Magnetic core-shell cellulose system for the oriented immobilization of a recombinant ß-galactosidase with a protein tag.

The objective of this study was to immobilize a recombinant ß-galactosidase (Gal) tagged with a cellulose-binding domain (CBD) onto a magnetic core-shell (CS) cellulose system. After 30 min of reaction, 4 U/capsule were immobilized (CS@Gal), resulting in levels of yield and efficiency exceeding 80 %. The optimal temperature for ß-galactosidase-CBD activity increased from 40 to 50 °C following oriented immobilization. The inhibitory effect of galactose decreased in the enzyme reactions catalyzed by CS@Gal, and Mg2+ increased the immobilized enzyme activity by 40 % in the magnetic CS cellulose system. The relative enzyme activity of the CS@Gal was 20 % higher than that of the soluble enzyme activity after 20 min at 50 °C. The CS support and CS@Gal capsules exhibited an average size of 8 ± 1 mm, with the structure of the shell (alginate-pectin-cellulose) enveloping and isolating the magnetic core. The immobilized ß-galactosidase-CBD within the magnetic CS cellulose system retained ∼80 % of its capacity to hydrolyze lactose from skim milk after 10 reuse cycles. This study unveils a novel and promising support for the oriented immobilization of recombinant ß-galactosidase using a magnetic CS system and a CBD tag. This support facilitates ß-galactosidase reuse and efficient separation, consequently enhancing the catalytic properties of the enzyme.

One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support: Rapid immobilization and high thermal stability.

For the first time, this work reported the one-step purification and targeted immobilization process of a ß-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after ß-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between ß-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. ß-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of ß-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using ß-galactosidase as a model.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Designing a Support for Lipase Immobilization Based On Magnetic, Hydrophobic, and Mesoporous Silica.

A mesoporous, magnetic, and hydrophobic material was designed step by step to act as a support for lipase immobilization. Its pore size (8.0 nm) is compatible with the size of lipase from Thermomyces lanuginosus (TLL), and its hydrophobic surface (contact angle of a water drop = 125°) was planned to interact with lipase on its interfacially activated form (open conformation). The presence of magnetite particles provides magnetic retrieval of the material and enables recyclability of the biocatalysts. Regarding immobilization parameters, the hydrophobic support was tested in comparison to the unmodified hydrophilic support in phosphate buffer solution (50 mmol L-1, pH 7.5) at 25 °C. Hydrophobicity was found to be critical for the amount of immobilized TLL (immobilization yield of 97% versus 36% for the hydrophilic support), whereas the hydrophilic support favors the native conformational state and substrate access to the enzyme's catalytic site (specific activity of 5.7 versus 4.7 U g-1 for the hydrophobic support, even when it has higher TLL content). Therefore, the hydrophobic support immobilizes higher amounts of TLL and the hydrophilic support keeps the enzyme hyperactivated. Last, due to the stronger interactions of TLL with hydrophobic surfaces, the hydrophobic support offers better preservation of enzyme activity in repeated cycles (76% of activity retained after three cycles versus 50% for the hydrophilic support).

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.

Immobilization of ß-Galactosidases on Magnetic Nanocellulose: Textural, Morphological, Magnetic, and Catalytic Properties.

We describe a process for obtaining nanocrystalline cellulose (NC) by either acidic (H-NC) or alkaline treatment (OH-NC) of microcrystalline cellulose, which was subsequently bonded to magnetic nanoparticles (H-NC-MNP and OH-NC-MNP) and used as support for the immobilization of Aspergillus oryzae (H-NC-MNP-Ao and OH-NC-MNP-Ao) and Kluyveromyces lactis (H-NC-MNP-Kl and OH-NC-MNP-Kl) ß-galactosidases. The mean size of magnetic nanocellulose particles was approximately 75 nm. All derivatives reached saturation magnetizations of 7-18 emu/g, with a coercivity of approximately 4 kOe. Derivatives could be applied in batch hydrolysis of lactose either in permeate or in cheese whey for 30× and it reached hydrolysis higher than 50%. Furthermore, using a continuous process in a column packed-bed reactor, the derivative OH-NC-MNP-Ao had capacity to hydrolyze over 50% of the lactose present in milk or whey after 24 h of reaction. Fungal ß-galactosidases immobilized on magnetic nanocellulose can be applied in lactose hydrolysis using batch or continuous processes.

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.

Magnesium surface enrichment of CoFe2O4 magnetic nanoparticles immobilized with gold: reusable catalysts for green oxidation of benzyl alcohol.

Gold nanoparticles have shown excellent activity for selective oxidation of alcohols; such catalytic systems are highly dependent on the initial activation of the substrates, which must occur on the catalyst surface in heterogeneous catalysts. In many cases, an extra base addition is required, although the basicity of the support may also be of significant importance. Here, we explored the intrinsic basicity of magnesium-based enrichments on CoFe2O4 magnetic nanoparticles for the oxidation of benzyl alcohol using molecular oxygen as oxidant. The MgO and Mg(OH)2 enrichments enabled gold impregnation, which was not possible on the bare CoFe2O4 nanoparticles. The Au/MgO/CoFe2O4 and Au/Mg(OH)2/CoFe2O4 catalysts reached 42% and 18% conversion, respectively without base promotion, in 2.5 hour and 2 bar of O2. When the catalysts were tested with sub-stoichiometric amounts of base, they became more active (>70% of conversion) and stable in successive recycling experiments without metal leaching, under the same reaction conditions. We also showed the oxide phases of the enrichments performed using Rietveld refinements and how the Mg(OH)2 phase interferes with the activity of MgO-based materials.