Magnetic Cross-linked Enzyme Aggregates Research Articles

A comparison of dual-enzyme immobilization by magnetic nanoparticles and magnetic enzyme aggregates for cascade enzyme reactions

Immobilized enzymes have been widely used for their efficiency, economy, and environment friendly. In this case, a simpler and effective immobilization method has been developed. In this study, the enzyme immobilized by magnetic beads with genipin (MGE) was prepared by mixing genipin-modified nano-Fe3O4 with (R)− 1-phenylethanol dehydrogenase ((R)-PEDH) and recombinant glucose dehydrogenase (rGDH), and the magnetic cross-linked enzyme aggregates (MCLEAs) was prepared by stirring a mixture of the sulfur precipitate of the (R)-PEDH and rGDH, poly-L-lysine, and cross-linkers. When o-phthalaldehyde (o-PA) was used as a cross-linking agent for the MCLEAs preparation, a protein loading of 99.9% and 62.93% recovery activity of (R)-PEDH and 70.12% of rGDH were obtained. Compared to the MGE, the MCLEAs possessed better enzyme activity recovery. Furthermore, this study tried the synthesis of (R)− 1-(furan-2-yl) ethanol by the immobilized enzymes and obtained a concentration of 1.10 mM for the MCLEAs and 0.24 mM for the MGE at 48 h. This study explored a facile method for co-immobilization of (R)-PEDH and rGDH to synthesize (R)− 1-(furan-2-yl) ethanol.

Magnetic Crosslinked Porcine Pancreatic Lipase Aggregates for Transesterification Process

Lipases have been used in industrial processes as biocatalysts for transesterification reactions. The synergism between enzymes and magnetic properties may be reached by using magnetic nanoparticles (MNPs) as support to immobilize them in aggregate structures, denominated by magnetic crosslinked enzyme aggregates (MCLEA). One of the advantages of such supports is the possibility of using magnetic separation for enzyme recovery, reducing costs and allowing reuse in continuous systems. Here, porcine pancreatic lipase (PPL) was immobilized onto functionalized magnetite support (Fe3O4-APTS) with a protein binding efficiency of 78.84%. Physical and chemical properties of the nanoparticles and immobilized lipase were characterized by X-ray diffraction (XDR), transmission electron microscopy (TEM), infrared spectroscopy (FTIR), dynamic light scattering (DLS), zeta potential, vibrating sample magnetometer measurements (VSM), and 57Fe Mössbauer spectroscopy. The immobilized lipase additionally exhibited improved stability across wide pH and temperature ranges compared with free lipase. The immobilized derivate also attained good reusability, maintaining 61.37% of its initial activity after 6 reaction cycles. Through magnetic behavior and also because of its surface modification to crosslinking the enzyme, the MCLEA produced in this work has enhanced the biocatalytic activities of PPL.

Enhanced Laccase Activity and Stability as Crosslinked Enzyme Aggregates on Magnetic Copper Ferrite Nanoparticles for Biotechnological Processes

AbstractHighly stable and reusable magnetic crosslinked enzyme aggregates (m‐CLEAS) of laccase are synthesized with simultaneous improved enzymatic activity. Magnetic copper ferrite nanoparticles (CFNPs) were synthesized by solvothermal procedure with an average size of ~8 nm. The nanometric m‐CLEAS were formed by co‐aggregation of enzyme with CFNPs and crosslinked using glutaraldehyde. Different mass ratios of CFNPs:Laccase were assayed (1 : 2, 1 : 3, and 1 : 6), where 1 : 6 resulted in the highest activity recovery (97 %). The m‐CLEAS showed an average size of ~239 nm, ~24 % enzyme immobilization efficiency, and loading as high as 1.75 g of protein per g of support. As expected, m‐CLEAS oxidized the substrate with a higher transformation rate (kcat) and catalytic efficiency (kcat/Km) than the free enzyme. m‐CLEAS showed superior storage and thermostability compared to free enzyme and non‐magnetic CLEAS. In particular, the m‐CLEAS showed ~150 % and ~100 % residual activity after 30 days of storage at 4 °C and room temperature, respectively. Furthermore, m‐CLEAS showed good recyclability, retaining ~78 % and ~54 % laccase activity after 5 and 10 cycles of reuse, respectively. This work highlights the facile and cost‐effective synthesis of nanometric m‐CLEAS with exceptional storage stability and simultaneously improved laccase activity, making them suitable for a range of green industrial processes.

Tyrosinase Magnetic Cross-Linked Enzyme Aggregates: Biocatalytic Study in Deep Eutectic Solvent Aqueous Solutions.

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free immobilization towards the preparation of both non-magnetic and magnetic cross-linked enzyme aggregates (CLEAs). The prepared biocatalyst was characterized and the biocatalytic and structural traits of free tyrosinase and tyrosinase magnetic CLEAs (mCLEAs) were evaluated in numerous DES aqueous solutions. The results showed that the nature and the concentration of the DESs used as co-solvents significantly affected the catalytic activity and stability of tyrosinase, while the immobilization enhanced the activity of the enzyme in comparison with the non-immobilized enzyme up to 3.6-fold. The biocatalyst retained the 100% of its initial activity after storage at -20 °C for 1 year and the 90% of its activity after 5 repeated cycles. Tyrosinase mCLEAs were further applied in the homogeneous modification of chitosan with caffeic acid in the presence of DES. The biocatalyst demonstrated great ability in the functionalization of chitosan with caffeic acid in the presence of 10% v/v DES [Bet:Gly (1:3)], enhancing the antioxidant activity of the films.

Tyrosinase Immobilization Strategies for the Development of Electrochemical Biosensors-A Review.

The development of enzyme biosensors has successfully overcome various challenges such as enzyme instability, loss of enzyme activity or long response time. In the electroanalytical field, tyrosinase is used to develop biosensors that exploit its ability to catalyze the oxidation of numerous types of phenolic compounds with antioxidant and neurotransmitter roles. This review critically examines the main tyrosinase immobilization techniques for the development of sensitive electrochemical biosensors. Immobilization strategies are mainly classified according to the degree of reversibility/irreversibility of enzyme binding to the support material. Each tyrosinase immobilization method has advantages and limitations, and its selection depends mainly on the type of support electrode, electrode-modifying nanomaterials, cross-linking agent or surfactants used. Tyrosinase immobilization by cross-linking is characterized by very frequent use with outstanding performance of the developed biosensors. Additionally, research in recent years has focused on new immobilization strategies involving cross-linking, such as cross-linked enzyme aggregates (CLEAs) and magnetic cross-linked enzyme aggregates (mCLEAs). Therefore, it can be considered that cross-linking immobilization is the most feasible and economical approach, also providing the possibility of selecting the reagents used and the order of the immobilization steps, which favor the enhancement of biosensor performance characteristics.

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst.

Cross-linked enzyme aggregates (CLEAs) represent an effective tool for carrier-free immobilization of enzymes. The present study promotes a successful application of functionalized magnetic nanoparticles (MNPs) for stabilization of cellulase CLEAs. Catalytically active CLEAs and magnetic cross-linked enzyme aggregates (mCLEAs) of cellulase from Trichoderma reesei were prepared using glutaraldehyde (GA) as a cross-linking agent and the catalytic activity and stability of the CLEAs/mCLEAs were investigated. The influence of precipitation agents, cross-linker concentration, concentration of enzyme, addition of bovine serum albumin (BSA), and addition of sodium cyanoborohydride (NaBH3CN) on expressed activity and immobilization yield of CLEAs/mCLEAs was studied. Particularly, reducing the unsaturated Schiff's base to form irreversible linkages is important and improved the activity of CLEAs (86%) and mCLEAs (91%). For increased applicability of CLEAs/mCLEAs, we enhanced the activity and stability at mild biochemical process conditions. The reusability after 10 cycles of both CLEAs and mCLEAs was investigated, which retained 72% and 65% of the initial activity, respectively. The thermal stability of CLEAs and mCLEAs in comparison with the non-immobilized enzyme was obtained at 30 °C (145.65% and 188.7%, respectively) and 50 °C (185.1% and 141.4%, respectively). Kinetic parameters were determined for CLEAs and mCLEAs, and the KM constant was found at 0.055 ± 0.0102 mM and 0.037 ± 0.0012 mM, respectively. The maximum velocity rate (Vmax) was calculated as 1.12 ± 0.0012 µmol/min for CLEA and 1.17 ± 0.0023 µmol/min for mCLEA. Structural characterization was studied using XRD, SEM, and FT-IR. Catalytical properties of immobilized enzyme were improved with the addition of reducent NaBH3CN by enhancing the activity of CLEAs and with addition of functionalized aminosilane MNPs by enhancing the activity of mCLEAs.

Magnetic CLEAs of β-Galactosidase from Aspergillus oryzae as a Potential Biocatalyst to Produce Tagatose from Lactose

β-galactosidase is an enzyme capable of hydrolysing lactose, used in various branches of industry, mainly the food industry. As the efficient industrial use of enzymes depends on their reuse, it is necessary to find an effective method for immobilisation, maintaining high activity and stability. The present work proposes cross-linked magnetic cross-linked enzyme aggregates (mCLEAs) to prepare heterogeneous biocatalysts of β-galactosidase. Different concentrations of glutaraldehyde (0.6%, 1.0%, 1.5%), used as a cross-linking agent, were studied. The use of dextran-aldehyde as an alternative cross-linking agent was also evaluated. The mCLEAs presented increased recovered activity directly related to the concentration of glutaraldehyde. Modifications to the protocol to prepare mCLEAs with glutaraldehyde, adding a competitive inhibitor or polymer coating, have not been effective in increasing the recovered activity of the heterogeneous biocatalysts or its thermal stability. The biocatalyst prepared using dextran-aldehyde presented 73.6% recovered activity, aside from substrate affinity equivalent to the free enzyme. The thermal stability at 60 °C was higher for the biocatalyst prepared with glutaraldehyde (mCLEA-GLU-1.5) than the one produced with dextran-aldehyde (mCLEA-DEX), and the opposite happened at 50 °C. Results obtained for lactose hydrolysis, the use of its product to produce a rare sugar (D-tagatose) and operational and storage stability indicate that heterogeneous biocatalysts have adequate characteristics for industrial use.

Bio-imprinted magnetic cross-linked polyphenol oxidase aggregates for enhanced synthesis of L-dopa, a neurodegenerative therapeutic drug

Bio-imprinted magnetic cross-linked enzyme aggregates (i-m-CLEAs) of polyphenol oxidase (PPO) obtained from potato peels were prepared using amino-functionalized magnetic nanoparticles. Bio-imprinting is being used to improve the catalytic efficiency and conformational stability of enzymes. For bio-imprinting, PPO was incubated with different imprint/template molecules (catechol, 4-methyl catechol and l-3,4-dihydroxy phenylalanine) before cross-linking with glutaraldehyde. CLEAs imprinted with 4-methyl catechol showed maximum activity as compared with non-bio-imprinted magnetic CLEAs (m-CLEAs). They were further characterized by scanning electron microscopy and confocal microscopy. In bio-imprinted m-CLEAs, half-life (t1/2) of PPO significantly improved (364.74 min) as compared to free PPO (43.58 min) and non-bio-imprinted m-CLEAs (266.54 min). Bio-imprinted m-CLEAs showed excellent thermal and storage stability as well as reusability. The CLEAs preparation were used for the synthesis of l-3,4-dihydroxyphenylalanine (L-dopa, a therapeutic drug to treat neurodegenerative disorder) and a remarkable increase in L-dopa yield (23.5-fold) was obtained as compared to free enzyme. A cost effective and reusable method has been described for the production of L-dopa.

Magnetic cross-linked lipase aggregates coupled with ultrasonic pretreatment for efficient synthesis of phytosterol oleate

In this study, coupling with substrate ultrasonic pretreatment technology, magnetic cross-linked enzyme aggregates (CRL-FIL-CLEAs@Fe3O4) was used as novel biocatalyst for efficient preparation of phytosterol oleate in solvent-free reaction system. The CRL loading was 0.16 mg protein/mg CRL-FIL- CLEAs@Fe3O4. The effects of ultrasonic parameters, catalyst dosage, substrate molar ratio, and reaction temperature on the synthesis of phytosterol esters were studied. The results showed that the catalytical properties of CRL-FIL-CLEAs@Fe3O4 (7.09±0.11 µmol phytosterol/mg protein. hour) was significantly improved compared with natural lipase from Candida rugosa (CRL, 5.13±0.21 µmol phytosterol/mg protein. hour). Simultaneously, ultrasonic pretreatment greatly enhanced substrate mass transfer, further reducing the esterification time. The max conversion rate 94.72% of phytosterol was obtained after 9 h reaction under optimum condition (the molar ratio of oleic acid to phytosterol was 6:1, the concentration of CRL-FIL-CLEAs@Fe3O4 was 10 wt% of the phytosterol mass). After five recycles, the activity of CRL-FIL-CLEAs@Fe3O4 (80.02%) was still 2.68 times higher than that of natural lipase system (29.91%). Meanwhile, the novel immobilized lipase (CRL-FIL-CLEAs@Fe3O4) before and after reaction was characterized by SEM, AFM, VSM, TEM, and TGA. The results showed that CRL-FIL-CLEAs@Fe3O4 was a robust and recyclable biocatalyst for phytosterol esterification.

Ethanolysis of soybean oil catalyzed by magnetic CLEA of porcine pancreas lipase to produce ecodiesel. Efficient separation of ethyl esters and monoglycerides

Porcine pancreas lipase (PPL, a 1,3-specific lipase) magnetic crosslinked enzyme aggregates have been used to produce Ecodiesel, a blend of fatty acid ethyl esters (FAEE) and acylglycerides (mainly monoacylglycerides, MAG), by ethanolysis of soybean oil. An Ecodiesel containing 67 wt% FAEE and 27.7 wt% acylglycerides was obtained using 750 tributyrin activity units (TBU) of PPL per gram of oil and an oil:ethanol molar ratio of 1:7 at 37 °C after 48 h of reaction. This product was fractioned into a FAEE-rich phase and a MAG-rich phase. To this goal, Soxhlet extractor and silica gel column chromatography were used. Soxhlet extraction for 60 cycles allowed recovering 91% of the ethylic esters fed into the extractor in a nonpolar phase containing around 90 wt% of FAEE, which could be further increased to around 92% by caustic or to 94% by enzymatic polishing treatments (using Lipozyme® CALB L), this product being close to the requirements of biodiesel. On the other hand, the separation in the silica gel column chromatography was more efficient to purify MAG, allowing the recovery of the 93% acylglycerides with emulsifying properties, with a final purity of 94 wt% MAG.

The Synthesis of (Magnetic) Crosslinked Enzyme Aggregates With Laccase, Cellulase, β-Galactosidase and Transglutaminase.

Immobilized enzymes have important aspects due to the fact that they possess higher stability, have the possibility to be easily removed from the reaction mixture, and are much easier to use when compared to free enzymes. In this research, the enzymes laccase, cellulase, β-galactosidase (β-gal), and transglutaminase (TGM) were immobilized by two different methods: crosslinked enzyme aggregates (CLEAs) and magnetic crosslinked enzyme aggregates (mCLEAs). The processes for CLEAs and mCLEAs preparation with different enzymes have been optimized, where the aim was to achieve the highest possible relative activity of the immobilized enzyme. The optimal conditions of the synthesis of CLEAs in mCLEAs are described, thus emphasizing the difference between the two types of immobilization based on different enzymes. This comparative study, which represents the synthesis of crosslinked enzyme aggregates using different enzymes, has not been performed so far. Moreover, the obtained activity of CLEAs and mCLEAs is presented, which is important for further use in different biocatalytic processes. Specifically, of a higher importance is the selection of enzymes involved in immobilization, as they belong to the three different most applicable enzymes (oxidoreductases, hydrolases, and transferases). The study confirmed that the resulting activity of the immobilized enzyme and the optimization of enzyme immobilization depended on the type of the enzyme. Moreover, the prepared CLEAs and mCLEAs were exposed to the supercritical carbon dioxide (scCO2) at different pressures to determine the effect of scCO2 on enzyme activity in immobilized form. Additionally, to demonstrate the reuse and stability of the immobilized enzyme, the stability and reusability tests of CLEAs and mCLEAs were performed. The catalytic performance of immobilized enzyme was tested, where the catalytic efficiency and long-term operational stability of mCLEAs were obviously superior to those of CLEAs. However, the higher activity observed for CLEAs compared to mCLEAs suggests a significant effect of magnetic nanoparticles in the stabilization of an enzyme crosslinked aggregate structure.

Enhancing stability and by-product tolerance of β-glucuronidase based on magnetic cross-linked enzyme aggregates

β-glucuronidase is an important catalyst which is highly specific for β-glucuronides. Here, we constructed magnetic cross-linking β-glucuronidase aggregates (MCLEAs) to for the production of glycyrrhetinic acid (GA). Before crosslinking via glutaraldehyde, we used carbodiimide to enhance the interaction between enzymes and carboxyl-functionalized Fe3O4, efficiently improving the activity recovery. Compared to free enzymes, both kcat and kcat/Km enhanced, indicating that crosslinking and aggregation brought higher catalytic efficiency to enzymes. MCLEAs enhanced pH and thermal stabilities and retained 63.3% of catalytic activity after 6 cycles. More importantly, it was first found that the glucuronic acid tolerance of β-glucuronidase after the formation of MCLEAs enhanced 221.5% in 10 mM of glucuronic acid. According to the Raman spectroscopy, the ordered structure of β-glucuronidase increased from 43.9% to 50.6% after immobilization, which explained the increased stability and tolerance. To sum up, MCLEAs provided an efficient strategy for immobilization of enzymes, which enhanced stability and glucuronic acid tolerance of enzymes. It might be an effective solution to the serious inhibition caused by by-products during the preparation of aglycone from natural glycosides, having a significant applied prospect in industry.

Immobilization and Biochemical Characterization of Keratinase 2S1 onto Magnetic Cross-Linked Enzyme Aggregates and its Application on the Hydrolysis of Keratin Waste

Immobilization and Biochemical Characterization of Keratinase 2S1 onto Magnetic Cross-Linked Enzyme Aggregates and its Application on the Hydrolysis of Keratin Waste

Determination of sarcosine based on magnetic cross-linked enzyme aggregates for diagnosis of prostate cancer

As a biomarker of prostate cancer, sarcosine plays an important role in the diagnosis of cancer. The specificity of enzymatic reaction between sarcosine oxidase and sarcosine could be applied for the rapid and specific assay of sarcosine. Enzyme-based sarcosine quantification method has attracted our attention due to its high specificity and selectivity, but its wide use is restricted by the poor stability and challenging reusability. In this study, sarcosine oxidase was used to synthesize magnetic cross-linked enzyme aggregates (MCLEAs) with great stability and recyclability. The MCLEAs were characterized by transmission electron microscope, X-ray photoelectron spectroscopy, thermogravimetric analysis, X-ray diffractometer, and vibrating sample magnetometer, respectively. Subsequently, a colorimetric assay of sarcosine was developed based on the enzymatic reaction between MCLEAs and the substrate. The method can be used to quantify sarcosine with a linear range from 0.3125 to 10 μg mL−1 and recoveries ranging 87.50–97.75 %, suggesting its potential for reliable analysis of sarcosine in urine.

Magnetic Cross-Linked Enzyme Aggregates of a Transpeptidase-Specialized Variant (N450D) of Bacillus licheniformis γ-Glutamyl Transpeptidase: An Efficient and Stable Biocatalyst for l-Theanine Synthesis

γ-Glutamyl transpeptidase (GGT) catalyzes the transfer of glutathione’s γ-glutamyl group and related γ-glutamyl amides to water, amino acids or peptides, and utilizes a conserved Thr residue to process its own polypeptide chain into a large and a small subunit that then assemble to produce a catalytically competent enzyme. In this study, the magnetic cross-linked enzyme aggregates (mCLEAs) of a transpeptidase-specialized variant (N450D) of Bacillus licheniformis GGT were successfully prepared with optimized process parameters viz.1.25:1 (v/v) of isopropanol to N450D (0.3 mg/mL) ratio/0.02:1 (w/w) of enzyme to 3-aminopropyl triethoxysilane (APTES)-coated magnetic nanoparticle ratio/20 mM of glutaraldehyde. The prepared magnetic nanoparticles and immobilized enzyme (N450D-mCLEAs) were characterized by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy, field-emission scanning electron microscope integrated with energy dispersive X-ray spectroscopy (FESEM/EDS), and superparamagnetic analysis. As compared with free enzyme, N450D-mCLEAs displayed significantly higher heat resistance at temperatures of 55 and 60 °C, and had a greater stability over a storage period of one month. The immobilized enzyme could also be reused for 10 consecutive biocatalytic cycles with no significant reduction in the percent yield of l-theanine. Conclusively, this immobilization strategy surely provides a meaningful glance of developing N450D-mediated biocatalysis for the production of physiologically important γ-glutamyl compounds.

Development of recyclable magnetic cross-linked enzyme aggregates for the synthesis of high value rare sugar d-tagatose in aqueous phase catalysis

In this study, a high value rare sugar d-tagatose was synthesized using recyclable magnetic catalysts.

Preparation and synthetic dye decolorization ability of magnetic cross-linked enzyme aggregates of laccase from Bacillus amyloliquefaciens.

Laccases are versatile oxidases that are capable of decolorizing various synthetic dyes. Recombinant Bacillus amyloliquefaciens laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) for application in dye decolorization. Several parameters influencing the activity recovery were evaluated during the synthesis of M-CLEAs. With ammonium sulfate as precipitant, maximum activity was recovered by cross-linking with 0.16% glutaraldehyde for 1h. The prepared M-CLEAs exhibited improved activity under alkaline conditions. It remained 74% activityafter incubation at 60°C for 5h. Enhanced tolerance towards NaCl was also observed for the M-CLEAs, with 68% activity remaining in the presence of 1M NaCl. The immobilized laccase could rapidly decolorize more than 93% of reactive black 5 and indigo carmine in 1h, while its catalytic efficiency towards reactive blue 19 was relatively low. After four cycles of consecutive reuse, the M-CLEAs could decolorize 92% of indigo carmine. The easy recovery and reusability of M-CLEAs facilitate the potential application of bacterial laccase in dye decolorization.

Enzymatic glycosylation of bioactive acceptors catalyzed by an immobilized fungal β-xylosidase and its multi-glycoligase variant

A recombinant β-xylosidase (rBxTW1) from the ascomycete Talaromyces amestolkiae and a mutant derived from it, with mostly synthetic activity, have been immobilized as magnetic cross-linked enzyme aggregates (mCLEAs). The mCLEAs of rBxTW1 kept the excellent hydrolytic and O-transxylosylating activities of the free enzyme and had improved thermal and pH stability. The mCLEAs of the mutant also maintained or improved the catalytic properties of the soluble enzyme, synthetizing the O-xylosides of vanillin and (−)-epigallocatechin gallate, and the N- and S-xyloside of 3,5-dibromo-1,2,4-triazole and thiophenol, respectively. The mCLEAs were recyclable across 4 cycles of synthesis of the O-xylosides through a green and highly selective process. The magnetic properties of the scaffold used for immobilization may allow the easy recovery and reuse of the biocatalyst even from reactions containing insoluble lignocellulosic biomass.

Immobilized laccase in the form of (magnetic) cross-linked enzyme aggregates for sustainable diclofenac (bio)degradation

Nowadays, major problems worldwide derive from the presence of pharmaceutically active compounds in the wastewater of hospitals and healthcare facilities. To address this issue, biological transformation of diclofenac using native and immobilized laccase is described in our study. Laccase stabilization is demonstrated, using two different types of immobilization: cross-linked enzyme aggregates (CLEAs) and magnetic cross-linked enzyme aggregates (mCLEAs). High diclofenac (DCF) removal capacity of CLEAs laccase and mCLEAs laccase in comparison to native laccase is determined. Under optimal conditions, the removal capacity of native laccase is 11.5 ± 0.3 μg DCF/glaccase, of CLEAs 15.6 ± 0.4 μg DCF/glaccase and of mCLEAs 13.6 ± 0.4 μg DCF/glaccase. Our immobilized laccase also possesses very good reusability, since the half-life of the immobilized laccase in the form of CLEAs is reached after the second cycle of biocatalyst reuse, while the half-life of the mCLEAs laccase can be detected at the fourth cycle. The improved stability of immobilized laccase in supercritical carbon dioxide (SC CO2) is shown. The promising results of DCF removal using CLEAs laccase and mCLEAs laccase show that both immobilized forms of laccase have the potential to be used in cleaner industrial technologies, e.g. wastewater treatment.

Preparation of CLEAs and magnetic CLEAs of a recombinant l-arabinose isomerase for d-tagatose synthesis

The insolubilization of a recombinant l-arabinose isomerase (l-AI) from Enterococcus faecium by cross-linked enzyme aggregates (CLEA) was investigated, aiming the biochemical production of d-tagatose from d-galactose. d-tagatose is a functional sweetener that has many health benefits, sweetening properties and lower calorific value. Different precipitants (ammonium sulfate, ethanol, acetone, polyethylene glycol 4000) were used in the first step of the protocol, in order to establish the precipitation conditions, and the best results of yield and activity were achieved with ammonium sulfate. In order to facilitate the recovery of the biocatalyst, a new strategy for immobilization of the multimeric enzyme l-arabinose isomerase was proposed. Magnetic cross-linked enzyme aggregates (m-CLEA) were obtained using ammonium sulfate as precipitant and magnetic nanoparticles (MNP) functionalized with APTES (3- Aminopropyltriethoxysilane). Another immobilization strategy was to immobilize the enzyme onto MNP-APTES, as a control. The best results were achieved when the m-CLEA was produced with 20 mg of MNP, 7.69 U. g−1 of enzymatic activity, 7.61 % of recovered activity, 99 % of yield of immobilization. On the other hand, the enzyme immobilized onto MNP-APTES, presented only 2.12 U. g−1 of enzymatic activity, 32.3 % of recovered activity, and 15 % of yield of immobilization.