Jorge Luiz Cavalcante Coelho

Screening of filamentous fungi for production of xylitol from D-xylose

Eleven filamentous fungi were screened for xylitol production in batch cultures. Production was generally low under the growth conditions used in this study. Penicillium crustosum presented the highest production, 0.52 g L-1 from 11.50 g L-1 of D-xylose, representing consumption of 76% of the original D-xylose.

Partial purification and characterization of xylanase produced by Penicillium expansum

An extracellular xylanase was found to be the major protein in the filtrate culture of Penicillium expansum when grown on 0.3 % wheat bran, which showed no xylanase multiplicity. The enzime was partial purified by.ammonium sulfate fractioning, molecular exclusion chromatography, ultrafiltration and anion exchange chromatography. The protein eluation profile showed only one form of xylanase that was partially characterized. The activity of purified xylanase was optimal at pH 5.5 and 40 oC. The enzyme was stable at pH between 5.5 and 6.5 and temperatures between 20-40 oC. The enzyme showed a Km of 3.03 mM and Vmax of 0.027 mmol min-1 mg -1 of protein. The enzymatic activity was increased 31 % by Mg2+ and 28 % by Al3+.

Production of pectin lyase by Penicillium griseoroseum cultured on sucrose and yeast extract for degumming of natural fibres

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.

Production of pectin lyase by Penicillium griseoroseum as a function of the inoculum and culture conditions.

Optimum activity of an extracellular pectin lyase produced by Penicillium griseoroseum in submerged culture was after 120 h using 0.1% (w/v) citrus pectin as substrate. Sucrose at 0.1% (w/v) stimulated enzyme production and citrus pectin gave the highest activity of enzyme per unit growth.

Cyclic AMP and low molecular weight effector(s) present in yeast extract are involved in pectin lyase production by Penicillium griseoroseum cultured on sucrose.

Pectin lyase (PL) induction by organic and inorganic components of yeast extract (YE) was evaluated in Penicillum 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. APL 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 in to 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 enzymes synthesis.

Sugar-cane juice induces pectin lyase and polygalacturonase in Penicillium griseoroseum

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.

Differential expression of polygalacturonase-encoding genes from Penicillium griseoroseum in different carbon sources.

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. Journal of Industrial Microbiology & Biotechnology (2002) 29, 145–148 doi:10.1038/sj.jim.7000296

Tea extract as an inexpensive inducer of pectin lyase in Penicillium griseoroseum cultured on sucrose

Extracts of tea, coffee, cocoa, and yeast induced pectin lyase (PL) in Penicillium griseoroseum cultured in a mineral medium with sucrose as the carbon source. PL activity and fungal growth were similar in the treatments with 0.5% tea extract, the highest concentration tested, and 0.03% yeast extract. When tea extract was added singly to the culture medium, P. griseoroseum produced 59% and 17% of the PL activity and mycelial mass, respectively, obtained in a treatment with tea extract and sucrose. These results suggest that the production of the enzyme was not proportional to mycelial growth. No PL was produced in the medium with sucrose and without inducers. The small amounts of pectic substances present in the tea extract could not be responsible for PL induction. PL activity was detected after 12 h of growth in the medium containing sucrose and tea extract added at time zero, and after 48 h of growth when tea extract was added at times 12 and 24 h. Mycelial mass in all treatments was similar after 48 h of incubation. However, the addition of tea extract at time zero increased PL activity by 20–25%. Cyclic AMP at 5 and 10 mM in the culture medium induced 20 and 30%, respectively, of the PL activity obtained with 0.03% yeast extract, suggesting that PL induction brought about by either yeast extract or tea extract might involve the intracellular metabolism of cAMP.

Methylxanthines as inducers of pectin lyase in Penicillium griseoroseum cultured on sucrose

Pectin lyase (PL) from Penicillium griseoroseum can be induced by xanthine, theobromine, theophylline and especially by caffeine and hypoxanthine (5 mmol l−1 with 0·01% yeast extract (YE)). For caffeine and hypoxanthine, PL activity was, respectively, 5·2 and 3·7 times higher than with YE alone. The simultaneous addition of caffeine or hypoxanthine (5 mmol l−1) and YE (0·1%) had a synergistic effect on PL activity as compared to the addition of these substances alone (0·2% YE; 10 mmol l−1 caffeine; 10 mmol l−1 hypoxanthine). Increasing caffeine concentrations (0–10 mmol l−1) for a constant YE content of 0·01%, resulted in an increase in PL activity and a decrease in mycelial mass. For a constant caffeine concentration (5 mmol l−1) and increasing YE contents (0–0·2%), a higher PL activity and mycelial mass were detected. The addition of caffeine (10 mmol l−1) at the beginning of incubation increased PL activity and decreased mycelial mass, while caffeine added after 12 and 24 h resulted in decreases in PL activity and increases in mycelial mass. The results presented here indicate that methylxanthines, especially caffeine, can induce PL in P. griseoroseum.

Pectin lyase production by Penicillium griseoroseum: Effect of tea extract, caffeine, yeast extract, and pectin

SummaryMycelial growth and production of extracellular pectin lyase by Penicillium griseoroseum at different concentrations of inducers were investigated. The fungus was cultured in mineral medium using sucrose as a carbon source and caffeine, yeast extract, tea extract or pectin as inducers. Caffeine, yeast extract and tea extract in the presence of sucrose, and tea extract alone were capable of inducing pectin lyase in P. griseoroseum, even at low concentrations.