Production of a xylanase by Trichoderma harzianum (Hypocrea lixii) in solid-state fermentation and its recovery by an aqueous two-phase system

Production of xylanase enzyme by fungi strains has gained interest in the recent years due to its high productivity, high catalytic power, as well as its potential applications in different areas such as feed, food, textile, and biofuel industries. The conventional methodologies, to produce enzymes, involve complex apparatus and chemical solvents and are associated with high costs and low- yield recovery. To obtain the high-yield recovery of the enzymes, modern enzyme extraction methods are taken into account. Aqueous two-phase systems (ATPS) are an alternative separative methodology for the purification and recovery of the enzymes and other biomolecules. The advantages of ATPS are easy scale-up and extraction, volume reduction, and rapid separation. The objective of this study was to produce Trichoderma harzianum xylanase by solid-state fermentation (SSF) using corn cobs as a support/substrate and employing ATPS for its partial recovery. In this study, the results showed the ability of a microorganism to grow on the corn cobs and to produce the xylanase enzyme. Xylanolytic activity reached 7.85 U/g of corn cobs. The enzyme was efficiently concentrated by ATPS. In addition, a high purification factor (10-fold) and considerable enzyme recovery (%ER) (84%) percentage were obtained

Purification of chymotrypsin from pancreas homogenate by adsorption onto non-soluble alginate beads

Chymotrypsin was purified from an activated homogenate of bovine pancreas by adsorption onto non-soluble alginate beads in 25 mM Tris-acetate-5 mM CaCl2 buffer at different adsorbate-adsorbent ratios and the pH values were assayed. Under all the experimental conditions, the enzyme has a positive net electrical charge whereas alginate is negatively charged. After performing steps of washing and desorption in 25 mM Tris-acetate-500 mM CaCl2 buffer ρH 7.0, chymotrypsin was purified 9 times with an enzyme recovery of 62%. The eluate fractions resulted in two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The method allows purification with suitable values from a raw sample like pancreas homogenate without a previous clarification step.Fil: Spelzini, Darío. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Picó, Guillermo Alfredo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Chitosan-bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin-BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan-BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.Fil: Boeris, Valeria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentin

Single method of purification for endoglucanase from Aspergillus niger by polyelectrolyte precipitation

The precipitation of endoglucanase from Aspergillus niger with synthetic and natural electrically charged polymers -poly vinyl sulfonate (PVS) and chitosan (CHS)- was characterized and applied to a simple method of purification of an enzymatic extract obtained from fungal culture under solid-state fermentation (SSF).The kinetics of complex formation was determined. The results of the kinetic profile obtained for CHS and PVS indicated an exothermic mechanism for the formation of the non-soluble complex. CHS exhibited a marked stabilizing effect on endoglucanase.The enzyme precipitated successfully with both polymers. The precipitation method applied to commercial endoglucanase and the fungal extract showed similar patterns with high purification factors. The recovery of the activity in the re-dissolved precipitate from the fungal extract was close to 40% at pH 5.3 using PVS (1% w/w) as precipitating agent and the purification factor was near 9. The purification factor of endoglucanase in the precipitate of the enzymatic extract from SSF with CHS (0.05% w/v) was around 7. These parameters make this precipitation method appropriate to be included in the last stages of a downstream process, with advantages such as simplicity, scalability and ability to concentrate and stabilize the enzyme.Fil: Boggione, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Becher, Ramiro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin

Aqueous two-phase extraction and polyelectrolyte precipitation combination: A simple and economically technologies for pepsin isolation from bovine abomasum homogenate

The combination of two bioseparation techniques, partition in aqueous two-phase systems and polyelectrolyte precipitation of the target enzyme from the phase where it is present, was assayed to purify pepsin from bovine abomasum homogenate. Pepsin was partitioned in favor of the polyethyleneglycol-rich phase in an aqueous two-phase system of polyethyleneglycol 600 and 1450-sodium phosphate; however, a great amount of impure proteins were present. Chitosan (a cationic natural polyelectrolyte) was added to precipitate this acid enzyme as a form of insoluble complex. The addition of this second step increased the purity of the enzyme significantly while the yield was not significantly decreased. The combination of both partition in polyethyleneglycol 1450-phosphate system and chitosan precipitation produced a pepsin recovery of 48.5% with a purification factor of 9.0. The biological activity of the recovered enzyme remained unaltered.Fil: Boeris, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Spelzini, Darío. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Picó, Guillermo Alfredo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Production of alpha-amylase from Aspergillus oryzae for several industrial applications in a single step

A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit® E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation. The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit® E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05 M) in the medium totally dissociates the protein-polymer complexes. When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing.Fil: Porfiri, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Milatich, Esteban J. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Romanini, Diana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin

Potential use of soybean hulls and waste paper as supports in SSF for cellulase production by Aspergillus niger

Cellulase has by vast applications in the biofuel, pulp and paper, detergent and textile industries. The three components of the enzyme complex (endoglucanase, exoglucanase and β-glucosidase) can effectively depolymerize the cellulose chains in lignocellulosic substrate. Solid-state fermentation (SSF) by fungi is a preferable production route for cellulase because of its low cost, among other advantages. This work describes the cellulase production by Aspergillus niger NRRL3 grown on SSF. SSF was carried out on soybean hulls and waste paper as supports. The effect of the support on cellulase production was assessed under a completely randomized factorial design. The support-time interaction was significant for all the variables studied. Both materials were characterized in terms of water absorption index and critical humidity point. Samples of culture were analyzed with scanning electron microscopy (SEM) to study spores and fungal growth. Maximum endoglucanase activity was found at 96 h using soybean hulls as support (5914.29 U L−1), being four times higher than that obtained using waste paper at the same fermentation time. The exoglucanase activity in soybean hulls was maximal at 96 h (4551.19 U L−1), being 9.6 times higher than that obtained in waste paper at the same time. The maximum β-glucosidase activity in soybean hulls (984.01 U L−1) was reached at 96 h, being 1.7 times greater than that obtained in waste paper. Besides, the use of soybean hulls provided high volumetric productivities at shorter times, which may decrease production costs considering a scaled process.Fil: Boggione, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Allasia, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Bassani, Georgina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin

Synthesis and characterization of chitosan membranes functionalized with amino acids and copper for adsorption of endoglucanase

Chitosan membranes were obtained and functionalized with amino acids and copper in order to improve adsorption selectivity of endoglucanase. The membranes were characterized by Fourier-transformed infrared spectroscopy with attenuated total reflectance device (FTIR-ATR) to monitor chemical changes. Scanning electron microscopy (SEM) was performed to compare the surface morphology of the membranes. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were carried out in order to analyze thermal stability of functionalized membranes and to identify the differences between functionalized chitosan membranes before and after endoglucanase adsorption. SEM results proved that functionalization had occurred since the surface of the chitosan membrane was modified. FTIR-ATR results confirmed an effective chemical modification of chitosan membranes with amino acids and copper and corroborated endoglucanase adsorption. The characteristic parameters of DSC and TGA also evidenced endoglucanase adsorption. The use of functionalized membranes as adsorbents increased 40-fold the percentage of endoglucanase adsorption as compared to unmodified membranes. Thus, chitosan membranes functionalized with amino acids and copper may represent a novel, low-cost adsorbent to be used in endoglucanase purification from complex systems.Fil: Boggione, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Mahl, Cynthia R.A.. Universidade Estadual de Campinas; BrasilFil: Beppu, Marisa M.. Universidade Estadual de Campinas; BrasilFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin

Valorization of corn cob for the obtention and purification of endoglucanase produced by SSF

Agro-food by-products contain valuable nutrients which are wasted. In this work, solid-state fermentation (SSF) was carried out using corn cob as substrate for endoglucanase production. The radial growth of three fungal strains -Trichoderma harzianum T104, Aspergillus niger GH1 and Aspergillus niger NRRL3- was analyzed in order to select the most appropriate. Radial growth data were analyzed with a mixed linear model for longitudinal data and no statistically significant differences were found between both A. niger strains. Endoglucanase was separated from the extract of A. niger GH1 by fast protein liquid chromatography (FPLC).The highest endoglucanase activity was detected in fraction number three collected from FPLC corresponding to 72 h of SSF. Seven bands in a range from 24 to 50 kDa, which correspond to endoglucanase from fungal extract, were detected by zymogram analysis. According to protein quantification performed by the ImageJ software, 85% of the proteins present in the samples collected by FPLC corresponded to endoglucanase proteins. The purified endoglucanase retained about 100% of its catalytic activity at 30 °C and 50 °C and was stable in a pH range between 4.00-6.00. These properties make this isolated enzyme suitable for industrial applications such as the saccharification process for bioethanol production.Fil: Boggione, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Allasia, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Matemática y Estadística; ArgentinaFil: Aguilar, Cristóbal Noé. Universidad Autónoma de Coahuila; MéxicoFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin

Synthesis of Polymeric Matrices for Adsorption and Purification of Endoglucanase

Chitosan (CHS), chitosan–Eudragit® EPO (CHS–EPO) and chitosan beads partially cross-linked with glutaraldehyde (CHS–GLUT) were obtained in order to improve the adsorption selectivity of endoglucanase from a fungal culture obtained under SSF by Aspergillus niger using soybean harvest residues as support. The matrices synthetized were characterized in terms of physical and chemical changes. Fourier-transformed infrared spectroscopy with attenuated total reflectance device (FTIR-ATR) was employed to verify the chemical changes on the CHS matrix after the synthesis of CHS–GLUT and CHS–EPO. Scanning electron microscopy (SEM) was performed to compare the surface morphology of the polymeric beads. Two variables, purification factor and yield percentage of the adsorption process, were analyzed using a bifactorial ANOVA considering the matrix–time first order interaction. SEM results exhibited greater surface roughness in the CHS–GLUT and CHS–EPO matrices which may enhance endoglucanase adsorption. FTIR-ATR results confirmed an effective chemical modification of the CHS matrix after crosslinking with GLUT and corroborated the efficiency of the synthesis of the CHS–EPO matrix by the presence of chemical groups of the EPO polymer. An endoglucanase purification factor close to 9 was achieved with the CHS–GLUT matrix and a yield percentage of 60% was obtained with the CHS–EPO matrix. Bifactorial ANOVA results showed the matrix–time interaction to be significant for both variables. The CHS–GLUT matrix with low crosslinking times and the novel CHS–EPO matrix could be included in the bioseparation stage of endoglucanase using a simple and a low-cost method such as batch adsorption.Fil: Boggione, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Zilli, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Allasia, María Belén. Universidad Nacional de Rosario. Facultad de Cs.económicas y Estadística. Escuela de Estadística; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin