Production of new nanobiocatalysts via immobilization of lipase B from C. antarctica on polyurethane nanosupports for application on food and pharmaceutical industries.

Nanobiocatalysts were produced via immobilization of CalB lipase on polyurethane (PU) based nanoparticles and their application on the synthesis of important industrial products was evaluated. Nanoparticles of polyurethane functionalized with poly(ethylene glycol) (PU-PEG) were synthetized through miniemulsion polymerization and the addition of crosslinking agents were evaluated. The nanoparticles were employed as support for CalB and the kinetic parameters were reported. The performance of new biocatalysts was evaluated on the hydrolysis reaction of p-NPB and on the enantioselective hydrolysis of (R,S)-mandelic acid. The esterification reaction was evaluated on the production of ethyl esters of Omega-3. The effect of poly(ethylene glycol) molar mass (400, 4000 or 6000 Da)on the biocatalyst activity was also analyzed. The PU-PEG6000-CalB showed the highest value of the kinetic parameters, highlighting the high reaction rate. The addition of trehalose as crosslinking agent improved the thermal stability of the biocatalysts. PU-PEG400-CalB was the most active nanobiocatalyst, exhibiting a ethyl esters production of 43.72 and 16.83 mM.U -1 using EPA and DHA, respectively. The nanobiocatalyst was also applied in enantiomeric resolution of mandelic acid, showing promising enantiomeric ratios. The results obtained in this work present alternative and sustainable routes for the synthesis of important compounds used on food and pharmaceutical industries.

Amino Acid Supplementation Improves the Production of Extracellular Peptidases by Aspergillus Section Flavi and their Ionic Immobilization

Abstract Bioprocess studies have been highlighted due to the importance of physiological processes and industrial applications of enzymes. The potential of peptidase production from Aspergillus section Flavi using different amino acids as a supplemental nitrogen source was investigated. A production profile revealed that amino acids had positive effects on peptidase production when compared to the control without amino acids. Optimal production (100 U/mL) was obtained with Arginine amino acid in 96 h of fermentation. Extracellular peptidase from Aspergillus section Flavi was identified in submerged bioprocesses by in situ activity. Biochemical studies revealed that the maximum activities of the enzyme extract were obtained at pH 6.5 and a temperature of 55°C. The inhibition by EDTA and PMSF suggests the presence of more than one peptidase while the Ni2+ and Cu2+ had a negative influence on the enzyme activity. When the crude extract was reversibly immobilized on ionic supports, DEAE-Agarose and MANAE-Agarose the derivative showed different profiles of thermal and pH stabilities. Hence, this study revealed the basic properties and biochemical characteristics that allowed the production improvement of this class of enzyme. Moreover, with known properties stabilization and immobilization process is required to further explore its biotechnological capacities.

Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support.

This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and Buriti oils by the derivatives was done in a biphasic (organic/aqueous) system, and the products were quantified in RP-HPLC. The results showed the potential of these immobilized lipases to obtain omegas-6 and 9 from Brazilian natural oils. This work may improve the enzymatic methodologies for obtaining foods and drugs enriched with fatty acids.

Bio-electro oxidation of indigo carmine by using microporous activated carbon fiber felt as anode and bioreactor support.

The bioremediation and electro-oxidation (EO) processes are included among the most promising cleaning and decontamination mechanisms of water. The efficiency of bioremediation is dictated by the biological actuator for a specific substrate, its suitable immobilization and all involved biochemical concepts. The EO performance is defined by the anode efficiency to perform the complete mineralization of target compounds and is highlighted by the low or null use of reagent. Recently, the combination of both technologies has been proposed. Thus, the development of high efficient, low cost and eco-friendly anodes for sustainable EO, as well as, supporting devices for immobilization of biological systems applied in bioremediation is an open field of research. Therefore, the aim of this work was to promote the bio-electrochemical remediation of indigo carmine dye (widely common in textile industry), using new anode based on a microporous activated carbon fiber felt (ACFF) and ACFF with immobilized Laccase (Lcc) from Pycnoporus sanguineus. The results were discolorations of 62.7% with ACFF anode and 83.60% with ACFF-MANAE-Lcc anode, both for 60 min in tap water. This remediation rates show that this new anode has low cost and efficiency in the degradation of indigo dye and can be applied for other organic pollutant.

Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii.

ß-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor ß-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70-75 °C, with higher stability in pH 3.0-4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb+2 and citrate and total with Cu+2, Hg+2, and Co+2. Partially purified BXYL II and BXYL I (the main ß-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-ß-D-xylopyranoside hydrolysis.

Optimization of the production and characterization of lipase from Candida rugosa and Geotrichum candidum in soybean molasses by submerged fermentation.

This present work describes the production and biochemical characterization of lipase by Candida rugosa and Geotrichum candidum in a culture supplemented with soybean molasses. After optimizing the fermentation times for both microorganisms, the effects of changing the soybean molasses concentration, the fermentative medium pH and the fermentation temperature were evaluated using the Central Composite Planning. When soybean molasses was used at a concentration of 200 g/L at 27 ± 1 °C and pH 3.5, the lipolytic activity measured in the broth was 12.3 U/mL after 12 h for C. rugosa and 11.48 U/mL after 24 h for G. candidum. The molecular masses were 38.3 kDa to G. candidum lipase and 59.7 kDa to C. rugosa lipase, determined by SDS-PAGE. The lipase from both microorganisms exhibited maximal hydrolytic activity at a temperature of 40 °C and were inhibited at pH 10.0. Using different concentration of p-nitrophenylbutyrate (p-NPB), the kinetic parameters were calculated, as follows: the Km of lipase from G. candidum was 465.44 µM and the Vmax 0.384 µmol/min; the Km and Vmax of lipase from C. rugosa were 129.21 µM and 0.034 µmol/min, respectively. Lipases activity were increased by metallic ions Mg(2+) and Na(+) and inhibited by metallic ion Cu(3+).

Characterization of a tannase from Emericella nidulans immobilized on ionic and covalent supports for propyl gallate synthesis.

The extracellular tannase from Emericela nidulans was immobilized on different ionic and covalent supports. The derivatives obtained using DEAE-Sepharose and Q-Sepharose were thermally stable from 60 to 75 °C, with a half life (t50) >24 h at 80 °C at pH 5.0. The glyoxyl-agarose and amino-glyoxyl derivatives showed a thermal stability which was lower than that observed for ionic supports. However, when the stability to pH was considered, the derivatives obtained from covalent supports were more stable than those obtained from ionic supports. DEAE-Sepharose and Q-Sepharose derivatives as well as the free enzyme were stable in 30 and 50 % (v/v) 1-propanol. The CNBr-agarose derivative catalyzed complete tannic acid hydrolysis, whereas the Q-Sepharose derivative catalyzed the transesterification reaction to produce propyl gallate (88 % recovery), which is an important antioxidant.