Enzyme Leaching Research Articles

Sustainable Biocatalytic System for the Enzymatic Epoxidation of Waste Cooking Oil.

The present study is integrated in a global effort to capitalize waste cooking oil (WCO) into versatile compounds by introducing an oxirane ring into the unsaturated carbon chain of fatty acid residues (the epoxidation of double bound). Therefore, an enzymatic method was set up for the epoxidation of artificially adulterated WCO (SFw) and WCO under real conditions (SFr) derived from sunflower biomass. Commercial lipase (Novozyme, NZ) was used as a biocatalyst for generating the peracid requested by the epoxidation pathway. Optimum experimental conditions (e.g., 1.5 wt% NZ, 1:1:0.5 = H2O2/double bonds/peracid precursor (molar ratio) and 12 h reaction time) allowed for the conversion of 90% of the SFw substrate into products with an oxirane ring. Octanoic acid was selected as the best peracid precursor. The versatility of the developed system was tested for olive, milk thistle, hemp and linseed oils as both fresh and WCO samples. The characterization of the oil samples before and after the enzymatic epoxidation allowed for the evaluation of the system performance. SFw/SFr exhibited a better susceptibility to enzymatic epoxidation. In addition, the reusability of the biocatalytic system was investigated. Furthermore, different strategies, such as biocatalyst coating and the addition of organic solvents/buffers were applied, limiting enzyme leaching, for the better recovery of the biocatalyst activity.

Green synthesis of NiO NPs for metagenome-derived laccase stabilization: Detoxifying pollutants and wastes

Laccases play a crucial role in neutralizing environmental pollutants, including antibiotics and phenolic compounds, by converting them into less harmful substances via a unique oxidation process. This study introduces an environmentally sustainable remediation technique, utilizing NiO nanoparticles (NPs) synthesized through green chemistry to immobilize a metagenome-derived laccase, PersiLac1, enhancing its application in pollutant detoxification. Salvadora persica leaf extract was used for the synthesis of NiO nanoparticles, utilizing its phytochemical constituents as reducing and capping agents, followed by characterization through different analyses. Characterization of NiO nanoparticles revealed distinctive FTIR absorption peaks indicating the nanoparticulate structure, while FESEM showed structured NiO with robust interconnections and dimensionality of about 50nm, confirmed by EDX analysis to have a consistent distribution of Ni and O. The immobilized PersiLac1 demonstrated enhanced thermal stability, with 85.55 % activity at 80 °C and reduced enzyme leaching, retaining 67.93 % activity across 15 biocatalytic cycles. It efficiently reduced rice straw (RS) phenol by 67.97 % within 210 min and degraded 70–78 % of tetracycline (TC) across a wide pH range (4.0–8.0), showing superior performance over the free enzyme. Immobilized laccase achieved up to 71 % TC removal at 40–80 °C, significantly outperforming the free enzyme. Notably, 54 % efficiency was achieved at 500 mg/L TC by immobilized laccase at 120 min. This research showed the potential of green-synthesized NiO nanoparticles to effectively immobilize laccase, presenting an eco-friendly approach to purify pollutants such as phenols and antibiotics. The durability and reusability of the immobilized enzyme, coupled with its ability to reduce pollutants, indicates a viable method for cleaning the environment. Nonetheless, the production costs and scalability of NiO nanoparticles for widespread industrial applications pose significant challenges. Future studies should focus on implementation at an industrial level and examine a wider range of pollutants to fully leverage the environmental clean-up capabilities of this innovative technology.

Exploring the use of supercritical carbon dioxide in enzymatic hydrolysis of cellulosic substrate

Enzymes are critical components for production of fermentable sugars in lignocellulosic biorefineries. Their activity could be influenced by various reaction parameters. Herein, a formulated cellulolytic enzyme cocktail was immobilized into mesoporous aerogel material and used under high pressure environment of supercritical CO2 (scCO2). The cocktail was immobilized at different enzyme loads (36.0 to 144.0 mg enzyme/g aerogel) using sol-gel entrapment method and scCO2 drying. The immobilized cocktail at 144.0 mg/g aerogel enzyme load retained a residual yield of >50.0 % after fourth cycle of reuse compared to first cycle under atmospheric pressure. Sugar yield by the cocktail aerogel was below 10 wt% in second cycle under scCO2 due to enzyme leaching. Additionally, surface area and porous volume of reused aerogels was decreased over the reuse cycles. The study suggests that scCO2 could be employed for aerogel preparation after optimizing the immobilization process for pure enzymes rather than crude enzymes.

Lactic acid production enhancement using metagenome-derived bifunctional enzyme immobilized on chitosan-alginate/nanocellulose hydrogel

Lactic acid (LA) production from renewable and abundant lignocellulosic resources has received great interest due to its various applications in food, cosmetic, pharmaceutical, and textile industries. Herein, the efficient immobilized bi-functional enzyme on an innovative hydrogel (nano)carrier with boosted stability/activity were prepared and utilized for hydrolyzing lignocellulosic biomass to enhance the fermentative production of LA. First, different samples of sodium alginate (SA)-chitosan (CH) hydrogel grafted to acrylic acid and N-isopropylacrylamide and reinforced with nanocellulose (NCs) were synthesized. Selected hydrogels of pristine SA-CH hydrogel and reinforced SA-CH/NCs hydrogel were utilized for the immobilization of a cellulase/xylanase enzyme (PersiCelXyn1). While free PersiCelXyn1 had the optimum temperature of 50 °C, the immobilized enzyme on SA-CH and SA-CH/NCs hydrogels showed optimum temperatures at 70, and 80 °C, respectively. Furthermore, the immobilization of PersiCelXyn1 onto both hydrogels shifted the optimum pH to alkaline pHs, and increased the enzyme specific activity. The enzyme immobilized on nanocarrier (PersiCelXyn1@SA-CH/NCs) showed higher reusability than PersiCelXyn1@SA-CH, after 14 cycles (70 % activity vs. 52 % activity, respectively), and less enzyme leaching. PersiCelXyn1@SA-CH/NCs showed higher efficiency in the hydrolysis of sugar beet pulp to obtain the hydrolysate containing fermentable sugar as a substrate for lactic acid (LA) production, than PersiCelXyn1@SA-CH hydrogel. After 96 h, the concentration of LA produced with the PersiCelXyn1@SA-CH/NCs was 33.1 g/L, while it was 26 g/L and 21.6 g/L, in the fermentation of hydrolysate with PersiCelXyn1@SA-CH hydrogel, and free PersiCelXyn1, respectively.

Cellulase immobilization on zeolitic imidazolate frameworks for boosting cellulose hydrolysis at high solids loading

This study investigates the cellulase immobilization onto zeolitic imidazolate frameworks (ZIFs) to enable efficient cellulose hydrolysis at high solids loading. Different ZIF carriers (ZIF-8, ZIF-8-NH2, Fe3O4@ZIF-8, and Fe3O4@ZIF-8-NH2) were prepared and characterized. The resulting ZIF-immobilized cellulases were subsequently analyzed for their loading capacity and enzyme activity, as well as their biochemical properties such as temperature stability, pH stability, storage stability, enzyme leaching, and enzyme kinetics. Results revealed that the cellulase immobilized on Fe3O4@ZIF-8-NH2 exhibited an exceptional loading capacity of 359.89 mg/g and a relative enzyme activity of 69.39%. Remarkably, at a solid loading of 15% (w/w) and an enzyme dosage of 5 FPU/g cellulose, the Fe3O4@ZIF-8-NH2 immobilized cellulase demonstrated a 20.44% higher hydrolysis yield compared to the free cellulase. Furthermore, this immobilized enzyme system displayed robust reusability, retaining 71.03% of its initial activity even after 10 cycles of reuse. A proposed mechanism of enhanced enzymatic hydrolysis by ZIF-immobilized cellulases was also presented. These findings have significant implications for the future application of ZIF-immobilized cellulases in efficient and cost-effective lignocellulosic bioconversion.

Enzyme immobilization on covalent organic framework supports.

Enzymes are natural catalysts with high catalytic activity, substrate specificity and selectivity. Their widespread utilization in industrial applications is limited by their sensitivity to harsh reaction conditions and difficulties relating to their removal and re-use after the reaction is complete. These limitations can be addressed by immobilizing the enzymes in solid porous supports. Covalent organic frameworks (COFs) are ideal candidate carriers because of their good biocompatibility, long-term water stability and large surface area. In post-synthetic immobilization, the enzyme is added to an existing COF; this has had limited success because of enzyme leaching and pore blockage by enzymes that are too large. Direct-immobilization methods-building the COF around the enzyme-allow tailored incorporation of proteins of any size and result in materials with lower levels of leaching and better mass transport of reactants and products. This protocol describes direct-immobilization methods that can be used to fabricate enzyme@COF (@ = engulfing) biocomposites with rationally programmed structures and functions. If COF construction requires harsh reaction conditions, the enzyme can be protected by using a removable metal-organic framework. Alternatively, a direct in situ approach, in which the enzyme and the COF monomers assemble under very mild conditions, can be used. Examples of both approaches are described: enzyme@COF-42-B/43-B capsules (enzymes including catalase, glucose oxidase, etc.) with ZIF-90 or ZPF-2 as protectors, and lipase@NKCOF-98/99 via in situ direct-immobilization methods (synthesis timing: 30-100 min). Example assays for physical and functional characterization of the COF and enzyme@COF materials are also described.

Enzyme-immobilized hierarchically porous covalent organic framework biocomposite for catalytic degradation of broad-range emerging pollutants in water

Efficient enzyme immobilization is crucial for the successful commercialization of large-scale enzymatic water treatment. However, issues such as lack of high enzyme loading coupled with enzyme leaching present challenges for the widespread adoption of immobilized enzyme systems. The present study describes the development and bioremediation application of an enzyme biocomposite employing a cationic macrocycle-based covalent organic framework (COF) with hierarchical porosity for the immobilization of horseradish peroxidase (HRP). The intrinsic hierarchical porous features of the azacalix[4]arene-based COF (ACA-COF) allowed for a maximum HRP loading capacity of 0.76 mg/mg COF with low enzyme leaching (<5.0 %). The biocomposite, HRP@ACA-COF, exhibited exceptional thermal stability (∼200 % higher relative activity than the free enzyme), and maintained ∼60 % enzyme activity after five cycles. LCMSMS analyses confirmed that the HRP@ACA-COF system was able to achieve > 99 % degradation of seven diverse types of emerging pollutants (2-mercaptobenzothiazole, paracetamol, caffeic acid, methylparaben, furosemide, sulfamethoxazole, and salicylic acid)in under an hour. The described enzyme-COF system offers promise for efficient wastewater bioremediation applications.

Co-immobilization of bienzyme HRP/GOx on highly stable hierarchically porous MOF with enhanced catalytic activity and stability: Kinetic and thermodynamic studies

Immobilization of enzymes onto solid carriers usually results in the reduction of enzyme catalytic activity. To solve this tricky problem, we proposed a novel strategy by accurately constructing the micro-mesoporous channel structure of highly stable hierarchically porous metal-organic framework (HP-Zr-MOF) for co-immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx). Notably, the precise construction and co-regionalization of HP-Zr-MOF with hierarchical structure can efficiently suppress enzyme leaching and significantly improve the catalytic efficiency, enzyme activity and the affinity for substrates of co-immobilized bienzyme reactor (GOx&HRP@HP-Zr-MOF). The Km value for GOx&HRP@HP-Zr-MOF was 0.2050 mM, which was 0.66-fold less than that of free GOx&HRP, while the Ka value for GOx&HRP@HP-Zr-MOF was superior to that of free GOx&HRP, manifesting the affinity of GOx&HRP@HP-Zr-MOF for substrates strengthened. Importantly, the GOx&HRP@HP-Zr-MOF exhibited favorable stability and reusability, maintaining 97.4% of the original activity after 1 h incubation at 60 °C and over 90% initial activity after 30 cycles. Moreover, the GOx&HRP@HP-Zr-MOF was applied to the colorimetric detection of glucose, manifesting outstanding detection capability. Finally, the GOx&HRP@HP-Zr-MOF exhibited excellent catalytic efficiency for the degradation of 2,4-dichlorophenol, which indicated that the bio-catalytic material has potential practical application value in wastewater treatment.

Eradication of ibuprofen and diclofenac via in situ synthesized and immobilized bacterial laccase to Cu-based metal organic framework

In this study, a simple in situ method was utilized for immobilization of laccase from Alcaligenes faecalis XF1. Protein loading of around 67.48 % was computed onto laccase@CuBTc. Immobilized laccase on CuBTc retained approximately 86 % activity after 14 days of storage at 4 °C. It was also observed that after 56 days of monitoring, residual activity was 32 % detected as a result of enzyme leaching. In comparison to free and immobilized laccase exhibited higher activity, stability and reusability. The characterization of the fabricated laccase@CuBTc was done using SEM, XRD and FT-IR. Laccase@Cu-BTc particles were used to degrade diclofenac (DCF) and Ibuprofen (IBF). In the absence of any mediator, laccase@CuBTc efficiently degraded around 95 % DCF within first 60 min of incubation and around 96 % IBF was degraded in 3 h. The findings showed that Laccase@CuBTc was a more stable, reusable, and storage-friendly method for removing pharmaceutical impurities such DCF and IBF.

Improved saccharification of rice straw by removing phenolic compounds using a stable immobilized metagenome-derived laccase on sodium alginate-based hydrogel

Laccases can biodegrade lignocellulosic agricultural waste, which has a recalcitrant structure hindering industrial biomass hydrolysis. In the context of biomass decomposition, introduction of a stabilized enzyme with enhanced performance is of critical importance. This study aimed to stabilize laccase, obtained from tannery metagenome (PersiLac2) on sodium alginate-based hydrogels. The immobilized laccase remained stable in the acidic and alkaline pH ranges and preserved over 79.39 % of its initial activity at pH 4.0–9.0. Similarly, immobilized enzyme improved laccase activity at high temperatures, which is crucial in biorefinery processes, and the immobilized enzyme demonstrated 88.35 % activity at 80 ºC. The immobilization of laccase on hydrogels effectively prevented enzyme leaching and rendered it easier to separate and recycle, showing 58.74 % laccase activity after 15 cycles. The efficiency of immobilized PersiLac2 in enzymatic hydrolysis of rice straw was evaluated to enhance the generation of fermentable sugars, saccharification, and phenol reduction. Laccase treatment of rice straw resulted in an excellent saccharification yield and liberation of reducing sugars and removed 77.19 % of phenols during hydrolysis. The depolymerization effect of free and immobilized laccase on rice straw was investigated by FTIR and SEM analyses which confirmed efficient structural disruption by immobilization. The results indicated the potential of immobilized Persilac2 to degrade the compact wrap of lignin–carbohydrate complex in lignocellulosic substrates and its application in biomass-based industries.

Conductive polymer blend based on polyaniline and galactomannan: Optical and electrical properties

Polyaniline (PAni) is a semiconductor polymer characterized by a wide conductivity range and can be used as a biosensor for glucose measurement via glucose oxidase (GOx) catalysis because PAni can immobilize GOx on its surface and allows charge transfer between GOx and its surface. However, PAni presents issues with enzyme leaching and high dielectric constant (104-105), which hinders its electrical response as a sensor. To solve both issues simultaneously, a blend of polyaniline and galactomannan (Gal) is presented. Gal is able to preserve the enzymatic activity and has insulating properties, making it suitable to enhance the immobilization of GOx on PAni and to improve the electrical response of the latter for biosensing. PAni was synthesized from the polymerization of aniline in HCl and ammonium persulfate and blended with a solution of Gal 2%, resulting in the polyaniline and galactomannan blend (PAni/Gal). Samples of Gal 2%, PAni and PAni/Gal were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and impedance spectroscopy (IS). The FTIR of the PAni/ AP blends detected the presence of the band associated to the deformation outside the binding plane (C-N) and the oxidizing agent responsible for the semiconducting property of PAni. The XRD patterns showed the crystalline structure of PAni and the amorphous structure of PAni/Gal. The IS showed that PAni/Gal exhibited a smaller dielectric constant and a higher loss tangent than PAni, indicating that PAni/Gal has better electrical behavior as a sensor compared to PAni and thus has solid potential for use in biosensors.

Continuous fixed-bed column studies to remove polycyclic aromatic hydrocarbons by degrading enzymes immobilized on polyimide aerogels

The main sources of polycyclic aromatic hydrocarbons (PAHs), entering aquatic environments are industrial discharges, petroleum spills, combustion of fossil fuels, urban runoff, and atmospheric deposition. For the biodegradation of PAHs, the use of biocatalysts, such as enzymes, is an environmentally friendly method. Despite this, it is necessary to immobilize the enzymes to facilitate their recovery and reusability, as well as to prevent their loss. Using covalent bonding, (PAHs) degrading enzymes including naphthalene and catechol 2,3 dioxygenase enzymes were immobilized on modified polyimide aerogels. Covalent immobilization of enzymes on surface-modified polyimide aerogels resulted in around 9- and 6-fold lower enzyme leaching for naphthalene and catechol 2,3 dioxygenase enzymes compared to enzyme immobilization using adsorption. The Fourier Transform Infrared Spectrum (FT-IR) confirmed the enzyme immobilization and aerogel modification. SEM images indicated completely different textures for surface-modified aerogel attributed to the presence of functional groups after surface modification that enhanced enzyme loading. The removal capacity (∼20mganthracenegaerogel) of enzyme loaded aerogel was determined to select an appropriate range for column tests. Finally, the effects of flow rate, size of aerogels and inlet concentration of anthracene on the removal efficiency of pollutants were examined. Using the derived model as a basis for prediction, in terms of removal efficiency, the highest result was achieved to be 84.01% at a flow rate of 22 mL min−1, initial concentration of 34 mg L−1and aerogel size of 2 cm while under these conditions, the removal efficiency was experimentally measured to be 87.14 %. A fixed-bed column packed with enzyme-loaded aerogel for the removal of PAHs provides novel insight into the application of aerogel base materials for water treatment and PAH removal with possibilities of scaling up for larger applications.

Laccase-Carrying Polylactic Acid Electrospun Fibers, Advantages and Limitations in Bio-Oxidation of Amines and Alcohols.

Laccases are oxidative enzymes that could be good candidates for the functionalization of biopolymers with several applications as biosensors for the determination of bioactive amine and alcohols, for bioremediation of industrial wastewater, and for greener catalysts in oxidation reactions in organic synthesis, especially used for non-phenolic compounds in combination with redox mediators in the so-called Laccase Mediator System (LMS). In this work, we describe the immobilization of Laccase from Trametes versicolor (LTv) in poly-L-lactic acid (PLLA) nanofibers and its application in LMS oxidation reactions. The PLLA-LTv catalysts were successfully produced by electrospinning of a water-in-oil emulsion with an optimized method. Different enzyme loadings (1.6, 3.2, and 5.1% w/w) were explored, and the obtained mats were thoroughly characterized. The actual amount of the enzyme in the fibers and the eventual enzyme leaching in different solvents were evaluated. Finally, the PLLA-LTv mats were successfully applied as such in the oxidation reaction of catechol, and in the LMS method with TEMPO as mediator in the oxidation of amines with the advantage of easier work-up procedures by the immobilized enzyme. However, the PLLA-LTv failed the oxidation of alcohols with respect to the free enzyme. A tentative explanation was provided.

Immobilization of horseradish peroxidase on hierarchically porous magnetic metal-organic frameworks for visual detection and efficient degradation of 2,4-dichlorophenol in simulated wastewater

Immobilization of enzymes onto solid carrier is an important way to improve the stability and reusability of enzymes. However, the enzymatic activity and catalytic efficiency are severely hampered due to enzyme leaching and increased substrate mass transfer resistance. Herein, for the first time, we reported a novel strategy of co-regionalization for enzyme immobilization using hierarchically porous magnetic metal-organic frameworks (HP-Zr-MOF@Fe3O4). The HP-Zr-MOF@Fe3O4 integrate magnetic characteristics and hierarchical porous structure for supporting horseradish peroxidase (HRP) via metal-ion affinity interaction, in which the mesoporous pores are suitable for entrapping enzyme while the microporous can be used to concentrate substrate to reduce the mass transfer resistance. Significantly, the prepared HRP@HP-Zr-MOF@Fe3O4 can efficiently suppress enzyme leaching from the HP-Zr-MOF@Fe3O4 skeleton and remain superior recovered activity (96.8 %) of the initial activity of free HRP, which exhibited excellent stability and reusability. Interestingly, the HRP@HP-Zr-MOF@Fe3O4 was used efficiently in the colorimetric detection and catalytic degradation of 2,4-dichlorophenol in simulated wastewater. The excellent property of HRP@HP-Zr-MOF@Fe3O4 was investigated through the analyses of kinetics and thermodynamics, demonstrating that the HRP@HP-Zr-MOF@Fe3O4 had significant specificity and binding affinity for the substrate compared with free HRP.

Systematic evaluation of agarose- and agar-based bioinks for extrusion-based bioprinting of enzymatically active hydrogels.

Extrusion-based 3D bioprinting enables the production of customized hydrogel structures that can be employed in flow reactors when printing with enzyme-containing inks. The present study compares inks based on either low-melt agarose or agar at different concentrations (3-6%) and loaded with the thermostable enzyme esterase 2 from the thermophilic organism Alicyclobacillus acidocaldarius (AaEst2) with regard to their suitability for the fabrication of such enzymatically active hydrogels. A customized printer setup including a heatable nozzle and a cooled substrate was established to allow for clean and reproducible prints. The inks and printed hydrogel samples were characterized using rheological measurements and compression tests. All inks were found to be sufficiently printable to create lattices without overhangs, but printing quality was strongly enhanced at 4.5% polymer or more. The produced hydrogels were characterized regarding mechanical strength and diffusibility. For both properties, a strong correlation with polymer concentration was observed with highly concentrated hydrogels being more stable and less diffusible. Agar hydrogels were found to be more stable and show higher diffusion rates than comparable agarose hydrogels. Enzyme leaching was identified as a major drawback of agar hydrogels, while hardly any leaching from agarose hydrogels was detected. The poor ability of agar hydrogels to permanently immobilize enzymes indicates their limited suitability for their employment in perfused biocatalytic reactors. Batch-based activity assays showed that the enzymatic activity of agar hydrogels was roughly twice as high as the activity of agarose hydrogels which was mostly attributed to the increased amount of enzyme leaching. Agarose bioinks with at least 4.5% polymer were identified as the most suitable of the investigated inks for the printing of biocatalytic reactors with AaEst2. Drawbacks of these inks are limited mechanical and thermal stability, not allowing the operation of a reactor at the optimum temperature of AaEst2 which is above the melting point of the employed low-melt agarose.

Green and Scalable Fabrication of High-Performance Biocatalysts Using Covalent Organic Frameworks as Enzyme Carriers.

Enzyme immobilization is essential to the commercial viability of various critical large-scale biocatalytic processes. However, challenges remain for the immobilization systems, such as difficulties in loading large enzymes, enzyme leaching, and limitations for large-scale fabrication. Herein, we describe a green and scalable strategy to prepare high-performance biocatalysts through in situ assembly of enzymes with covalent organic frameworks (COFs) under ambient conditions (aqueous solution and room temperature). The obtained biocatalysts have exceptional reusability and stability and serve as efficient biocatalysts for important industrial reactions that cannot be efficiently catalyzed by free enzymes or traditional enzyme immobilization systems. Notably, this versatile enzyme immobilization platform is applicable to various COFs and enzymes. The reactions in an aqueous solution occurred within a short timeframe (ca. 10-30 min) and could be scaled up readily (ca. 2.3 g per reaction).

Green and Scalable Fabrication of High‐Performance Biocatalysts Using Covalent Organic Frameworks as Enzyme Carriers

AbstractEnzyme immobilization is essential to the commercial viability of various critical large‐scale biocatalytic processes. However, challenges remain for the immobilization systems, such as difficulties in loading large enzymes, enzyme leaching, and limitations for large‐scale fabrication. Herein, we describe a green and scalable strategy to prepare high‐performance biocatalysts through in situ assembly of enzymes with covalent organic frameworks (COFs) under ambient conditions (aqueous solution and room temperature). The obtained biocatalysts have exceptional reusability and stability and serve as efficient biocatalysts for important industrial reactions that cannot be efficiently catalyzed by free enzymes or traditional enzyme immobilization systems. Notably, this versatile enzyme immobilization platform is applicable to various COFs and enzymes. The reactions in an aqueous solution occurred within a short timeframe (ca. 10–30 min) and could be scaled up readily (ca. 2.3 g per reaction).

Co-immobilization of lipase and laccase on agarose-based supports via layer-by-layer strategy: Effect of diffusional limitations

Co-immobilized enzymes present desirable advantages to performing cascade reactions when employed in one-pot processes, mainly because of the shorter lag times owing to better reagent diffusion across the active sites of the different enzymes. But high product yields require high enzyme loads, usually achieved with high surface area and porous supports, where diffusional limitations may be introduced or increased. Heterogeneous biocatalysts were prepared by co-immobilizing Pseudomonas fluorescens lipase (PFL) and Aspergillus sp. Laccase (Novozymes 51003) on two agarose-based supports, DEAE-Sepharose and Octyl-agarose. A layer-by-layer strategy was used by adding polyethyleneimine (PEI) to allow the attachment of the upper enzyme layer onto the lower adjacent layer. A final step, crosslinking with glutaraldehyde, was performed to prevent enzyme leaching under harsh environmental conditions. The heterogeneous biocatalysts showed lower temperature dependence and a significant increase in thermal stability at 50 ºC. No internal mass transfer effects were observed when a low protein load was used during co-immobilization (2 mgprotein/gsupport of each enzyme). However, increasing the enzyme load (more than 10 mgprotein/gsupport) promoted internal diffusion limitations. The layer-by-layer strategy defined in this work enabled the preparation of a multi-enzymatic heterogeneous biocatalyst with high activity, thermal stability, and no diffusional limitations, depending on enzyme concentration.

Precipitation-based microscale enzyme reactors coupled with porous and adhesive elastomer for effective bacterial decontamination and membrane antifouling on-demand

Bacterial contamination of water environments can cause various troubles in various areas. As one of potential solutions, we develop enzyme-immobilized elastomer, and demonstrate the uses of enzyme reactions on-demand for effective microbial decontamination and antifouling. Asymmetrically-structured elastomer is prepared by combining two polydimethylsiloxane (PDMS) layers with different degrees of crosslinking: highly-crosslinked and lightly-crosslinked PDMS layers. At the surface of highly-crosslinked PDMS layer, porous structure with average diameter of 842 nm is formed by dissolving pre-packed and entrapped latex beads. Lightly-crosslinked PDMS on the other side, due to its adhesive nature, enables iterative attachments on various materials under either dry or wet condition. Glucose oxidase (GOx) is immobilized by using the pores at the surface of highly-crosslinked PDMS matrix via a ship-in-a-bottle protocol of precipitation-based microscale enzyme reactor (p-MER), which consists of GOx adsorption, precipitation and chemical crosslinking (EAPC). As a result, crosslinked enzyme aggregates (CLEAs) of GOx not only are well entrapped within many pores of highly-crosslinked PDMS layer (ship-in-bottle) but also cover the external surface of matrix, both of which are well connected together. Highly-interconnected network of CLEAs themselves effectively prevents enzyme leaching, which shows the 25% residual activity of GOx under shaking at 200 rpm for 156 days after 48% initial drop of loosely-bound p-MER after 4 days. In presence of glucose, the underwater attachment of biocatalytic elastomer demonstrates the generation of hydrogen peroxide via p-MER-catalyzed glucose oxidation, exhibiting effective biocidal activities against both gram-positive S. aureus and gram-negative E. coli. Adhesion-induced GOx-catalyzed reaction also alleviates the biofouling of membrane, suggesting its extendibility to various engineering systems being suffered by biofouling. This study of biocatalytic elastomer has demonstrated its new opportunities for the facile and on-demand enzyme-catalyzed reactions in various environmental applications, such as bactericidal treatment, water treatment/purification, and pollutant degradation.

One-pot laccase@MOF biocatalysts efficiently remove bisphenol A from water

The presence of bisphenol A (BPA) in wastewater, its toxicity and the hardness for its complete degradation constitutes a serious environmental problem and a scientific challenge. The enzyme laccase is an oxidoreductase capable of oxidizing phenolic compounds, being one of the most efficient biocatalysts for BPA removal. The immobilization of laccases to lead solid biocatalysts is essential for their applications as it allows their stabilization and easy recovery from the liquid reaction media and subsequent reuse. This work compares the efficiency of siliceous-type supports, which have traditionally been used for this purpose, with the use of MOF-type supports. Thanks to the versatility in the synthesis methods offered by MOFs, MOF-type biocatalysts can be prepared in a one-pot using an in-situ procedure (Lac@NH2-MIL-53(Al)) under conditions compatible with enzymatic activity (room temperature, aqueous solution and moderate pH values). The resulting biocatalyst displays a high enzyme loading of 100 mg.g-1 which is also permanent: free of enzyme leaching. As opposed, post-synthesis in two steps: Lac#NH2-MIL-53(Al) (with low enzyme loading) and Lac#Amorphous silica-NH2 (100 mg.g-1 enzyme loading, but high rate of enzyme leaching) were also prepared and compared.In contrast to the elimination of 90% of BPA in 60 min obtained with Lac#Amorphous silica-NH2, the one-pot immobilization of laccase on MOFs resulted much more efficient. The own enzyme-free MOF-type support (NH2-MIL-53(Al)) removed more than 90% of BPA from aqueous solution in 30 min, but such performance was widely surpassed by the biocatalyst Lac@NH2-MIL-53(Al) (100% in just 3 min). The significant improvement of BPA degradation was attributed to the mutual and simultaneous contribution of both support and enzyme. In addition, the biocatalyst Lac@NH2-MIL-53(Al) was able to remove BPA in five successive cycles with efficiency higher than 85% at the end of the last batch.