Free Enzyme In Solution Research Articles

Tuning Multistep Biocatalysis through Enzyme and Cofactor Colocalization in Charged Porous Protein Macromolecular Frameworks.

Spatial organization of biocatalytic activities is crucial to organisms to efficiently process complex metabolism. Inspired by this mechanism, artificial scaffold structures are designed to harbor functionally coupled biocatalysts, resulting in acellular materials that can complete multistep reactions at high efficiency and low cost. Substrate channeling is an approach for efficiency enhancement of multistep reactions, but fast diffusion of small molecule intermediates poses a major challenge to achieve channeling in vitro. Here, we explore how multistep biocatalysis is affected, and can be modulated, by cofactor-enzyme colocalization within a synthetic bioinspired material. In this material, a heterogeneous protein macromolecular framework (PMF) acts as a porous host matrix for colocalization of two coupled enzymes and their small molecule cofactor, nicotinamide adenine dinucleotide (NAD). After formation of the PMF from a higher order assembly of P22 virus-like particles (VLPs), the enzymes were partitioned into the PMF by covalent attachment and presentation on the VLP exterior. Using a collective property of the PMF (i.e., high density of negative charges in the PMF), NAD molecules were partitioned into the framework via electrostatic interactions after being conjugated to a polycationic species. This effectively controlled the localization and diffusion of NAD, resulting in substrate channeling between the enzymes. Changing ionic strength modulates the PMF-NAD interactions, tuning two properties that impact the multistep efficiency oppositely in response to ionic strength: cofactor partitioning (colocalization with the enzymes) and cofactor mobility (translocation between the enzymes). Within the range tested, we observed a maximum of 5-fold increase or 75% decrease in multistep efficiency as compared to free enzymes in solution, which suggest both the colocalization and the mobility are critical for the multistep efficiency. This work demonstrates utility of collective behaviors, exhibited by hierarchical bioassemblies, in the construction of functional materials for enzyme cascades, which possess properties such as tunable multistep biocatalysis.

Acoustomicrofluidic synthesis of ZIF-8/HRP metal–organic framework composites with enhanced enzymatic activity and stability

Encapsulating enzymes within metal–organic frameworks (MOFs) constitute a promising strategy to circumvent their fragile nature in harsh environments, although deleterious molecular conformational changes and decreased substrate accessibility within the pores of the MOF during enzyme loading can often result in decreased catalytic activity. Herein, we report a rapid (within seconds) and facile one-pot aerosol-based method for simultaneous MOF crystallisation and enzyme encapsulation for the synthesis of ZIF-8/horseradish peroxidase (HRP) biocomposites using a high frequency acoustomicrofluidic nebulisation platform. As a consequence of the enhanced mixing and fast crystallisation afforded by the acoustic coupling into the precursors, the resultant combination of enhanced porosity, missing-linker/metal-node defect creation, pore activation and favourable conformational changes to the enzyme secondary structure allows for more efficient enzyme loading and greater enzyme–substrate interactions, and hence stronger enzyme binding within the MOF structure. Together, these culminate in a biocomposite that possesses four times greater enzymatic activity compared to that for the same material synthesized using conventional bulk-solution techniques, and endows the enzyme with increased protective effect (by approximately three to seven times) against high temperature and organic solvents, even after several exposure cycles. Such a possibility facilitates better potential for the enzyme to be recycled—an important factor given their typically high costs and the difficulty in recovering free enzymes in solution.

Immobilization of Tyrosinase and Its Application

Immobilized enzymes are more robust and resistant to environmental changes than free enzymes in solution. More crucially, the immobilized enzyme systems' heterogeneity enables facile recovery of enzymes and products, multiple reuses, continuous enzymatic processes, quick reaction termination, and a more comprehensive range of bioreactor designs. This paper examines recent findings on enzyme immobilization using diverse approaches for various uses. The information gathered from the reactions catalyzed by the encapsulated tyrosinase provided a good view of hetero-biocatalysts in the phenol biosensor industries. This review proposes an effective method for immobilizing tyrosinase biomolecules into a silica aerogel matrix. Silica matrix has been utilized to encapsulate a wide range of biomolecules, mainly in sol-gel composites. We also discovered that silica aerogel synthesized from sol-gel method retains all the immobilized enzyme activity. The use of a silica matrix for enzyme immobilization, in conjunction with a moderate immobilization method, results in the successful retention of enzyme activity. Future studies should explore practical encapsulating approaches and inventively modified supports to enhance the commercialization of immobilized enzymes and offer fresh perspectives to the industrial sector.

Adenylate Kinase Fused to Spidroin as a Catalyst for Decreasing Leakage out of 3D-Bioprinted Hydrogels and for ATP Regeneration.

Numerous metabolic reactions and pathways use adenosine 5'-triphosphate (ATP) as an energy source and as a phosphorous or pyrophosphorous donor. Based on three-dimensional (3D)-printing, enzyme immobilization can be used to improve ATP regeneration and operability and reduce cost. However, due to the relatively large mesh size of 3D-bioprinted hydrogels soaked in a reaction solution, the lower-molecular-weight enzymes cannot avoid leaking out of the hydrogels readily. Here, a chimeric adenylate-kinase-spidroin (ADK-RC) is created, with ADK serving as the N-terminal domain. The chimera is capable of self-assembling to form micellar nanoparticles at a higher molecular scale. Although fused to spidroin (RC), ADK-RC remains relatively consistent and exhibits high activity, thermostability, pH stability, and organic solvent tolerance. Considering different surface-to-volume ratios, three shapes of enzyme hydrogels are designed, 3D bioprinted, and measured. In addition, a continuous enzymatic reaction demonstrates that ADK-RC hydrogels have higher specific activity and substrate affinity but a lower reaction rate and catalytic power compared to free enzymes in solution. With ATP regeneration, the ADK and ADK-RC hydrogels significantly increase the production of d-glucose-6-phosphate and obtain an efficient usage frequency. In conclusion, enzymes fused to spidroin might be an efficient strategy for maintaining activity and reducing leakage in 3D-bioprinted hydrogels under mild conditions.

Spatial Localization of Two Enzymes at Pickering Emulsion Droplet Interfaces for Cascade Reactions.

Developing biocatalytic cascades in abiological conditions is of utmost significance, but such processes often suffer from low reaction efficiency because of incompatible reaction environments and suppressed intermediate transportation. Herein we report a new type of biocatalytic cascade by localizing two different enzymes separately in the outer and inner interfacial layers of Pickering emulsion droplets. This versatile approach enables the localization of two enzymes in their preferred reaction microenvironments and simultaneously in nanoscale proximity of each other. The thus-designed interfacial biocatalytic cascades show outstanding catalytic efficiency in alkene epoxidation and thioether oxidation with in situ generation of hydrogen peroxide under mild conditions, 6.9-13.6 times higher than the catalytic efficiency of the free enzymes in solution and their multi-enzymatic counterparts. The remarkable interfacial effect of Pickering droplets was found to be responsible for the significantly enhanced cascading efficiency.

Spatial Localization of Two Enzymes at Pickering Emulsion Droplet Interfaces for Cascade Reactions

AbstractDeveloping biocatalytic cascades in abiological conditions is of utmost significance, but such processes often suffer from low reaction efficiency because of incompatible reaction environments and suppressed intermediate transportation. Herein we report a new type of biocatalytic cascade by localizing two different enzymes separately in the outer and inner interfacial layers of Pickering emulsion droplets. This versatile approach enables the localization of two enzymes in their preferred reaction microenvironments and simultaneously in nanoscale proximity of each other. The thus‐designed interfacial biocatalytic cascades show outstanding catalytic efficiency in alkene epoxidation and thioether oxidation with in situ generation of hydrogen peroxide under mild conditions, 6.9–13.6 times higher than the catalytic efficiency of the free enzymes in solution and their multi‐enzymatic counterparts. The remarkable interfacial effect of Pickering droplets was found to be responsible for the significantly enhanced cascading efficiency.

3D Printing: An Emerging Technology for Biocatalyst Immobilization.

Employment of enzymes as biocatalysts offers immense benefits across diverse sectors in the context of green chemistry, biodegradability, and sustainability. When compared to free enzymes in solution, enzyme immobilization proposes an effective means of improving functional efficiency and operational stability. The advance of printable and functional materials utilized in additive manufacturing, coupled with the capability to produce bespoke geometries, has sparked great interest toward the 3-dimensional (3D) printing of immobilized enzymes. Printable biocatalysts represent a new generation of enzyme immobilization in a more customizable and adaptable manner, unleashing their potential functionalities for countless applications in industrial biotechnology. This review provides an overview of enzyme immobilization techniques and 3D printing technologies, followed by illustrations of the latest 3D printed enzyme-immobilized industrial and clinical applications. The unique advantages of harnessing 3D printing as an enzyme immobilization technique will be presented, alongside a discussion on its potential limitations. Finally, the future perspectives of integrating 3D printing with enzyme immobilization will be considered, highlighting the endless possibilities that are achievable in both research and industry.

Are enzymes transported in soils by water fluxes?

Transport of extracellular hydrolytic enzymes in soils has always been a subject of doubt. The considerations against its importance are that (i) enzymes benefit their producers the most when they remain in close proximity; and (ii) enzymes are large molecules with low mobility due to high affinity to fine soil particles and organic matter. However, soil mineral colloids (SMC), to which extracellular enzymes also have an affinity and which are known to facilitate transport of a broad variety of chemicals and microorganisms in soils, can serve as vehicles for enzyme transport as well. Since current literature lacks information on enzyme transport in soils, our goal was to determine whether enzymes are transported and, if so, whether they are transported in a free- or in a colloid-associated form. We conducted column transport experiments with four hydrolytic enzymes, namely, β-glucosidase, acid-phosphatase, cellobiohydrolase, and xylosidase, in soils with contrasting textures. The eluents containing enzymes were applied on top of soil columns, while enzyme activities, SMC, and electrical conductivity were measured in the effluents from the columns. Our results provided evidence of joint enzyme transport with soil colloids. The enzymes associated with the coarse SMC (1 μm < Ø) contributed 52–88% of the total enzyme activity in the effluents. The remaining enzyme activity was attributed to the enzymes associated with organic colloids, fine SMC (Ø < 1 μm) and free enzymes in solution. This study suggested a dual effect of ionic strength in the soil suspension on enzyme activity and their release from soils with soil colloids.

Identification of bioactive natural products using yeast：Application to monoacylglycerol lipase inhibitor extraction from Corydalis Rhizoma

Identification of bioactive principles from natural products is considered a challenging task in drug discovery. Recently, ligand fishing has been growing in interest as a sustainable strategy. In this study, a yeast-based drug discovery strategy was investigated to screen and fish active ingredients from natural products. Human monoacylglycerol lipase (MAGL) was first displayed on the cell wall of Pichia pastoris. The expression of MAGL on the cell surface was confirmed by immunofluorescence analysis. The activity toward 7-HCA which was consistent with free enzymes in solution. Recombinant yeast strains were used to screen the potential inhibitors from traditional Chinese medicines. Preliminary screening showed that the extracts of 12 herbs showed inhibition on MAGL activity, among which Corydalis Rhizoma had the highest inhibition rate of 60.66 ± 2.44%. Recombinant yeast fishing combined with HPLC-Q-TOF-MS/MS analysis was further used to identify the potential MAGL inhibitors. Finally, dehydrocorydaline (DHC) in Corydalis Rhizoma was determined as a ligand to MAGL with the IC50 value at 154.7 μΜ. Corydalis Rhizoma has been used as a pain reliever in TCM. Intraperitoneal injection of 7 mg kg− 1 DHC in chronic constriction injury model rats significantly attenuated the mechanical allodynia and thermal hyperalgesia. Meanwhile, 2-arachidonoylglycerol, the major MAGL substrate in the brain, was significantly increased both in the hippocampus and striatum. In conclusion, yeast-based ligand fishing combined with HPLC-Q-TOF-MS/MS is a powerful strategy for drug discovery in complex mixtures and DHC from Corydalis Rhizoma was confirmed with high inhibitory activity to MAGL either in vitro or in vivo .

Polyethyleneimine-Functionalized Carbon Nanotubes Enabling Potent Antimycotic Activity of Lyticase.

In this work, the positively-charged polymer polyethyleneimine was used to functionalize carbon nanotubes and activated carbon to load antimycotic enzyme lyticase. Interestingly, polyethyleneimine played a dual role functionalizing carbon materials to synergistically enhance antimycotic activity of loaded lyticase as well as exhibiting its own apparent antimycotic activity, where the enhanced enzymatic activity of loaded lyticase on functionalized carbon nanotubes was more than 2.8 times as high as the activity of free enzyme in solution. The actual activity of loaded lyticase on functionalized carbon nanotubes was applied with Penicillium janthinellum, exhibiting much faster digesting lysis of the bacteria in comparison with free lyticase. The synergistic and potent antimycotic activities from combined action of antimycotic lyticase and polyethyleneimine on carbon nanotubes provides a new antimycotic protection for medicine, food industry, and other biochemical processes.

Hierarchically encapsulating enzymes with multi-shelled metal-organic frameworks for tandem biocatalytic reactions

Biocatalytic transformations in living organisms, such as multi-enzyme catalytic cascades, proceed in different cellular membrane-compartmentalized organelles with high efficiency. Nevertheless, it remains challenging to mimicking biocatalytic cascade processes in natural systems. Herein, we demonstrate that multi-shelled metal-organic frameworks (MOFs) can be used as a hierarchical scaffold to spatially organize enzymes on nanoscale to enhance cascade catalytic efficiency. Encapsulating multi-enzymes with multi-shelled MOFs by epitaxial shell-by-shell overgrowth leads to 5.8~13.5-fold enhancements in catalytic efficiencies compared with free enzymes in solution. Importantly, multi-shelled MOFs can act as a multi-spatial-compartmental nanoreactor that allows physically compartmentalize multiple enzymes in a single MOF nanoparticle for operating incompatible tandem biocatalytic reaction in one pot. Additionally, we use nanoscale Fourier transform infrared (nano-FTIR) spectroscopy to resolve nanoscale heterogeneity of vibrational activity associated to enzymes encapsulated in multi-shelled MOFs. Furthermore, multi-shelled MOFs enable facile control of multi-enzyme positions according to specific tandem reaction routes, in which close positioning of enzyme-1-loaded and enzyme-2-loaded shells along the inner-to-outer shells could effectively facilitate mass transportation to promote efficient tandem biocatalytic reaction. This work is anticipated to shed new light on designing efficient multi-enzyme catalytic cascades to encourage applications in many chemical and pharmaceutical industrial processes.

Tailoring surface properties of functionalized graphene papers aiming to enzyme immobilization

The use of enzymes as catalysts requires recovery and reuse to make the process viable. Enzymatic immobilization changes enzyme stability, activity, and specificity. It is very important to explore new substrates for immobilization with appropriate composition and structure to improve the efficiency of the immobilized enzymes. This work explores the use of two different graphene oxide papers, one produced by oxidation route (GO) and the other by electrochemical synthesis (EG), aiming for β-galactosidase immobilization. The chemical and structural properties of these two papers were characterized by Raman spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction. Atomic force microscopy images showed that EG paper ensured more efficient immobilization of the enzymes on the surface of the paper. Cyclic voltammetry was used to monitor the reaction of conversion of lactose to glucose in the free enzyme solution and graphene paper immobilized enzyme solutions. The cyclic voltammetry analysis showed that immobilized enzymes on GO paper showed an improvement in the activity of β-galactose when compared to free enzyme solution, as well as enzyme immobilized on a glassy carbon electrode.

Dual confinement of high–loading enzymes within metal–organic frameworks for glucose sensor with enhanced cascade biocatalysis

The intrinsically fragile nature and leakage of the enzymes is a major obstacle for the commercial sensor of a continuous glucose monitoring system. Herein, a dual confinement effect is developed in a three dimensional (3D) nanocage-based zeolite imidazole framework (NC-ZIF), during which the high-loading enzymes can be well encapsulated with unusual bioactivity and stability. The shell of NC-ZIF sets the first confinement to prevent enzymes leakage, and the interior nanocage of NC-ZIF provides second confinement to immobilize enzymes and offers a spacious environment to maintain their conformational freedom. Moreover, the mesoporosity of the formed NC-ZIF can be precisely controlled, which can effectively enhance the mass transport. The resulted GOx/Hemin@NC-ZIF multi-enzymes system could not only realize rapid detection of glucose by colorimetric and electrochemical sensors with high catalytic cascade activity (with an 8.3-fold and 16-fold enhancements in comparison with free enzymes in solution, respectively), but also exhibit long-term stability, excellent selectivity and reusability. More importantly, the based wearable sweatband sensor measurement results showed a high correlation (>0.84, P < 0.001) with the levels measured by commercial glucometer. The reported dual confinement strategy opens up a window to immobilize enzymes with enhanced catalytic efficiency and stability for clinical-grade noninvasive continuous glucose sensor.

Comparisons of Catalytic Efficiency during the Reduction of Lignocellulosic Substrates between Free Enzyme and Enzyme bound to Mobile Enzyme Sequestration Platforms (MESP)

The cellulolytic bacterium Clostridium thermocellum employs a flexible cellulosome that can bind a variety of enzymes to breakdown cellulosic biomass. A key feature of the cellulosome is a cohesin (Type 1) – dockerin (Type 1) interaction that binds dockerin-endowed enzymes to the cohesin-ladened scaffoldin backbone. Multiple enzymes of various enzyme classes (e.g., exocellulases, endocellulases, xylanases) can bind to the cellulosome structure for synergistic deconstruction of cellulosic substrates. Several “artificial cellulosomes” have been engineered in an attempt to mimic this system for abiotic decomposition of lignocellulosic substrates. One such engineered system is called a mobile enzyme sequestration platform (MESP). The prototype of this platform was called a “rosettasome”. MESPs are chimera of an archaeal group II chaperonin permutant fused to C. thermocellum cohesin via a 9-12 amino acid linker. The archaeal chaperonin complex is thermotolerant. In prior work our lab demonstrated that binding cellulolytic enzymes to MESPs enhances lignocellulose breakdown efficiency over free enzyme in solution (sans platform). Interestingly, our prior data indicated that MESP systems may also be acidotolerant since substrate was treated with mild sulfuric acid. In this study, we tested the acidotolerance of MESP technology as well as the efficiency of carbohydrate reduction on other feedstock (e.g., rice straw). We also tested our MESPs against the prototype rosettasome to determine if this next-generation platform outperforms the original system. Here we present the results of our comparisons. Understanding the limits of MESP enhancement will direct future engineering of this system for industrial and/or agricultural applications.

THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity

BackgroundConventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass.ResultsIn this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation.ConclusionsEnzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.

Cellulase immobilization on TEOS encapsulated and APTES functionalized magnetic nanoparticles (MNPs)

Immobilized enzymes are more stable and more resistant to environmental changes in contrast to free enzymes in solution. Magnetic nanoparticles (MNPs) are potential immobilization supports materials for enzymes and easily recovered for reuse. It has low toxicity effects and fast separation from reaction mixtures simply by use of external magnets. Enzyme immobilization technology also helps to boost the enzyme’s properties by using the technique to keep it in separate support or matrix. The aim of immobilization is to render the enzyme immobile so that it is reusable during the reaction, which leads to a more efficient process and easy separation from the substance. The aims of this investigation were to encapsulate (coat) and functionalize MNPs by using TEOS and APTES respectively for reusable cellulase immobilization on that as a supports matrix. The silicon encapsulation with TEOS and amino functionalization of synthesized MNPs was confirmed by the FT-IR and XRD. Cellulase was immobilized successfully on that nanoparticles and also confirmed by FT-IR, cellulase assay. The immobilized enzymes showed a satisfactory level of reusability (51%) after 10 consecutive reaction cycles.

Catalytic properties of variously immobilized mushroom tyrosinase: A kinetic study for future development of biomimetic amperometric biosensors

Mushroom tyrosinase was immobilized by direct embedding into electrode material (modified carbon paste electrode), incorporation of cross-linked enzyme aggregates into a polymer membrane (glassy carbon electrode covered by thin layer of Nafion®), and covalent attachment using self-assembled monolayers (gold electrode with the chemically bound enzyme). Both, standard UV–Vis spectrophotometry and amperometry in a batch configuration are presented as complementary methods to study the tyrosinase enzyme kinetics, whose catecholase activity results in electroactive products (ortho-quinones). Due to higher sensitivity of amperometric detection, evident advantage in the enzyme consumption was obtained. Prepared amperometric tyrosinase biosensors were characterized using cyclic voltammetry and atomic force microscopy. The Michaelis constant values of immobilized and unbound tyrosinase (free enzyme solution) towards dopamine and catechol were compared. The apparent Michaelis constant values for immobilized tyrosinase are significantly lower than the declared value of 0.840 mmol L−1 dopamine for the unbound enzyme. The enzyme tyrosinase arranged in self-assembled monolayer serves as an efficient sensor due to low apparent Michaelis constant of 0.061 mmol L−1 dopamine and high maximum reaction velocity of 0.458 μA s−1. This fact reflects the ideal arrangement of enzyme molecules causing high availability of the binding site. Tris-glycine sodium dodecyl sulphate polyacrylamide gel electrophoresis and atomic force microscopy clarified that the protein of molecular weight 25 kDa is bound preferably on chemically modified gold electrode. A sensor prepared by the immobilization of tyrosinase on gold electrode results in higher catecholase activity towards dopamine than in case of CPE and GC electrodes, where enzyme is immobilized physically.

Function and structural stability of protein adsorbed to swellable organosilica

Abstract Swellable organically modified silica (SOMS) is a mesoporous sol-gel derived material that spontaneously swells >6 mL/g upon addition organic liquids. Adsorption of 8 different proteins to swollen-state SOMS was studied as a function of adsorbate molecular size and solution pH. Adsorption capacity was dependent on molecular size with the highest capacity of 690 mg/g measured for wheat germ acid phosphatase (58 kDa), a mid-size protein. Adsorption capacities were generally independent of pH suggesting protein-protein interactions do not play a major role in the adsorption mechanism. Rate of adsorption was measured at different protein loadings and fit to a pseudo second-order rate equation. Adsorption rates were independent of loading state when the pH was near a protein's isoelectric point (pI), but varied when the pH differed from the pI of adsorbate. Adsorption was found to irreversible for all proteins when rinsed with buffer solution or ethanol. Infrared spectroscopy of adsorbed cytochrome c and myoglobin indicated that both proteins maintain a native folded state that is stabilized to thermal denaturation. Adsorbed streptavidin was found to bind FITC-biotin (63% binding site saturation) suggesting that adsorbed proteins are accessible to small molecules in bulk solution. Acid phosphatase showed catalytic activity when adsorbed to swollen SOMS, however, specific activity of the bound enzyme was 10-fold lower compared to free enzyme in solution. Adsorption to SOMS appears to be a one-step, reagent-free method to irreversibly immobilize small to mid-sized proteins. Data supports an adsorption mechanism where protein molecules become sterically entrapped in the flexible mesoporous material.

Sequential Co-immobilization of Enzymes in Metal-Organic Frameworks for Efficient Biocatalytic Conversion of Adsorbed CO2 to Formate.

The main challenges in multienzymatic cascade reactions for CO2 reduction are the low CO2 solubility in water, the adjustment of substrate channeling, and the regeneration of co-factor. In this study, metal-organic frameworks (MOFs) were prepared as adsorbents for the storage of CO2 and at the same time as solid supports for the sequential co-immobilization of multienzymes via a layer-by-layer self-assembly approach. Amine-functionalized MIL-101(Cr) was synthesized for the adsorption of CO2. Using amine-MIL-101(Cr) as the core, two HKUST-1 layers were then fabricated for the immobilization of three enzymes chosen for the reduction of CO2 to formate. Carbonic anhydrase was encapsulated in the inner HKUST-1 layer and hydrated the released CO2 to . Bicarbonate ions then migrated directly to the outer HKUST-1 shell containing formate dehydrogenase and were converted to formate. Glutamate dehydrogenase on the outer MOF layer achieved the regeneration of co-factor. Compared with free enzymes in solution using the bubbled CO2 as substrate, the immobilized enzymes using stored CO2 as substrate exhibited 13.1-times higher of formate production due to the enhanced substrate concentration. The sequential immobilization of enzymes also facilitated the channeling of substrate and eventually enabled higher catalytic efficiency with a co-factor-based formate yield of 179.8%. The immobilized enzymes showed good operational stability and reusability with a cofactor cumulative formate yield of 1077.7% after 10 cycles of reusing.

The phage display of Bacillus subtilis Lipase A significantly enhances catalytic activity due to altered nanoscale distribution in colloidal solution.

Screening libraries of mutant proteins by phage display is now relatively common. However, one unknown factor is how the bacteriophage scaffold itself influences the properties of the displayed protein. This communication evaluates the effect of solution parameters on the catalytic activity of phage displayed Bacillus subtilis Lipase A (BSLA), compared to the free enzyme in solution. While the pH- and temperature-activity profiles of BSLA were not intrinsically affected by phage display, the nanoscale distribution of BSLA within the micellar assay buffer was. This lead to a pronounced increase of activity of phage-BSLA relative to the free enzyme, owing to the accumulation of phage-BSLA at the substrate-rich micelles. Considering this result obtained for BSLA, caution is warranted and similar effects should be considered when selecting other enzymes/proteins by phage display, as the activity of the displayed protein may differ from that of the free protein.