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).

Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives.

Glycoside hydrolases (GH) are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes ß-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented.

Genipin crosslinked porous chitosan beads as robust supports for ß-galactosidase immobilization: Characterization, stability, and bioprocessing potential.

This study aimed to modify the porosity of chitosan beads using Na2CO3 as a porogen agent and to crosslink them with genipin for the immobilization of ß-galactosidase from Aspergillus oryzae. Immobilization was performed under four different pH conditions (4.5, 6.0, 7.5, and 9.0), resulting in biocatalysts named B4, B6, B7, and B9, respectively. The immobilized enzymes were characterized for immobilization parameters and stability, including thermal, pH, storage, and operational stability. The optimal conditions for the support were determined as 50 mM Na2CO3. The biocatalyst exhibited nearly 100 % retention of initial activity after 5 h of incubation at different pH conditions and showed improved thermal stability compared to the free enzyme across all pH conditions. After 50 cycles of lactose hydrolysis, all biocatalysts retained at least 71 % of their initial activity, with B6 retaining nearly 100 %. Scanning electron microscopy revealed structural modifications, particularly in B4, leading to weakened support structure after reuse. Continuous lactose hydrolysis showed increased productivity from 41.3 to 48.1 g L-1 h-1 for B6, with 78.1 % retention of initial capacity. All biocatalysts retained >95 % activity when stored at 4 °C for 20 weeks, highlighting their suitability for enzyme immobilization in continuous and discontinuous bioprocesses.

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.

Antimicrobial membrane cellulose acetate containing ionic liquid and metal nanoparticles.

Stable metallic Au(0), Ag(0) and Pt(0) nanoparticle-containing membrane films (20 microm thickness) were obtained by combining irregularly shaped nanoparticles of monomodal size distributions (11 +/- 1.5 nm Au(0), 8.9 +/- 2.1 nm Ag(0) and 2.8 +/- 0.4 nm Pt(0)) in the ionic liquid (IL) 1-n-butyl-3-methylimidazolium bis(trifluoromethane sulfonyl)imide (BMI x (NTf)2) with a syrup of cellulose acetate (CA) in acetone. The presence of small and stable Au(0), Ag(0) or Pt(0) nanoparticles induced an augmentation in the CA/IL film surface areas. The addition of the IL to the membrane resulted in an increase of its elasticity and a decrease in its tenacity and toughness, whereas its stress at break was not influenced. High antimicrobial activity was observed in membranes containing Au(0), Ag(0) and Pt(0) metal concentrations as low as 1 mg of metal per 5 g of CA. The CA/IL/nanoparticle combination enhanced the activity and durability of the metal nanoparticles and provided greater antimicrobial activity against E. coli and S. aureus bacteria.

Purification and characterization of an extracellular beta-glucosidase from Monascus purpureus.

An extracellular beta-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A 22 central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at 50 degrees and pH 5.5. The beta-glucosidase showed moderate thermostability, was inhibited by HgCl2, K2CrO4, and K2Cr2O7, whereas other reagents including beta- mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-beta-D-glucopyranoside, and maltose indicates that the beta-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-beta-Dcellobiose. beta-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

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.

Development of active biofilms of quinoa (Chenopodium quinoa W.) starch containing gold nanoparticles and evaluation of antimicrobial activity.

Active biofilms of quinoa (Chenopodium quinoa, W.) starch were prepared by incorporating gold nanoparticles stabilised by an ionic silsesquioxane that contains the 1,4-diazoniabicyclo[2.2.2]octane chloride group. The biofilms were characterised and their antimicrobial activity was evaluated against Escherichiacoli and Staphylococcusaureus. The presence of gold nanoparticles produces an improvement in the mechanical, optical and morphological properties, maintaining the thermal and barrier properties unchanged when compared to the standard biofilm. The active biofilms exhibited strong antibacterial activity against food-borne pathogens with inhibition percentages of 99% against E. coli and 98% against S. aureus. These quinoa starch biofilms containing gold nanoparticles are very promising to be used as active food packaging for the maintenance of food safety and extension of the shelf life of packaged foods.

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.

Characterization and antioxidant potential of Brazilian fruits from the Myrtaceae family.

The objective of this study is to evaluating the Brazilian biodiversity through physicochemical characterization and determination of antioxidant potential of three species from the Myrtaceae family, namely yellow guava (Psidium cattleyanum Sabine), guabiroba (Campomanesia xanthocarpa O. Berg), and uvaia ( Eugenia pyriformis Cambess). Guabiroba had the greater quantity of phenolic compounds (9033 mg chlorogenic acid/100 g) and vitamin C (30.58 mg/g) and showed the best TSS/TTA (total soluble solid/total titratable acid) ratio (45.12). For the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic) method, the guabiroba (507.49 µM Trolox/g) presented the highest antioxidant potential; however, in the DPPH (2,2-diphenyl-1-picrylhydrazyl) method, uvaia (170.26 g/g DPPH) and guabiroba (161.29 g/g DPPH) were not statistically different. The uvaia outranked the other fruits with respect to its high carotenoid (909.33 µg/g) and vitamin A (37.83 µg/g) contents, and the yellow guava, although showing a lower bioactive compound content and antioxidant activity, nevertheless presented much higher values than many traditionally consumed fruits.

Lipoamide dehydrogenase from Corynebacterium glutamicum: molecular and physiological analysis of the lpd gene and characterization of the enzyme.

Lipoamide dehydrogenase (LPD) is an essential component of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, both playing a crucial role within the central metabolism of aerobic organisms. Using oligonucleotides designed according to conserved regions of LPD amino acid sequences from several organisms, the lpd gene from Corynebacterium glutamicum was identified and subsequently subcloned. The cloned lpd gene expressed in C. glutamicum cells harbouring the gene on a plasmid showed a 12-fold higher specific LPD activity when compared to the wild-type strain. DNA sequence analysis of a 4524 bp segment containing the lpd gene and adjacent regions revealed that the lpd gene is not flanked by genes encoding other subunits of the pyruvate or 2-oxoglutarate dehydrogenase complexes and predicted an LPD polypeptide of 469 amino acids with an M(r) of 50619. The amino acid sequence of this polypeptide shows between 26 and 58% identity when compared to LPD enzymes from other organisms. Transcriptional analyses revealed that the lpd gene from C. glutamicum is monocistronic (1.45 kb mRNA) and that its transcription is initiated exactly at the nucleotide defined as the translational start. LPD was purified and biochemically characterized. This analysis revealed that the enzyme catalyses the reversible reoxidation of dihydrolipoic acid and NADH:NAD(+) transhydrogenation, and is able to transfer electrons from NADH to various redox-active compounds and quinones. An in vivo participation of C. glutamicum LPD in facilitation of quinone redox cycling is proposed.

Cellulase and xylanase productions by isolated Amazon Bacillus strains using soybean industrial residue based solid-state cultivation

In Brazil, a large amount of a fibrous residue is generated as result of soybean (Glycine max) protein production. This material, which is rich in hemicellulose and cellulose, can be used in solid state cultivations for the production of valuable metabolites and enzymes. In this work, we studied the bioconversion of this residue by bacteria strains isolated from water and soil collected in the Amazon region. Five strains among 87 isolated bacteria selected for their ability to produce either celullases or xylanases were cultivated on the aforementioned residue. From strain BL62, identified as Bacillus subtilis, it was obtained a preparation showing the highest specific cellulase activity, 1.08 UI/mg protein within 24 hours of growth. Concerning xylanase, the isolate BL53, also identified as Bacillus subtilis, showed the highest specific activity for this enzyme, 5.19 UI/mg protein within 72 hours of cultivation. It has also been observed the production of proteases that were associated with the loss of cellulase and xylanase activities. These results indicated that the selected microorganisms, and the cultivation process, have great biotechnological potential