Design and characterization of immobilized biocatalyst with lipase activity onto magnetic magnesium spinel nanoparticles: A novel platform for biocatalysis.

Lipases (EC 3.1.1.3) are very used industrial enzymes but presents drawbacks such as lack of stability, and poor recyclability. Most of these obstacles can be solved by lipase immobilization. The objective of this work was evaluated to magnetic magnesium spinel nanoparticles as support for lipase immobilization by covalent bound. The techniques used for nanoparticles synthesis presented advantages in the size selection of the nanoparticles obtained (60-100 nm). The immobilization of Candida rugosa lipase (CRL) was optimized. The optimal conditions were determined to be pH 3.7, enzyme concentration of 1.1 mg/mL at 4 °C and an ionic strength of 100 mM. The CRL@MgFe2O4 activity obtained was 3.2 times over the starting conditions (4.03 U/mL). The immobilization of the lipase on Fe3O4 was evaluated and compared. The activity of the CRL@MgFe2O4 was 61% higher than CRL@Fe3O4 and 22% higher than free enzyme. CRL@MgFe2O4 improved the lipase stability at alkaline pH, hydrophilic solvent and high temperatures. The thermogravimetric analysis showed that this new biocatalyst was more stable compared to the free enzyme. Additionally, the immobilized lipase was recycled by magnetic force and used in ten catalysis cycles. The performance of the recycle was improved using butanol or Triton X 100 during washing. Finally, CRL@FeMg2O4 showed hydrolysis and synthesis activity. Thus, CRL@FeMg2O4 as a novel biocatalyst generation presents interesting properties for industrial applications.

Designing cross-linked xylanase aggregates for bioconversion of agroindustrial waste biomass towards potential production of nutraceuticals.

Immobilized biocatalysts design has the potential to efficiently produce valuable bioproducts from lignocellulosic biomass. Among them, the carrier-free immobilization through the cross-linked enzyme aggregates technology is a simple and low-cost alternative. A two steps statistical approach was utilized to evaluate the synthesis of a cross-linked enzyme aggregate from a xylanolytic preparation, which was produced by Cohnella sp. AR92 grown in a peptone-based culture medium. The resulting immobilized biocatalyst, Xyl-CLEA, was significate more stable (25 to 45%) towards temperatures up to 50°C with respect to the free enzyme, and retained over 50% of its initial activity after 5 consecutive cycles of reuse. By means of infrared spectroscopy and electron microscopy, the Xyl-CLEA showed architectural features described as signature of type I and type II of protein aggregates. These, were the result of the simultaneous aggregation of a multiplicity of proteins from the crude enzymatic extract. The enzymatic activity was assessed using alkali pretreated sugar cane bagasse as substrate. Whereas the free enzymatic preparation released xylose as the main product, the immobilized xylanase produced xylooligosaccharides, thus showing that the immobilization procedure modified the potential application of the extracellular xylanase from Conhella sp. AR92.

Two amplicon sequencing strategies revealed different facets of the prokaryotic community associated with the anaerobic treatment of vinasses from ethanol distilleries.

The prokaryotic consortium from a pilot-scale UASB reactor fed with vinasses from ethanol distilleries was evaluated by means of amplicon sequencing of the 16S rRNA gene. Two different sets of primers targeted to overlapping regions of the V4-16S region were used to gain a broad picture of such community and to perform a comparative analysis. From the two datasets obtained, prevalent phyla were Firmicutes, Verrucomicrobia and Thermotogae. Interestingly, one set of primers captured variability in both the bacterial and archaeal portions of the community, whilst the other one revealed a more diverse community structure, but only in the Bacteria domain. Although a certain level of agreement between the two strategies was observed, sharp differences indicate that different facets of the community were disclosed by each approach.

pH control of the production of recombinant glucose oxidase in Aspergillus nidulans.

AIMS: Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. METHODS AND RESULTS: Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg(-1) protein and a maximum volumetric productivity of 29.9 U l(-1) h(-1) were obtained at pH 5.5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. CONCLUSIONS: Specific GOX activity obtained at pH 5.5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies.

Fluidized bed design parameters affecting novel lactic Acid downstream processing.

Lactic acid purification was directly done from fermentation utilizing a fluidized bed column refilled with a strong anionic exchange resin. The purpose of this work was to study the influence of two important design parameters, bed-diameter (D) and bed-height (H), in the lactic acid binding and elution capacity of the matrix. By changing the settled bed height from 2.5 to 5 cm for each diameter of column analyzed it was possible to obtain an 50% increase in the binding capacity of the resin in all experiments. This fact was attributed to a higher contact time between the culture broth and the anionic resin produced by the increase of back mixing and lactic acid residence time.

Modeling of direct recovery of lactic acid from whole broths by ion exchange adsorption.

Lactic acid fermentation process with L. casei CRL 686 was performed. The static adsorption isotherm over a strong anionic exchange resin, Amberlite IRA-400 was measured, and the static binding capacity parameters were quantified. Early recovery of lactic acid from this lactate producer from unclarified culture broth was performed in a liquid solid fluidized bed, with the resin as the solid adsorbent, and the dynamic adsorption capacity was calculated. Good agreement was found between static and dynamic binding capacity values. The fluidized bed height was twice the settled bed height and the overall process was controlled by the liquid solid mass transfer. This operation was also simulated by continuously well stirred tanks arranged in series and superficial solid deactivation as in a gas solid catalytic reactor. The deactivation process takes into account liquid channeling and agglomerations of solid induced by the viscosity of the broth and also by the cells during the adsorption. These patterns were also verified by experimental observations, and are in agreement with the results found in the literature. The breakthrough data together with others from previous works were satisfactorily fitted until the 90% dimensionless concentration was reached for both culture broths. The model could be used in future studies on predictions about the liquid solid fluidized bed behavior and other different operating conditions.

Rapid screening of textile dyes employed as affinity ligands to purify enzymes from yeast.

A rapid method for screening potential dye ligands for use in affinity chromatography is described. Textile dyes were non-covalently coupled to a cross-linked polysaccharide Sepharose(R) matrix. Yeast alcohol dehydrogenase (ADH) was used as the model protein for evaluating the screening system. A homogenate from baker's yeast was used as the crude source of enzyme. Batchwise adsorption and elution were used to evaluate the individual dyes. The influence of pH and ionic strength in the binding and elution steps was evaluated. Batch isotherms were used to evaluate parameter characteristics. Experimental data obtained were fitted to Langmuir isotherms to determine the maximum binding capacity and the dissociation constant for each dye evaluated in this study. A dynamic binding capacity of 107.6 units of ADH/g of resin was determined for Procion Turquoise MXG dye by frontal analysis. Specific elution with NAD+ and non-specific elution with 50 mM Tris/HCl buffer, pH 8.5, were tested when Procion Turquoise MXG was used, giving purification factors of 53.5 and 4.4 respectively. This screening technique is inexpensive and can be performed in a few hours. It was possible to predict the performance of different reactive dyes in this way, and the influence of pH and salt on the binding behaviour was demonstrated.

Determination of radial growth rate of colonies of Sclerotium rolfsii F-6656 for the evaluation of culture medium, optimum incubation temperature, osmo- and halotolerance.

The measurement of the colony radial growth rate (Kr) on solid medium of colonies of Sclerotium rolfsii Proimi F-6656 for the evaluation of scleroglucan production medium and other different media, incubation temperature and tolerance to diverse concentrations of sucrose and NaCl were studied. The optimum growth temperature observed was 30 degrees C. The Kr value reached on the Production Medium used (0.66 mm.h-1) showed no differences compared with those of the other media tested, indicating that all the requirements for growth were provided. Poor growth was only observed on Soil Extract Agar. The fungus tolerated concentrations of sucrose from 0.15 to 1.17 M, on both Czapek and production medium. Growth was limited by the highest concentrations of sucrose tested (0.88 and 1.17 M), as indicated by a slower increase in colony size. Addition of 0.86 M NaCl to the production medium and YM agar did not inhibit growth completely, but decreased the radial growth rate considerably (80 and 70% respectively).

Epitope contiguous chains and antibody recognition in HIV-1 synthetic peptide antigens.

Five peptides derived from human immuno deficiency virus (HIV-1) gp41 transmembrane protein have been synthesized: M9 (610-618), M12 (598-609), M15 (600-614), M21 (584-604) and M23 (587-609). These sequences partially overlap in the region vicinal to the immunodominant epitope CSGKLIC, between two cysteine residues 603-609 and three of them (M12, M15 and M23) include this complete heptapeptide. M23, the longer peptide, includes an hydrophilic chain in addition to the heptapeptide loop. The purpose of this work was to determine the influence of contiguous chains to the heptapeptide loop on antibody recognition in fluid and solid phases, and dissociation constants (KD) of each sequence with human anti-HIV-1 antibodies. Two peptides, M13 and M23, overlapped on this loop, were found to be more reactive. Antigen-antibody dissociation constants were determined for both peptides by competition enzyme-linked immunosorbent assay, using each peptide alternatively as the solid phase-immobilized antigen. In addition to the influence of solid-phase antigen on calculated dissociation constants (a phenomenon described by Seligman, 1994), the inhibitory effect of M15 in liquid phase on antibody binding to solid phase M23 was higher than exerted by M23 in solution over antibody binding to M15 on solid phase. On the basis of peptide sequence and predicted antigenicity, this behavior appeared to be contradictory. It is assured that the possible origin of this phenomenon is due to unfavorable conformation of the longer peptide. Even though synthetic peptides mimic mainly sequential epitopes, conformational preferences in fluid or solid phase play an important role in epitope functionality. In particular, addition of residues to known immunodominant sequences may not always amplify antibody recognition if conformation provokes steric hindrance in the native epitope.

Bacterial protein production from corn cob pre-treated with NaOH at room temperature.

Corn cobs were treated at room temperature with NaOH at a ratio of 100:3 (w/w), but with different volumes of water from 3 to 0.25 ml/g corn cob. The biomasses obtained from a mixture culture of Cellulomonas sp. and Bacillus subtilis under each condition were similar (5.5 to 6.0 g/l) and protein only varied between 30% and 40% (w/w biomass) or 1.9 and 2.2 g/l. Low volumes and low amounts of NaOH can therefore be used in a cost-effective manner.