Sebastian Fernando Cavalitto
National University of La Plata
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Biotechnology Letters | 1996
Sebastian Fernando Cavalitto; Jorge A. Areas; Roque Alberto Hours
SummarySolid-state cultures of a pectinase-producing fungus (Aspergillus foetidus NRRL 341) were performed under different acidic conditions. Glass bottles containing 5 g of wheat bran and 7.5 mL of 0.2, 0.3, 0.4, or 0.5 N HCl were autoclaved (15 min, 121°C), inoculated with a spore suspension appropriately diluted to achieve an initial concentration of 4 × 104 spores per gram of wet substrate (with a 60 % humidity, on wet basis) and incubated at 30°C.Time course of pH and of different pectinase activities were determined in culture extracts. Total pectinase activity (TPA), expressed in terms of viscosimetric units per gram of wet substrate (VU.g−1), was affected by the initial culture acidity. The higher the HCl concentration used, the higher the TPA achieved, but after longer cultivation times. On the other hand, when 0.5 HCl was used, no fungal growth was observed. Nevertheless, enzyme productivity increased with culture acidity. When 0.4 HCl was used, TPA reached its maximum after 36 h of cultivation (2,535 VU.g−1). With 0.2 and 0.3 N HCl, TPA was the highest at 24 h (733 VU.g−1) and at 30 h (1,860 VU.g−1) respectively.The composition of the pectinase pool was also affected by culture acidity. The higher the acidity, the lower the pectinesterase activity and the higher both the polymethylgalacturonate lyase and polygalacturonase activities.
Journal of Industrial Microbiology & Biotechnology | 2000
Sebastian Fernando Cavalitto; Roque Alberto Hours; Carlos F. Mignone
Geotrichum klebahnii ATCC 42397 produces a protopectinase (PPase-SE) with polygalacturonase (PGase) activity. The microorganism was aerobically cultivated in synthetic media. Glucose, fructose and xylose yielded the highest enzyme levels (10–11 PGase units ml−1). Galacturonic acid repressed enzyme production and no growth was obtained with disaccharides and pectin. Specific enzyme activity obtained in an O2-limited culture was similar to that found in nonlimited ones. A growth yield (Yx/s) of 0.49 g of cell dry weight per gram of glucose consumed was obtained in a typical batch bioreactor culture. Enzyme production was growth associated, and no major products other than biomass and CO2 were detected. The volumetric enzyme activity reached a maximum around D=0.3–0.4 h−1 in glucose-limited continuous cultures. However, it varied strongly (together with microorganism morphology) even after retention times ≥8 at any D tested (0.035–0.44 h−1) though the rest of the culture variables remained fairly constant. No correlation between morphology and enzyme activity could be obtained. Enzyme production was poor in urea- and vitamin-limited continuous cultures. In all cases, biomass and CO2 accounted for ≅100% of carbon recovery though Yx/s values were different. Journal of Industrial Microbiology & Biotechnology (2000) 25, 260–265.
Bioresource Technology | 2013
Yanina N. Martinez; Ivana Alejandra Cavello; Roque Alberto Hours; Sebastian Fernando Cavalitto; Guillermo R. Castro
A keratinase isolated from Paecilomyces lilacinus (LPS #876) was tested against proteins present in the skin but the high enzyme activity was detected on collagen. Keratinase was physically immobilized onto PVA-pectin cryogels and enzyme release was 20.8±2.1%, 63.8±0.2%, 41.5±3.5% and 26.0±3.5% in cryogels containing pectins with esterification degrees (DE) 33.0%, 55.0%, 62.7% and 71.7% respectively at 37°C after 3h incubation. In presence of 0.75 M NaCl, the percentage of enzyme release changed to: 57.5±1.5, 65.8±3.8, 57.3±0.2 and 34.0±4.0 for the four pectins respectively. In-vitro studies of enrofloxacin release from PVA-pectin cryogels at pH close to the human skin (pH=5.5) showed 15.0% free antibiotic following first order kinetic at 37°C after 5h incubation. However, in the presence of keratinase only 6.9% of enrofloxacin was released under the same experimental conditions.
Biotechnology Techniques | 1999
Sebastian Fernando Cavalitto; Roque Alberto Hours; Carlos F. Mignone
Pectin releasing activity of protopectinase SE (PPase-SE) from Geotrichum klebahnii (= G. penicillatum = Trichosporon penicillatum) ATCC 42397 was determined using different batches of lemon protopectin as substrate. Results obtained showed a high degree of variability depending on the batch of protopectin used. As PPase-SE also shows polygalacturonase (PGase) activity, a method for the assay of this activity was optimised. The best assay conditions were: substrate (polygalacturonic acid) concentration of 2.0 g 1−1, reaction time of 10 min and up to 0.17 PGase units per test tube.
Biotechnology Techniques | 1997
Sebastian Fernando Cavalitto; Roque Alberto Hours; Carlos F. Mignone
A method for quantification of a pectin releasing enzyme (PPase-SE) from Geotrichum klebahnii (= Geotrichum penicillatum = Trichosporon penicillatum) ATCC 42397 is reported. PPase activity was determined by measuring the amount of soluble pectin released from lemon protopectin. Particle size of the substrate, reaction time and linearity range of the assay, were analysed. The best assay conditions were a reaction time of 30 min, 20 mg substrate (mesh 60) and up to 0.045 units PPase activity per test tube.
Colloids and Surfaces B: Biointerfaces | 2014
Yanina N. Martinez; Ivana Alejandra Cavello; Sebastian Fernando Cavalitto; Andrés Illanes; Guillermo R. Castro
Polyvinyl alcohol-pectin (PVA-P) films containing enrofloxacin and keratinase were developed to treat wounds and scars produced by burns and skin injuries. However, in order to prevent enzyme inactivation at the interface between the patch and the scars, crosslinked enzyme aggregates (CLEAs) from a crude extract of keratinase produced by Paecilomyces lilacinus (LPSC#876) were synthesized by precipitation with acetone and crosslinking with glutaraldehyde. Soluble vs. CLEA keratinase (K-CLEA) activities were tested in 59% (v/v) hydrophobic (isobutanol and n-hexane) and hydrophilic (acetone and dimethylsulfoxide) solvents mixtures. K-CLEA activity was 1.4, 1.7 and 6.6 times higher in acetone, n-hexane and isobutanol than the soluble enzyme at 37 °C after 1 h of incubation, respectively. K-CLEA showed at least 45% of enzyme residual activity in the 40-65 °C range, meanwhile the soluble biocatalyst was fully inactivated at 65 °C after 1h incubation. Also, the soluble enzyme was completely inactivated after 12 h at pH 7.4 and 45 °C, even though K-CLEA retained full activity. The soluble keratinase was completely inactivated at 37 °C after storage in buffer solution (pH 7.4) for 2 months, meanwhile K-CLEAs kept 51% of their activity. K-CLEA loaded into polyvinyl alcohol (PVA) and PVA-P cryogels showed six times lower release rate compared to the soluble keratinase at skin pH (5.5). Small angle X-ray scattering (SAXS) analysis showed that K-CLEA bound to pectin rather than to PVA in the PVA-P matrix.
Electronic Journal of Biotechnology | 2008
Natalia Lorena Rojas; Sebastian Fernando Cavalitto; Carlos F. Mignone; Roque Alberto Hours
Protopectinases (PPases) constitute a heterogeneous group of extracellular enzymes able to release soluble pectin from insoluble protopectin in plant tissues. Geotrichum klebahnii (ATCC 42397) produces PPase-SE with endopolygalacturonase activity. PPase-SE has been used for pectin extraction and maceration of plant tissues. Here, the capacity of G. klebahnii to use different pectins as carbon and energy sources (CES) was studied, in addition to PPase-SE capacity to release pectin from lemon peel. The strain was unable to use pectin from different origins as CES. When G. klebahnii was cultivated with mixtures of different amounts of glucose and citrus pectin as CES, the biomass obtained was proportional to the initial concentration of glucose, which was completely consumed. In addition, it produced PPase-SE in a glucose-containing medium. A culture was used for the extraction of pectin from lemon peels. Pectin was enzymatically extracted simultaneously with tissue maceration, yielding 3.7 g of (dry) pectin per 100 g of (wet) lemon peel. Extracted pectin was not metabolized by the strain. It was concluded that G. klebahnii uses PPase-SE to macerate, invade and colonize plant tissues, thus releasing soluble sugars to be used as CES without metabolizing solubilized pectin.
Environmental Technology | 2015
Nihan Gogus; Ezgi Evcan; Canan Tari; Sebastian Fernando Cavalitto
The potential of important agro-industrial wastes, apple pomace (AP) and orange peel (OP) as C sources, was investigated in the maximization of polygalacturonase (PG), an industrially significant enzyme, using an industrially important microorganism Aspergillus sojae. Factors such as various hydrolysis forms of the C sources (hydrolysed-AP, non-hydrolysed-AP, hydrolysed-AP + OP, non-hydrolysed-AP + OP) and N sources (ammonium sulphate and urea), and incubation time (4, 6, and 8 days) were screened. It was observed that maximum PG activity was achieved at a combination of non-hydrolysed-AP + OP and ammonium sulphate with eight days of incubation. For the pre-optimization study, ammonium sulphate concentration and the mixing ratios of AP + OP at different total C concentrations (9, 15, 21 g l−1) were evaluated. The optimum conditions for the maximum PG production (144.96 U ml−1) was found as 21 g l−1 total carbohydrate concentration totally coming from OP at 15 g l−1 ammonium sulphate concentration. On the other hand, 3:1 mixing ratio of OP + AP at 11.50 g l−1 ammonium sulphate concentration also resulted in a considerable PG activity (115.73 U ml−1). These results demonstrated that AP can be evaluated as an additional C source to OP for PG production, which in turn both can be alternative solutions for the elimination of the waste accumulation in the food industry with economical returns.
Colloids and Surfaces B: Biointerfaces | 2017
Nelida.Y. Martinez; Patricia Fernanda Andrade; Nelson Durán; Sebastian Fernando Cavalitto
In the present work, a double emulsion was developed for the encapsulation of Bovine Serum Albumin (BSA) as a model protein for the future encapsulation of viral proteins. The first emulsion polydispersity index (PDI) was studied with increasing concentrations of poly (ε-caprolactone) (PCL) as stabilizer (from 16% w/v to 5% w/v) and polyvinyl alcohol (PVA) as the surfactant in the second emulsion at 1.5% w/v. Results suggest that at decreasing concentrations of PCL the PDI of the emulsion also decrease, indicating that viscosity of the emulsion is crucial in the homogeneity of the resultant size distribution of the nanoparticles. When PVA concentration in the second emulsion was increased from 1.5% w/v to 2.5% w/v the PDI also increased. To study the relationship between the structure of the surfactant in the second emulsion and the resultant BSA encapsulation, emulsions were prepared with Pluronic F68 and PVA both at 1.5% w/v and PCL in the first emulsion at 5% w/v. Results indicated that Pluronic F68 was a better stabilizer because at the same experimental conditions encapsulation of BSA was 1.5 higher than PVA. FTIR studies confirmed the presence of BSA in the nanoparticles. SEM and TEM microscopies showed a size distribution of 300nm-500nm size of nanoparticles. Circular dichroism studies demonstrated that the secondary structure of the protein was conserved after the encapsulation into the nanoparticles.
Journal of Molecular Microbiology and Biotechnology | 2017
Dante Fratebianchi; Ivana Alejandra Cavello; Sebastian Fernando Cavalitto
An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.