture for 24 h in 2% (w/v) sucrose or in solid substrate culture on passion fruit peel, lemon or orange pulp pellets after 3-6 days of incubation. Chromatographic profiles in a CM-Sepharose column of liquid and solid cultures were very similar, consisting of one endopoligalacturonase (endo-PG I) and one pectinolytic complex constituted by an endopoligalacturonase (endo-PG II) and pectinesterase. Pectin and pectate lyases were undetectable in both media. In Talaromyces flavus the synthesis of pectinases was repressed by glucose and finally controlled by the concentration of products from pectic enzymes degradation.

Regulation of pectic enzymes from the exo-1 mutant strain of Neurospora crassa: effects of glucose, galactose, and galacturonic acid.

The exo-1 mutant of Neurospora crassa produced and secreted pectolytic activities when incubated in the presence of pectin-containing biological materials. This study shows that polygalacturonase, pectate lyase and pectin lyase activities were induced in media supplemented with galactose or galacturonic acid, indicating that these sugars induced the synthesis of pectinases. Pectinesterase activity was undetectable. Polygalacturonase activity was better induced by galactose than by galacturonic acid. The reverse was true for lyase activities. The inducing effect of galactose and galacturonic acid seemed to be different: (i) a mixture of galactose and galacturonic acid synergistically increased the production of pectic enzymes, as compared to that in the presence of one of these sugars; (ii) the inducing effect of galacturonic acid was partially repressed by glucose; (iii) in contrast, the inducing effect of galactose, rather than repressed, was enhanced by the presence of glucose. Altogether, these data point out to a complex mechanism of regulation of pectolytic enzymes by pectin-containing organic substances.