Biocatalytic Performance Research Articles

Evaluating Immobilization of Hemoglobin onto Nanomesoporous MCM-41

Introduction: The development and applications of biological products have been seriously hindered by conventional immobilization for its low efficiency and high cost. The immobilization on solid supports for enzyme is an area of intense research due to their widespread use in synthetic chemistry and various industries. This paper highlights a great interest in the adsorption of an enzyme on the solid porous structured material and the design of new controlled delivery systems. The enzyme immobilized on solid supports and synthetic materials of the reaction mixture with strong mechanical force and easy separation serves as a high selective catalyst. Objective: A novel protein delivery system for hemoglobin (Hb) enzyme was proposed by incorporating the molecular molecules into the mesopores of well-ordered hexagonal nanometer MCM (Mobil Composition of Matters) - 41. The prepared adsorbents were successfully applied to the design and synthesis of new functionalization materials. Various parameters affecting adsorption process, such as adsorption time, adsorption isotherm, and the reusability of adsorbent, were investigated. Methods: The interaction between Hb and MCM-41 was investigated using powder X-ray diffraction (XRD), Fourier infrared spectroscopy, UV-visible solid diffuse reflectance spectroscopy and 77 K lowtemperature N2 adsorption-desorption study. Results: The experimental parameters were optimized, including the concentrations of Hb, the MCM-41 amount, and the interior surface of phenyl-functionalized Ph-MCM-41 materials. Under the optimized conditions, the biocatalytic performance was studied for Hb/MCM-41 and Hb/Ph-(MCM-41). The adsorption process of Hb by MCM-41 / Ph-(MCM-41) was in agreement with the quasi-two-order kinetic model. Process of Hb adsorption by MCM-41 / Ph-(MCM-41) belongs to an exothermic reaction, the reaction is not reversible at 4°C and it is a spontaneous reaction. The Freundlich model can better describe the adsorption of hemoglobin on MCM-41 / Ph-(MCM-41). During the desorption process of composite (MCM-41)-Hb/[Ph-(MCM-41)]-Hb in 0.1 mol/L NaOH solution, the desorption rate can reach above 70% at 2 min. At 60 min, the desorption reached equilibrium and the desorption rates were 99.58% and 91.36%, respectively. The reuse activity experimental results indicated that the immobilized enzyme exhibited high catalytic activity. Reusability stability studies suggested that the prepared composites retained their activity even after five recycling runs. This shows that the phenylation of MCM-41 reduced the "leakage" of enzyme in the main material. Conclusion: The results of the present study demonstrated that Hb/MCM-41 and Hb/Ph-(MCM-41) are highly efficient potential nanobiocatalysts for the immobilization of enzymes onto mesoporous materials.

Rational Design of a Biocatalyst Based on Immobilized CALB onto Nanostructured SiO2

The adsorption of the lipase B from Candida antarctica (CALB) over nanostructured SiO2 (Ns SiO2 from now on) with and without the addition of polyols (sorbitol and glycerol) was investigated. The isotherms of adsorption made it possible to establish that the maximum dispersion limit was 0.029 µmol of protein per surface area unit of Ns SiO2 (29.4 mg per 100 mg of support), which was reached in 30 min of exposure. The studies through SDS-PAGE of the immobilization solutions and infrared spectroscopy of the prepared solids determined that CALB (from a commercial extract) is selectively adsorbed, and its secondary structure distribution is thus modified. Its biocatalytic activity was corroborated through the kinetic resolution of rac-ibuprofen. Conversions of up to 70% and 52% enantiomeric excess toward S-ibuprofen in 24 h of reaction at 45 °C were achieved. The biocatalytic performance increased with the increase in protein loading until it leveled off at 0.021 µmol.m−2, reaching 0.6 µmol.min−1. The biocatalyst containing the lipase at the maximum dispersion limit and co-adsorbed polyols presented the best catalytic performance in the kinetic resolution of rac-ibuprofen, an improved thermal resistance (up to 70 °C), and stability under long-term storage (more than 2 years).

Electrochemical characterization of a dual cytochrome-containing lactate dehydrogenase

Flavin-dependent L-lactate dehydrogenase (LDH) from baker’s yeast (Saccharomyces cerevisiae) reversibly catalyzes the oxidation of L-lactate to L-pyruvate. In this study, four different enzymatic constructs were generated, and their catalytic and electrochemical properties were compared. Specifically, a truncated form of the native enzyme that includes only the catalytic domain, the native enzyme that includes an intrinsic electron-transferring cytochrome b2, a novel artificial enzyme containing a minimal cytochrome c and a version of the enzyme containing a fusion between two cytochromes were designed. All four variants were successfully expressed in Escherichia coli and presented properly matured heme domains. Assessing in vitro biocatalytic performance as reflected by lactate oxidation revealed the fusion-containing enzyme to be ∼ 12 times more active than the native enzyme. Electrochemical studies of electrode drop-casted enzyme variants also showed the superior performance of the dual-cytochrome construct, which displayed a lower average redox-potential for lactate oxidation, oxygen insensitivity in the lactate oxidation potential range and a wider dynamic range for lactate sensing, relative to the native enzyme. Moreover, product inhibition of this variant occurred at much higher lactate concentrations than with the native enzyme. In addition, when lower potentials were scanned using cyclic voltammetry, lactate-dependent oxygen reduction was measured for the dual-cytochrome fusion enzyme.

Improvement of Laccase Production by Thielavia terrestris Co3Bag1. Enhancing the Bio-Catalytic Performance of the Native Thermophilic TtLacA via Immobilization in Copper Alginate Gel Beads.

A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.

Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum

Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO2) into multicarbon biochemicals. Genotype-phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic performance; however, systemic investigation on the fitness contribution of individual gene has been limited. Here, we report genome-scale CRISPR interferencescreening using 41,939 guide RNAs designed from the E.limosum genome, one of the model acetogenic species, where all genes were targeted for transcriptional suppression. We investigated the fitness contributions of 96% of the total genes identified, revealing the gene fitness and essentiality for heterotrophic and autotrophic metabolisms. Our data show that the Wood-Ljungdahl pathway, membrane regeneration, membrane protein biosynthesis, and butyrate synthesis are essential for autotrophic acetogenesis in E.limosum. Furthermore, we discovered genes that are repression targets that unbiasedly increased autotrophic growth rates fourfold and acetoin production 1.5-fold compared to the wild-type strain under CO2-H2 conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.

Thermodynamics, Conformation, and Biocatalytic Performance of Glucose Oxidase Combined with Black Phosphorus Quantum Dots.

Glucose oxidase (GOD) has a wide range of applications in biosensing and cancer treatment as a result of its unique biocatalytic properties. More importantly, GOD could synergistically enhance the cancer therapeutic effect when combined with other cancer therapeutic strategies. However, the interaction of GOD with a cancer therapeutic agent has not been well-studied. Herein, the thermodynamic properties of the interaction between black phosphorus quantum dots (BPQDs) and GOD were systematically elucidated, and the dose-dependent conformational and enzymatic activity changes of BPQDs on GOD were quantitatively and qualitatively analyzed. The results indicated that the stoichiometric ratio of BPQDs to GOD was approximately 1:1. In particular, fluorescence spectroscopy, circular dichroism spectra, and Fourier transform infrared spectra have synergistically studied the changes in secondary and tertiary conformations of GOD induced by BPQDs. Higher doses of BPQDs resulted in a loose structure of GOD but still maintained the native conformation and preserved effective enzymatic activity, effectively catalyzing the production of H2O2 from glucose in a cell. The interaction mechanism between BPQDs and GOD provides a theoretical basis for the design of GOD-based multimodal synergistic cancer therapy and its clinical translation analysis.

Tailoring a highly conductive and super-hydrophilic electrode for biocatalytic performance of microbial electrolysis cells

Bioelectrochemical hydrogen production via microbial electrolysis cells (MECs) has attracted attention as the next generation of technology for the hydrogen economy. MECs work by electrochemically active bacteria reducing organic compounds at the anode. However, the hydrophobic nature of carbon-based anodes suppresses the release of the produced gas and water penetration, which significantly reduces the possibility of microbial attachment. Consequently, a limited surface area of the anode is used, which decreases hydrogen production efficiency. In this study, the bifunctional material poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was applied to the surface of a three-dimensional carbon felt anode to enhance the hydrogen production efficiency of an MEC owing to the high conductivity of PEDOT and super-hydrophilicity of PSS. In experiments, the PEDOT:PSS-modified anode almost doubled the hydrogen production efficiency of the MEC compared with the control anode owing to the increased capacitance current (239.3 %) and biofilm formation (220.7 %). The modified anode reduced the time required for the MEC to reach a steady state of hydrogen production by 14 days compared to the control anode. Microbial community profiles demonstrated that the modified anode had a greater abundance of electrochemically active bacteria than the control anode. This simple method could be widely applied to various bioelectrochemical systems (e.g., microbial fuel cells and solar cells) and to scaling up MECs.

Extracting extremophilic lipases from aqueous streams by using biocompatible ionic liquids

In this work, biocompatible ionic liquids based on aminoacids were employed as extractants to separate extremolipases from aqueous streams. First, the influence of aminoacid and dipeptide-based ionic liquids (cholinium glycinate, ChGly, and cholinium glycylglycinate, ChGlygly) on the lipolytic activity of a commercial lipase from Candida antarctica (CaLB) and in-house synthesized extremophilic lipases from Thermus thermophilus HB27 (TtHB27L) and Halomonas sp. LM1C (HL) was investigated. The combination of thermophilic enzyme with ChGly turned out to be the optimum combination for maximizing the biocatalytic performance, clearly improving the levels attained when water was exclusively employed as solvent and also surpassing the activity levels provided for the commercial enzyme CaLB. The salting out capacity of ChGly in aqueous solutions of biodegradable surfactants Tergitol 15S7 and Tergitol 15S9 was discussed, recording immiscibility areas almost covering all the ternary diagrams. The aqueous biphasic systems were experimentally characterized by determining both tie-lines and solubility curves at several temperatures and the data was modelled with relevant equations like Merchuk, Othmer-Tobias and Bancroft ones, as they are the most common ones to describe this kind of equilibrium data. So, ChGly was applied to extract thermophilic and commercial lipases from aqueous solutions at 313.15 K, achieving very high extraction levels (about 100 %) for TtHB27L, which clearly surpasses the maximum extraction values observed for the commercial enzyme (about 80 %). Finally, the process was simulated at real scale through SuperPro Designer v.8.5 for the production of 385 Kg/year of extremolipase.

One-step immobilization of β-glucosidase in crude enzyme solution by recyclable UCST-responsive polymer with enhanced uniformly biocatalytic performance

In this study, the upper critical solution temperature (UCST)-responsive polymers poly (ethylene oxide) monomethyl ether-block-poly(acrylamide-co-acrylonitrile) (PEG-b-p(AAM-co-AN) were synthesized and successfully utilized to immobilize β-glucosidase in crude enzyme solution. These UCST-responsive β-glucosidase biocatalysts (PEG-b-p(AAM-co-AN@LytA-Glu) have specific UCST with tunable transition temperature, which could be tuned the separation temperature to the desired temperature range. The P2 @ LytA-Glu with an UCST of about 42.9 ℃ was exploited by one-step covalent immobilization of β-glucosidase in crude enzyme solution. The prepared P2 @ LytA-Glu exhibited significantly improved temperature, pH, storage, and operation stabilities compared with that of free enzyme. The catalytic rate of P2 @ Glu-LytA was 14.5% higher than that of P2-Glu (immobilized pure β-glucosidase), which indicated that one-step immobilization of crude enzyme directly from crude enzyme solution was feasible, and it can greatly save the purification step and reduce the experimental cost. The engineered UCST-responsive immobilized enzymes are potentially useful for the practical green biocatalysis.

Assessing Peroxygenase-Mediated Oxidations in the Presence of High Concentrations of Water-Miscible Co-Solvents

The use of water-miscible organic co-solvents in biocatalysis is a simple procedure for obtaining higher enzymatic activities toward hydrophobic substrates. However, effects on activity and stability have to be carefully evaluated, also with regard to the type and concentration of the respective co-solvent. In this contribution, we investigated and evaluated the effect of some common water-miscible co-solvents on the biocatalytic performance of the recombinant unspecific peroxygenase rAaeUPO from Agrocybe aegerita. rAaeUPO showed promising activities in the presence of high concentrations of the best co-solvent acetonitrile, which enabled to use higher substrate concentrations (≥100 mM). Employing high acetonitrile concentrations for UPO-mediated oxidation of ethylbenzene to (R)-1-phenylethanol was demonstrated under preparative scale conditions and led to product accumulation rates of 31 mM h−1.

Protein Mediated Enzyme Immobilization.

Enzyme immobilization is an essential technology for commercializing biocatalysis. It imparts stability, recoverability, and other valuable features that improve the effectiveness of biocatalysts. While many avenues to join an enzyme to solid phases exist, protein-mediated immobilization is rapidly developing and has many advantages. Protein-mediated immobilization allows for the binding interaction to be genetically coded, can be used to create artificial multienzyme cascades, and enables modular designs that expand the variety of enzymes immobilized. By designing around binding interactions between protein domains, they can be integrated into functional materials for protein immobilization. These materials are framed within the context of biocatalytic performance, immobilization efficiency, and stability of the materials. In this review, supports composed entirely of protein are discussed first, with systems such as cellulosomes and protein cages being discussed alongside newer technologies like spore-based biocatalysts and forizymes. Protein-composite materials such as polymersomes and protein-inorganic supraparticles are then discussed to demonstrate how protein-mediated strategies are applied to many classes of solid materials. Critical analysis and future directions of protein-based immobilization are then discussed, with a particular focus on both computational and design strategies to advance this area of research and make it more broadly applicable to many classes of enzymes.

Kurthia gibsonii Mb126 immobilised chitinase against Aspergillus flavus, a fungal pathogen linked to lemon postharvest deterioration

Background of the study: As fungi become resistant to commonly used pesticides, fungicides are becoming not only more expensive, but also less effective in controlling postharvest infections. New chitinase immobilisation techniques are urgently needed to minimize enzyme costs while enhancing bio catalytic performance, antifungal activity, enzyme stability, and reusability. Objectives: The objective of this study was to immobilise chitinase of Kurthia gibsonii Mb126 by entrapping it in calcium alginate beads and to analyse its antifungal activities against Aspergillus flavus. Methods: The optimal parameters influencing the immobilization process and the characteristics of soluble and immobilised chitinase of K. gibsonii Mb126 were analysed. The antifungal activities of immobilised and free chitinase of K. gibsonii Mb126 against A. flavus, which was isolated from decayed lemon fruit, were performed using the agar-disk diffusion method. Free chitinase 25.0 mL (0.8 U/mL) and immobilised chitinase 0.06 g (specific activity 124–192 U/mg) were treated on separate lemon fruits for testing antifungal activity against A. flavus. Findings: K. gibsonii Mb126 chitinase was immobilised perfectly in calcium alginate beads. After optimising process parameters of immobilisation (sodium alginate concentration 3%, calcium chloride 0.2 M, 120 min. curing time), the specific activity of K. gibsonii Mb126 immobilised chitinase improved to 11.9-fold greater than the free form of chitinase and the immobilisation yield increased to 84%. It was observed that the thermal stability and storage stability of immobilised chitinase were better than those of free enzymes. The immobilised chitinase could be reused, and it retained 78% activity even after 16 cycles. The surface morphology of immobilised chitinase was observed in a scanning electron microscope at different magnification powers. Enzyme kinetics was studied and compared with that of its chitinase soluble counterpart. An in vitro study demonstrated that immobilised chitinase of K. gibsonii Mb126 has higher antifungal activity against A. flavus. In vivo experimental study of theantifungal nature of chitinase in lemon fruits infected by A. flavus indicated that immobilised chitinase significantly decreased the incidence of postharvest decay. Conclusion: The immobilised chitinase of K. gibsonii Mb126 can be utilised as an important source of sustainable and environmentally friendly fungicides to prevent pathogens and saprophytic fungi from food. Novelty: To the best of our knowledge, this is the one and only study report about the antifungal nature of immobilised chitinase against a fungus that causes postharvest decay in lemon fruits. Keywords: Kurthia gibsonii Mb126; Aspergillus flavus; immobilised chitinase; postharvest decay of lemon; sodium alginate

Cold-Active Lipase-Based Biocatalysts for Silymarin Valorization through Biocatalytic Acylation of Silybin

Extremophilic biocatalysts represent an enhanced solution in various industrial applications. Integrating enzymes with high catalytic potential at low temperatures into production schemes such as cold-pressed silymarin processing not only brings value to the silymarin recovery from biomass residues, but also improves its solubility properties for biocatalytic modification. Therefore, a cold-active lipase-mediated biocatalytic system has been developed for silybin acylation with methyl fatty acid esters based on the extracellular protein fractions produced by the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania). The extracellular production of the lipase fraction was enhanced by 1% olive-oil-enriched culture media. Through multiple immobilization approaches of the cold-active putative lipases (using carbodiimide, aldehyde-hydrazine, or glutaraldehyde coupling), bio-composites (S1–5) with similar or even higher catalytic activity under cold-active conditions (25 °C) have been synthesized by covalent attachment to nano-/micro-sized magnetic or polymeric resin beads. Characterization methods (e.g., FTIR DRIFT, SEM, enzyme activity) strengthen the biocatalysts’ settlement and potential. Thus, the developed immobilized biocatalysts exhibited between 80 and 128% recovery of the catalytic activity for protein loading in the range 90–99% and this led to an immobilization yield up to 89%. The biocatalytic acylation performance reached a maximum of 67% silybin conversion with methyl decanoate acylating agent and nano-support immobilized lipase biocatalyst.

A robust biocatalyst based on laccase immobilized superparamagnetic Fe3O4@SiO2–NH2 nanoparticles and its application for degradation of chlorophenols

The presence of chlorophenols in water and wastewater is considered a serious environmental issue. To eliminate these micropollutants, biodegradation of chlorophenols using enzyme-nanoparticle conjugated biocatalyst, is proposed as an economical and eco-friendly method. Herein, amino-functionalized superparamagnetic Fe3O4@SiO2–NH2 nanoparticles with core-shell structure were constructed as a promising carrier for immobilization of laccase from Trametes versicolor. Compared with free laccase, Fe3O4@SiO2–NH2-Laccase displayed remarkable outcomes in all major areas such as temperature and storage stabilities, and tolerance to organic solvents and metal ions. The biocatalytic performance and reusability of Fe3O4@SiO2–NH2-Laccase were evaluated for the degradation of 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP) in repeated cycles. Even after 10 successive reuses, the degradation rate of 2,4-DCP and 2,4,6-TCP were found to be 54.9% and 68.7%, respectively. The influences of solution pH, initial chlorophenol concentration, and temperature on the degradation rate of these two chlorophenols were evaluated. The degradation intermediate products including dimers, trimers, and tetramers of 2,4-DCP and 2,4,6-TCP were identified. Release of chloride ions was observed during the enzymatic degradation of these two chlorophenols. Based on the determination of intermediate products and released chloride ions, the degradation pathway that was involved in dehydrogenation, reactive radical intermediates formation, dechlorination, self-coupling and oligomers/polymers formation was proposed. The toxicity of these two chlorophenols and their intermediates was substantially reduced during the enzymatic degradation. The results of this study might present an alternative clean biotechnology for the remediation of 2,4-DCP and 2,4,6-TCP contaminated water matrices.

One-Pot Surface Modification of β-Cu2O NPs for Biocatalytic Performance against A-549 Lung Carcinoma Cell Lines through Docking Analysis.

The physicochemical approaches and biological principles in bio-nanotechnology favor specially functionalized nanosized particles. Cuprous oxide nanoparticles (β-Cu2O NPs) of cuprite phase with a little tenorite (CuO) may be very effective in the development of novel therapeutic approaches against several fatalities including A-549 lung carcinoma cell lines. Consequently, the synthesis of β-Cu2O NPs for the improvement in the therapeutic index and drug delivery application is becoming an effective strategy in conventional anticarcinoma treatment. Hence, surface-enhanced nanosized spherical cuprous oxide nanoparticles (β-Cu2O NPs) of cuprite phase were successfully prepared using poly(ethylene glycol) (PEG) as an amphiphilic nonionic surfactant and l-ascorbic acid (K3[Cu(Cl5)]@LAA-PEG) reduced to cuprites β-Cu2O NPs via the sonochemical route. Less improved toxicity and better solubility of β-Cu2O NPs compared with Axitinib were a major reason for producing β-Cu2O NPs from K3[Cu(Cl5)]@LAA-PEG (LAA, l-ascorbic acid, PEG, poly(ethylene glycol) (PEG)). These nanoparticle syntheses have been suggested to influence their cytotoxicity, free-radical scavenging analysis, and reactive oxygen species (ROS) using poly(ethylene glycol) (PEG) and l-ascorbic acid (LAA) as coated and grafted materials due to their dose-dependent nature and IC50 calculations. The surface morphology of the formed β-Cu2O NPs has been examined via UV–vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy with energy diffraction scattering spectroscopy (SEM@EDS), field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM) analysis. X-ray diffraction (XRD) and Brunauer–Emmett–Teller (BET) surface analysis results confirm the presence of pure cuprite with a very little amount of tenorite (CuO) phase, Dynamic light scattering (DLS) confirms the negative ζ-value with stable nature. Docking was performed using PDB of lung carcinomas and others, as rigid receptors, whereas the β-Cu2O NP cluster was treated as a flexible ligand.

Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones.

Enantiopure α‐hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine‐diphosphate‐dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild‐type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co‐solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T50) and a different co‐solvent‐dependent activity. Using the most stable variant immobilized in packed‐bed reactors via the SpyCatcher/SpyTag system, (R)‐benzoin was synthesized from benzaldehyde over a period of seven days with a stable space‐time‐yield of 9.3 mmol ⋅ L‐1 ⋅ d−1. Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α‐hydroxy ketones and APIs.

Surface Modulation of Graphene Oxide for Amidase Immobilization with High Loadings for Efficient Biocatalysis.

As a type of important and versatile biocatalyst, amidase immobilization on solid materials has received broad attention with its relatively easy procedure and available reusability. However, current porous supports have suffered from limited loadings, and it is highly desired to develop a new type of material with abundant space so as to ensure a high loading of amidase. Here, graphene oxide was adopted as the support for amidase immobilization, which showed the highest loading capacity for amidase (~3000 mg/g) to date. To the best of our knowledge, it is the first case of amidase immobilized on graphene oxide. Through surface modulation via reducing the contents of oxygen-containing functional groups, activity recovery of immobilized amidase increased from 67.8% to 85.3%. Moreover, surface-modulated graphene oxide can efficiently uptake amidase under a wide range of pH, and the maximum loading can reach ~3500 mg/g. The resultant biocomposites exhibit efficient biocatalytic performance for asymmetric synthesis of a chiral amino acid (i.e., L-4-fluorophenylglycine, an intermediate of aprepitant).

Challenges and Opportunities for the Encapsulation of Enzymes over Porous Solids for Biodiesel Production and Cellulose Valorization into Glucose

AbstractDifferent porous supports of enzyme catalysts are reviewed here for the valorization of biomass and production of biodiesel, given the high yields and mild reaction conditions in the presence of the biocatalysts. Two main types of enzymes have been widely employed for such endeavors, i. e. lipase and cellulase, which are able to produce fatty acid alkyl esters and glucose from fats and cellulose, respectively. Robust and versatile porous silica, carbon, polymers and metal‐organic frameworks are among the most employed supports of the enzymes, via simple adsorption or covalent grafting. Herein, we provide a comprehensive review by comparing the catalytic performance of the enzymes supported over porous solids with respect to the bulk/free enzymes in terms of yields, stability and recycling, highlighting the benefits of the porous support for an optimal biocatalytic performance in the biodiesel production and cellulose valorization into glucose. The final section provides our views for further developments in this field.

Magnetic Nanoparticle-Containing Supports as Carriers of Immobilized Enzymes: Key Factors Influencing the Biocatalyst Performance.

In this short review (Perspective), we identify key features of the performance of biocatalysts developed by the immobilization of enzymes on the supports containing magnetic nanoparticles (NPs), analyzing the scientific literature for the last five years. A clear advantage of magnetic supports is their easy separation due to the magnetic attraction between magnetic NPs and an external magnetic field, facilitating the biocatalyst reuse. This allows for savings of materials and energy in the biocatalytic process. Commonly, magnetic NPs are isolated from enzymes either by polymers, silica, or some other protective layer. However, in those cases when iron oxide NPs are in close proximity to the enzyme, the biocatalyst may display a fascinating behavior, allowing for synergy of the performance due to the enzyme-like properties shown in iron oxides. Another important parameter which is discussed in this review is the magnetic support porosity, especially in hierarchical porous supports. In the case of comparatively large pores, which can freely accommodate enzyme molecules without jeopardizing their conformation, the enzyme surface ordering may create an optimal crowding on the support, enhancing the biocatalytic performance. Other factors such as surface-modifying agents or special enzyme reactor designs can be also influential in the performance of magnetic NP based immobilized enzymes.

Design and Construction of Bioreactor Based on Hybrid Microcapsules and its Bio-catalytic Performance

Design and Construction of Bioreactor Based on Hybrid Microcapsules and its Bio-catalytic Performance