Suraia Said

Endophytic Fungi: Natural Products, Enzymes and Biotransformation Reactions

Endophytes exhibit a complex web of interactions with host plants and with other endophytic microorganisms and therefore they have been intensively studied over the last several years as prolific sources of new and bioactive natural products. In fact, an impressive number of natural products have been produced by endophytic microorganisms. In addi- tion, some studies have shown endophytes to be good producers of useful enzymes to improve industrial processes. More recently, endophytes have also received attention as biocatalysts in the chemical transformation of natural products and drugs. Results have shown their ability to modify chemical structures with a high degree of stereospecificity. Some reac- tions are similar to those catalyzed by mammal phase I metabolism; therefore endophytes could be used as models for drug metabolism studies. This paper summarizes recent data on endophytes research as a source of novel and bioactive natural products, as producers of enzymes and their use on biotransformation processes.

Pectic enzymes production in solid-state fermentation using citrus pulp pellets byTalaromycesflavus,Tuberculariavulgaris andPenicilliumcharlessi

In order to select fungi with capacity to produce pectic enzymes when citrus pulp pellets were used in solid cultures we screened a collection of one hundred fungi

Comparative study of intracellular and extracellular pectinases produced by Penicillium frequentans

The filamentous fungus Penicillium frequentans synthesized eleven polygalacturonases (PGs) and two pectinesterases (PEs) when grown in liquid culture supplemented with pectin. Seven PGs and the two PEs were secreted in the medium, whereas four PGs were not secreted. Among the secreted PGs, the endo‐PG (band 10) and exo‐PGs (band 5) were the enzymes secreted at the highest levels. All secreted PGs bound to lectin and their secretion and/or enzymic activities were inhibited by tunicamycin (TM), except for the constitutive and inducible endo‐PG (band 10). Studies on the affinity for concanavalin A (ConA) and the effect of TM suggested that the secreted endo‐PG and exo‐PG differed in level and process of glycosylation. The exo‐PG was characterized as a N‐glycoprotein, whereas the endo‐PG is probably an O‐glycoprotein. The PGs (bands 3 and 4) were neither bound to ConA nor secreted and their enzymic activities were inhibited by TM, suggesting that they are probably N‐glycoproteins with complex oligosaccharides of type three and tetra‐antennary structure. The other PGs (bands 6 and 8) that were not secreted and did not bind to ConA were not inhibited by TM. These enzymes presented chromatographic characteristics and effects with TM that were similar to endo‐PG (band 10), because these PGs might be unglycosylated or/and aggregate forms of the endo‐PG (band 10).

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH--and temperature optima of 3.9 and 50 degrees C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-alpha-1,4-polygalacturonase (exo-PG I). Km and Vmax for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U x mg(-1), respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.

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 endopolygalacturonase (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.

The antimicrobial activity of Aspergillus fumigatus is enhanced by a pool of bacteria

In a screening program for new antibiotic producers, a strain of Aspergillus fumigatus was isolated from Brazilian soil samples. A pool of autoclaved bacteria was added to part of the fungus culture on the second day of fermentation to increase antibiotic production. The chloroform extract from the culture broth to which the pool of autoclaved bacteria was added showed an increase of 55%, 63% and more than 100% in activity against Staphylococcus aureus, Candida albicans and Micrococcus luteus, respectively. Also, the HPLC chromatographic profiles of the chloroform extracts from both culture conditions were different. Two active compounds were isolated from the broth of the culture grown in the presence of pooled bacteria and were identified as 3,4-dimethoxyphenol and 1,3,5-trimethoxybenzene.

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, β-cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5°C and 40% at 25°C after 8 months. The lipase dried with 10% of β-cyclodextrin retained 72% of activity at 5°C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.

Antimicrobial activity from endophytic fungi Arthrinium state of Apiospora montagnei Sacc. and Papulaspora immersa

Papulaspora immersa and Arthrinium state of Apiospora montagnei Sacc. were isolated from the roots of Smallanthus sonchifolius (yacon). The crude extracts from their cultures inhibited the growth of Staphylococcus aureus, Kocuria rhizophila, Pseudomonas aeruginosa and Escherichia coli. The more relevant results were observed in the ethyl acetate extract from P immersa against P aeruginosa (90 µg/mL) and ethyl acetate extract from Arthrinium state of A montagnei Sacc. against P aeruginosa (160 µg/mL). The two endophytic fungi isolated from yacon roots as well as their antimicrobial activity detected in the crude extracts cultures were being reported for the first time.

Partial purification and characterization of exopolygalacturonase II and III of Penicillium frequentans

Previous studies from our laboratory have demonstrated that the fungus Penicillium frequentans produces high levels of polygalacturonase and pectinesterase. Endopolygalacturonase I (Endo-PG I) and Exopolygalacturonase I (Exo-PG I) were previously purified and characterized. In the present study two extracellular polygalacturonases were separated, partially purified and biochemically characterized. Both were characterized as exopolygalacturonases so they were named exopolygalacturonase II (Exo-PG II) and exopolygalacturonase III (Exo-PG III) which had a molecular mass of 63 kDa (Exo-PG II) and 79 kDa (Exo-PG III). The Km values were 1.6 and 0.059 g/L and the Vmax values were 2571 and 185 U/mg, respectively. The optimum temperature was 50oC for both enzymes, while the optimum pH was 5.0 for Exo-PG II and 5.8 for Exo-PG III.

Extracellular polygalacturonases from Penicillium frequentans : separation and regulatory aspects

Extracellular polygalacturonase activities of Penicillium frequentans were induced by pectin and repressed, but not inactivated, by glucose. The absence of a carbohydrate source derepressed 50% of viscosity-diminishing activity and 36% of reducing-groups-releasing activity compared to production in the presence of pectin. High concentrations (30 mM and 50 mM) of D-galacturonic acid reduced only the viscosity-diminishing activity, and under these conditions the fungus grew poorly. Neither effect was observed if the mycelium had been previously induced by pectin. Polygalacturonase activities produced in the presence of pectin were separated by ion exchange chromatography. These enzymes eluted in six peaks, characterized as exopolygalacturonases I, II and III and endopolygalacturonases, I, II and III.