Pectin lyase (PL) induction by organic and inorganic components of yeast extract (YE) was evaluated in Penicillium griseoroseum, cultured in a mineral medium containing sucrose, by determining PL activity (A235) and mycelial growth (mycelial dry weight). The lowest YE concentration that promoted significant PL induction without acting as a carbon source for the fungus corresponded to 0.0075%. Neither calcined YE nor a nutrient solution containing micronutrients induced PL production, indicating that the inducer was an organic compound. Vitamins, phospholipid components, amino acids, and nitrogenous bases were tested in place of YE and promoted no significant PL induction. A PL inducer compound was found to be soluble in the nucleotide fraction obtained during extraction of YE. The inducer was shown to be a thermostable polar substance dialyzable at 2000 Daltons, hydrolyzable by HCl, and activated by boiling for up to 60 min. Cyclic AMP (cAMP) exogenously added to the culture medium at 5 and 10 mM was capable of inducing PL in P. griseoroseum grown on sucrose, suggesting that at least one compound may be present in YE acting in a cooperative fashion for the maintenance of high levels of cAMP into the cell. PL activity and the level of cAMP inside the fungal cells increased after the addition of YE to the culture medium, suggesting the participation of this messenger in this enzyme's synthesis.
Sucrose, a non‐pectic carbon source, with yeast extract (YE) added was able to support the production of pectin lyase (PL) by Penicillium griseoroseum Dierckx. However, in the absence of YE, the fungus did not produce PL but grew and caused a marked reduction in culture medium pH. Furthermore, in the absence of YE, only a culture medium with a high buffering capacity permitted the production of PL in the presence of pectin. On the other hand, in the presence of 0.06% YE and of 0.1% pectin, the fungus produced maximum growth and specific PL activity during a 48‐h period of culture, with a small variation in medium pH. In the absence of sucrose, YE concentrations from 0 to 0.6% did not support enzyme production, indicating synergism between sucrose and YE for production of the enzyme.
The use of other inducers as substitutes for pectin was studied aiming to reduce the production costs of pectic enzymes. The effects of sugar-cane juice on the production of pectin lyase (PL) and polygalacturonase (PG) by Penicillium griseoroseum were investigated. The fungus was cultured in a mineral medium (pH 6.3) in a rotary shaker (150 rpm) for 48 h at 25oC. Culture media were supplemented with yeast extract and sucrose or sugar-cane juice. Sugar-cane juice added singly to the medium promoted higher PL activity and mycelial dry weight when compared to pectin and the use of sugar-cane juice and yeast extract yielded levels of PG activity that were similar to those obtained with sucrose-yeast extract or pectin. The results indicated that, even at low concentrations, sugar-cane juice was capable of inducing pectin lyase and polygalacturonase with no cellulase activity in P. griseoroseum.
A second polygalacturonase-encoding gene (pgg2) of Penicillium griseoroseum was cloned and consists of an opening reading frame of 1107 bp after removal of two introns. The gene encodes a protein of 369 amino acids with a predicted molecular mass of 38.3 kDa. The deduced protein sequence exhibited high homology with other fungal endopolygalacturonases. A polymerase chain reaction (PCR)-based strategy was used to study the expression patterns of pgg1 and pgg2 genes under different culture conditions and our results show that both genes are regulated by the carbon source at the transcriptional level. The pgg1 transcript was detected at 76 h of fungal growth in pectin while the pgg2 transcript was also induced by sucrose. The addition of yeast extract to glucose medium abolished the repressive effect of glucose, suggesting that the transcription of these genes is controlled by different mechanisms.
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