Dynamics of yeast immobilized-cell fluidized-bed bioreactors systems in ethanol fermentation from lactose-hydrolyzed whey and whey permeate.

We studied the dynamics of ethanol production on lactose-hydrolyzed whey (LHW) and lactose-hydrolyzed whey permeate (LHWP) in batch fluidized-bed bioreactors using single and co-cultures of immobilized cells of industrial strains of Saccharomyces cerevisiae and non-industrial strains of Kluyveromyces marxianus. Although the co-culture of S. cerevisiae CAT-1 and K. marxianus CCT 4086 produced two- to fourfold the ethanol productivity of single cultures of S. cerevisiae, the single cultures of the K. marxianus CCT 4086 produced the best results in both media (Y EtOH/S = 0.47-0.49 g g(-1) and Q P = 1.39-1.68 g L(-1) h(-1), in LHW and LHWP, respectively). Ethanol production on concentrated LHWP (180 g L(-1)) reached 79.1 g L(-1), with yields of 0.46 g g(-1) for K. marxianus CCT 4086 cultures. Repeated batches of fluidized-bed bioreactor on concentrated LHWP led to increased ethanol productivity, reaching 2.8 g L(-1) h(-1).

Effect of the support size on the properties of ß-galactosidase immobilized on chitosan: advantages and disadvantages of macro and nanoparticles.

The effect of the support size on the properties of enzyme immobilization was investigated by using chitosan macroparticles and nanoparticles. They were prepared by precipitation and ionotropic gelation, respectively, and were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), transmission electron microscopy (TEM), light scattering analysis (LSA), and N(2) adsorption-desorption isotherms. ß-Galactosidase was used as a model enzyme. It was found that the different sizes and porosities of the particles modify the enzymatic load, activity, and thermal stability of the immobilized biocatalysts. The highest activity was shown by the enzyme immobilized on nanoparticles when 204.2 mg protein·(g dry support)(-1) were attached. On the other hand, the same biocatalysts presented lower thermal stability than macroparticles. ß-Galactosidase immobilized on chitosan macro and nanoparticles exhibited excellent operational stability at 37 °C, because it was still able to hydrolyze 83.2 and 75.93% of lactose, respectively, after 50 cycles of reuse.

Pectin lyase immobilization using the glutaraldehyde chemistry increases the enzyme operation range.

Pectin lyase (from Rohapect 10 L) was immobilized on glutaraldehyde supports at low ionic strength at pH 5, 6.5 or 8 and later incubated at pH 8 for 48 h. The activity recovery of the biocatalysts versus pectin was quite low, under 10% for all of the immobilized biocatalyst at 20 °C. However, a high stabilization was found when the enzyme was immobilized at pH 5, (e.g., the immobilized enzyme kept 83% of the activity when the free enzyme was fully inactivated (pH 4.8 and 55 °C in 5 h)). This biocatalyst increased the activity versus pectin in an almost exponential way when temperature increased until reach the maximum temperature used in the study (90 °C), conditions where the free enzyme was almost inactive. The immobilized biocatalyst was also active even at pH 9, where the free enzyme was fully inactive. This biocatalyst could be reused for pectin hydrolysis 5 times for 72 h reaction cycles at 40 °C maintaining more than 90% of the initial activity.

Optimized immobilization of polygalacturonase from Aspergillus niger following different protocols: Improved stability and activity under drastic conditions.

Polygalacturonase (PG) from Aspergillus niger was immobilized using glyoxyl, vinylsulfone or glutaraldehyde-activated supports. The use of supports pre-activated with glutaraldehyde presented the best results. The immobilization of PG on glutaraldehyde-supports was studied under different conditions: at pH 5 for 24 h; at pH 5, 6.5 or 8 for 3 h and then incubated at pH 8 for 24 h; at pH 8 in the presence of 300 mM NaCl for 24 h, to prevent ion exchange. The immobilization under all conditions showed a significant increase in the enzyme thermal stability under inactivation conditions at pH 4-10. As a result, at temperatures over 70 °C or pH values over 7, the immobilized PG maintained significant levels of activity while the free PG was fully inactivated. The immobilization conditions presented a clear effect on enzyme activity, thermostability and operational stability, suggesting that the different conditions permitted to get immobilized PG having different orientations. Varying the immobilization protocol it is possible to achieve high activity or stability, and the optimal biocatalyst depends on the conditions where it will be utilized. The immobilized PG biocatalysts could be reused 10 times without a significant decrease in enzyme activity and offered very linear reaction courses.

Stability/activity features of the main enzyme components of rohapect 10L.

Rohapect 10L is an enzyme cocktail commercialized for juice clarification. Here, we characterized the activity and stability of five enzymatic activities present in this cocktail: total pectinase (PE), polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), and total cellulase (CE) activities. All these enzyme activities have the maximum activity and stability at pH 4, conditions near those found in most fruit juices. However, if the enzymes need to be handled under different conditions (e.g., to immobilize them), their stability becomes extremely low in some cases, just at pH values slightly higher than the optimal one. For example, at pH 10 only CE was reasonably stable at 25°C, while many other enzyme activities were rapidly almost inactivated, even at 4°C. For these cases, different additives were evaluated, and we found that polyethylene glycol was positive or very positive for all enzyme stabilities, allowing keeping reasonable activities after several hours at pH 10 and 25°C. Another additive, that is, dextran, has a small positive effect for PE, PG, and CE, and a very positive effect for PL, albeit significantly destabilizing PME. Thus, the handling and use of this extract requires some care when is performed out of optimal conditions.

Immobilization of pectinase on chitosan-magnetic particles: Influence of particle preparation protocol on enzyme properties for fruit juice clarification.

Magnetic-chitosan particles were prepared following three different protocols enabling the preparation of particles with different sizes - nano (Nano-CMag, Micro (Micro-CMag) and Macro (Macro-CMag) - and used for pectinase immobilization and clarification of grape, apple and orange juices. The particle size had a great effect in the kinetic parameters, Nano-CMag biocatalyst presented the highest Vmax value (78.95 mg. min-1), followed by Micro-CMag and Macro-CMag, with Vmax of 57.20 mg.min-1 and 46.03 mg.min-1, respectively. However, the highest thermal stability was achieved using Macro-CMag, that was 8 and 3-times more stable than Nano-CMag and Micro-CMag biocatalysts, respectively. Pectinase immobilized on Macro-CMag kept 85% of its initial activity after 25 batch cycles in orange juice clarification. These results suggested that the chitosan magnetic biocatalysts presented great potential application as clarifying catalysts for the fruit juice industry and the great importance of the chitosan particles preparation on the final biocatalyst properties.

Efficient enzyme-assisted extraction of genipin from genipap (Genipa americana L.) and its application as a crosslinker for chitosan gels.

Enzyme-assisted extraction in liquid-liquid two-phase aqueous system was applied for the first time in order to extract genipin from genipap. The effect of different commercial enzymes, their concentrations, and extraction parameters were investigated. Moreover, chitosan gels were prepared, crosslinked with glutaraldehyde or genipin and characterized by their textural and rheological properties. The crosslinked chitosan was used as support for the immobilization of model ß-galactosidases. Among the different commercial enzymes tested for extraction, Celluclast 10% (36 °C and pH 3.7) provided an extraction of 196 mg.g-1 of genipin. Chitosan gels crosslinked with genipin 0.5% showed better textural and similar rheological properties when compared to the chitosan crosslinked with glutaraldehyde 3%. The percentage of lactose hydrolysis by the immobilized K. lactis ß-galactosidase using genipin as a crosslinker was 87%. Thus, the genipin obtained in this work proved to be an excellent alternative to the use of glutaraldehyde in chitosan crosslinking applications.

Magnetic biocatalysts of pectinase and cellulase: Synthesis and characterization of two preparations for application in grape juice clarification.

In the present study, we prepared two different magnetic biocatalysts of pectinase and cellulase: carrier-free magnetic CLEAs (CLEA-MP*) and immobilization on glutaraldehyde-activated magnetite (Enz-Glu-MP*). The biocatalysts were compared to their magnetic properties, immobilization parameters, stability and grape juice clarification. Enz-Glu-MP* presented higher magnetic properties than CLEA-MP*, whereas this presented higher surface area and pore volume. The KM of the enzyme immobilized on Enz-Glu-MP* was 25.65mM, lower in comparison to the CLEA-MP* (33.83mM). On the other hand, CLEA-MP* was the most active and stable biocatalyst, presenting higher recovered activity (33.4% of cellulase), higher thermal stability (2.39 stabilization factor) and improved reusability (8cycles). The integration of magnetic technology with enzymatic immobilization emerges as a possibility to increase the recover and reuse of biocatalysts for application in juice technology.

Chitosan crosslinked with genipin as support matrix for application in food process: Support characterization and ß-D-galactosidase immobilization.

In order to develop safer processes for the food industry, we prepared a chitosan support with the naturally occurring crosslinking reagent, genipin, for enzyme. As application model, it was tested for the immobilization of ß-D-galactosidase from Aspergillus oryzae. Chitosan particles were obtained by precipitation followed by adsorption of the enzyme and crosslinking with genipin. The particles were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The immobilization of the enzyme by crosslinking with genipin provided biocatalysts with satisfactory activity retention and thermal stability, comparable with the ones obtained with the traditional methodology of immobilization using glutaraldehyde. ß-D-Galactosidase-chitosan-genipin particles were applied to galactooligosaccharides synthesis, evaluating the initial lactose concentration, pH and temperature, and yields of 30% were achieved. Moreover, excellent operational stability was obtained, since the immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. Thus, the food grade chitosan-genipin particles seem to be a good alternative for application in food process.

Fructooligosaccharides synthesis by highly stable immobilized ß-fructofuranosidase from Aspergillus aculeatus.

The enzymatic synthesis of fructooligosaccharides (FOS) was carried out using a partially purified ß-fructofuranosidase from the commercial enzyme preparation Viscozyme L. Partial purification of ß-fructofuranosidase from Viscozyme L was done by batch adsorption using ion-exchange resin DEAE-Sepharose, showing a 6-fold increase in specific activity. The biocatalyst was then covalently immobilized on glutaraldehyde-activated chitosan particles. Thermal stability of the biocatalyst was evaluated at 50 °C and 60 °C, being around 100 times higher at 60 °C when compared to the free enzyme. The immobilized biocatalyst was reused 50 times for FOS production (100 min per batch at 50 °C and pH 5.5) without significant loss of activity. The average yield (grams of FOS per grams of initial sucrose) was 55%. The immobilization process combined with partial purification method resulted in a derivative with activity of 1230 Ut/g, which is among the best for FOS production.

High operational stability of invertase from Saccharomyces cerevisiae immobilized on chitosan nanoparticles.

Invertase (E.C.3.2.1.26) from Saccharomyces cerevisiae was covalently immobilized on chitosan nanoparticles prepared by the ionotropic gelation method and activated with glutaraldehyde. The support was characterized and it was studied its load capacity, the influence of the presence of substrate during immobilization, and determined the biocatalyst kinetic parameters and stabilities. The light scattering analysis (LSA) and transmission electron microscopy (TEM) techniques indicated a mixture of chitosan nano and aggregated nanoparticles, providing high superficial area for enzyme immobilization. The thermal and storage stabilities, the optimal pH and temperature of the enzyme were not altered. K(m) increased 3-fold, while V(max) remained unaltered. The immobilized biocatalyst was reused for 59 batches with maximal invertase activity, the highest operational stability so far described in the literature. These results fulfill some important aspects for the enzyme immobilization: the simplicity of the protocols, the conservation of the enzyme activity, and the high operational stability.

High stability of immobilized ß-D-galactosidase for lactose hydrolysis and galactooligosaccharides synthesis.

ß-D-Galactosidase from Kluyveromyces lactis was immobilized on glutaraldehyde-activated chitosan and used in a packed-bed reactor for the continuous hydrolysis of lactose and the synthesis of galactooligosaccharides (GOS). The biocatalyst was tested for its optima pH and temperature, thermal stability in the presence of substrate and products, and operational stability. Immobilization increased the range of operational pH and temperature, and the enzyme thermal stability was sharply increased in the presence of lactose. Almost complete lactose hydrolysis was achieved for both milk whey and lactose solution at 37 °C at flow rates up to 2.6 mL min(-1). Maximal GOS concentration of 26 g L(-1) was obtained at a flow rate of 3.1 mL min(-1), with a productivity of 186 g L(-1) h(-1). Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.