Biological Catalysts Research Articles

Electric Field-Induced Sequential Prototropic Tautomerism in Enzyme-like Nanopocket Created by Single Molecular Break Junction.

Mastering the control of external stimuli-induced chemical transformations with detailed insights into the mechanistic pathway is the key for developing efficient synthetic strategies and designing functional molecular systems. Enzymes, the most potent biological catalysts, efficiently utilize their built-in electric field to catalyze and control complex chemical reactions within the active site. Herein, we have demonstrated the interfacial electric field-induced prototropic tautomerization reaction in acylhydrazone entities by creating an enzymatic-like nanopocket within the atomically sharp gold electrodes using a mechanically controlled break junction (MCBJ) technique. In addition to that, the molecular system used here contains two coupled acylhydrazone reaction centers, hence demonstrating a cooperative stepwise electric field-induced reaction realized at the single molecular level. Furthermore, the mechanistic studies revealed a proton relay-assisted tautomerization showing the importance of external factors such as solvent in such electric field-driven reactions. Finally, single-molecule charge transport and energetics calculations of different molecular species at various applied electric fields using a polarizable continuum solvent model confirm and support our experimental findings. Thus, this study demonstrates that mimicking an enzymatic pocket using a single molecular junction's interfacial electric field as a trigger for chemical reactions can open new avenues to the field of synthetic chemistry.

Mechanism of Action of Formate Dehydrogenases.

The molybdenum- and tungsten-containing formate dehydrogenases from a variety of microorganisms catalyze the reversible interconversion of formate and CO2; several, in fact, function as CO2 reductases in the reverse direction under physiological conditions. CO2 reduction catalyzed by these enzymes occurs under mild temperature and pressure rather than the elevated conditions required for current industrial processes. Given the contemporary importance of remediation of atmospheric CO2 to address global warming, there has been considerable interest in the application of these enzymes in bioreactors. Equally important, understanding the detailed means by which these biological catalysts convert CO2 to formate, a useful and easily transported feedstock chemical, might also inspire the development of a new generation of highly efficient, biomimetic synthetic catalysts. Here we have examined the ability of the FdsDABG formate dehydrogenase from Cupriavidus necator to catalyze the exchange of solvent oxygen into product CO2 during the course of formate oxidation under single-turnover conditions. Negligible incorporation of 18O is observed when the experiment is performed in H218O, indicating that bicarbonate cannot be the immediate product of the enzyme-catalyzed reaction, as previously concluded. These results, in conjunction with the observation that the reductive half-reaction exhibits mildly acid-catalyzed rather than base-catalyzed chemistry, are consistent with a reaction mechanism involving direct hydride transfer from formate to the enzyme's molybdenum center, directly yielding CO2. Our results are inconsistent with any mechanism in which the initial product formed on oxidation of formate is bicarbonate.

Biological Waste-Derived Dual-Site Catalyst Empowers Electro-Fenton Systems to Sustainably Decontaminate Livestock Wastewater

Biological Waste-Derived Dual-Site Catalyst Empowers Electro-Fenton Systems to Sustainably Decontaminate Livestock Wastewater

Homogeneous HER electrocatalysis using monothiolate ligand-based {FeS} complexes: A review

Renewable energy sources are considered as efficient, low cost and sustainable energy systems with the capability of achieving a decarbonized world. Of particular interest has been the use of hydrogen as an affordable and clean energy for achieving a sustainable (renewable) energy economy. The hydrogenases discovered almost 90 years ago and found in green algae and anaerobic bacteria, are known to be highly efficient biological catalysts for hydrogen evolution in nature. Out of the three types of hydrogenases ([FeFe], [NiFe] and [Fe] only) the [FeFe] hydrogenases have drawn particular attention due to their higher activity/efficiency and their ability to reversibly reduce protons to dihydrogen. Several models of the [FeFe] hydrogenase enzymes have been reported over the last three decades. This review article mainly describes in details the characteristics of mono- and di-nuclear {FeS} complexes with monothiolates (-SR) as the bridging ligands that have been reported as electrocatalysts for the hydrogen evolution reaction (HER).

Production and characterization of acidophilic xylanase from wood degrading white rot fungus by solid-state fermentation of wheat straw

Xylanases (EC 3.2.1.8) catalyze the breakdown of xylan, which is the second most abundant polysaccharide in plant cell walls. Biological catalysts have gained greater global attention than chemical catalysts in different industrial processes because they are highly selective, easy to control and have a negligible environmental impact. The aim of this study was to investigate the xylanolytic potential of white-rot fungi, optimize their physicochemical conditions and characterize the resulting xylanase. Sixty-eight white-rot fungus (WRF) isolates were screened for their xylanolytic potential and growth conditions for maximal xylanase production using cheap agricultural residue (wheat straw) as the sole carbon source. Five WRF isolates with high xylanase yields (73.63 ± 0.0283–63.6 ± 0.01247 U/ml) were selected by qualitative and quantitative screening methods. The optimum xylanase production occurred at pH 5.0 and 28 °C. Solid-state fermentation (SSF) yielded a high amount of xylanase. The highest xylanase activity (80.9–61.274 U/mL) was recorded in the pH range of 5.0–6.5 and at 50 °C. The metal ions Mg2+, Ca2+ and Mn2+ enhanced the activity of xylanase (127.28–110.06 %), while Cu2+, Fe2+ and K+ inhibited the activity with 43.4–17 % losses. The km and Vmax were 0.32–0.545 mg/mL and 86.95–113.63 μmol/min/mg, respectively. This finding indicates that wheat straw can be used for large-scale xylanase production under SSF conditions. The pH and temperature profiles and stabilities indicate that the xylanase produced in the present study can be applied in food and animal feed industries.

Screening for the economic production of hydrolytic enzymes from locally-isolated fungi

Background Enzymes are complex proteins serving as biological catalysts to facilitate reactions in mild and environment-friendly conditions. Saprophytic fungi have long been harnessed for the efficient production of several industrially-significant enzymes whose market is still growing to cope with the increase in demand and natural resources’ depletion. Objective This investigation was performed with respect to the economic viewpoint of terrestrial fungi utilization and their hydrolytic enzymes’ biosynthetic potential. Materials and methods Several terrestrial fungi were isolated, cultivated on cheap agricultural wastes, and evaluated for industrial relevance. Solid-state fermentation was conducted to further boost the economic value and sustainability. The enzymatic productivity was estimated through solid-phase radial diffusion correlating the zones’ diameters to the enzymatic activity. Results and conclusion Six soil fungi were isolated, five belonging to the order Eurotiales and one to Mucorales. The molds belonged to four different genera; Aspergillus sydowii, Aspergillus versicolor, Aspergillus ustus, Fennelia flavipes (anamorph: Aspergillus flavipes), Cunninghamella elegans and Paecilomyces lilacinus. Many of the tested agricultural wastes were able to support the biosynthesis of the explored constitutive enzymes, recording better activity than the standard synthetic medium. Under the test conditions, L-asparaginase and protease were the most frequently detected enzymes while banana and mandarine peels led to the highest enzymes’ activity. In light of the global direction towards sustainability, enzymes can have immense prospects to sustain the industrial sectors innocuously. The cost-effectiveness of the manufacturing processes can be enhanced by accommodating the fiscal challenges for operating conditions. Using agrarian residues as raw material, highly productive enzyme producers, and cheaper solid-state fermentation processes are factors that may contribute to the efficacy, efficiency and economic feasibility of the enzyme-based processes.

Identifying Oncogenic Missense Single Nucleotide Polymorphisms in Human SAT1 Gene Using Computational Algorithms and Molecular Dynamics Tools.

The human SAT1 gene encodes spermidine/spermine N1-acetyltransferase 1 (SSAT1), a regulatory biological catalyst of polyamine catabolism. Numerous essential biological processes, such as cellular proliferation, differentiation, and survival, depend on polyamines like spermidine and spermine. Thus, SSAT1 is involved in key cellular activities such as proliferation and survival of cells and mediates various diseases including cancer. A plethora of studies established the involvement of missense single nucleotide polymorphisms (SNPs) in numerous pathological conditions due to their ability to adversely affect the structure and subsequent function of the protein. To date, an in silico study to identify the pathogenic missense SNPs of the human SAT1 gene has not been accomplished yet. This study aimed to filter the missense SNPs that were functionally detrimental and pathogenic. The rs757435207 (I21N) was ascertained to be the most deleterious and pathogenic by all algorithmic tools. Stability and evolutionary conservation analysis tools also stated that I21N variant decreased the stability and was located in the highly conserved residue. Molecular dynamics simulation revealed that I21N caused substantial alterations in the conformational stability and dynamics of the SSAT1 protein. Consequently, the I21N variant could disrupt the native functional roles of the SSAT1 enzyme. Therefore, the I21N variant was identified and concluded to be an oncogenic missense variant of the human SAT1 gene. Overall, the findings of this study would be a great directory of future experimental research to develop personalized medicine.

Pickering Emulsions Biocatalysis: Recent Developments and Emerging Trends.

Biocatalysis within biphasic systems is gaining significant attention in the field of synthetic chemistry, primarily for its ability to solve the problem of incompatible solubilities between biocatalysts and organic compounds. By forming an emulsion from these two-phase systems, a larger surface area is created, which greatly improves the mass transfer of substrates to the biocatalysts. Among the various types of emulsions, Pickering emulsions stand out due to their excellent stability, compatibility with biological substances, and the ease with which they can be formed and separated. This makes them ideal for reusing both the emulsifiers and the biocatalysts. This review explores the latest developments in biocatalysis using Pickering emulsions. It covers the structural features, methods of creation, innovations in flow biocatalysis, and the role of interfaces in these processes. Additionally, the challenges and future directions are discussed in combining chemical and biological catalysts within Pickering emulsion frameworks to advance synthetic methodologies.

Selection of a promiscuous minimalist cAMP phosphodiesterase from a library of de novo designed proteins

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To probe how unevolved sequences can develop new functions, we use ultrahigh-throughput droplet microfluidics to screen for phosphoesterase activity amidst a library of more than one million sequences based on a de novo designed 4-helix bundle. Characterization of hits revealed that acquisition of function involved a large jump in sequence space enriching for truncations that removed >40% of the protein chain. Biophysical characterization of a catalytically active truncated protein revealed that it dimerizes into an α-helical structure, with the gain of function accompanied by increased structural dynamics. The identified phosphodiesterase is a manganese-dependent metalloenzyme that hydrolyses a range of phosphodiesters. It is most active towards cyclic AMP, with a rate acceleration of ~109 and a catalytic proficiency of >1014 M−1, comparable to larger enzymes shaped by billions of years of evolution.

Eutectogels as Promising Materials in Biocatalysis

AbstractThe entrapment of deep eutectic solvents (DES) and eutectic systems into porous scaffolds renders a new class of soft and nonvolatile materials called eutectogels that have recently stepped into the spotlight in different areas ranging from electronics to drug delivery. Recent progress in the use of DES in biocatalysis, where they have been demonstrated to improve substrate supply, conversion, and enzyme stability, has opened an unparalleled opportunity to exploit the merits of eutectogels for immobilizing biological catalysts. The resulting functional materials could outperform traditional hydrogels and ionic liquid gels, offering fresh perspectives to broaden the application scope of many enzymes. In this perspective, we go into the potential of eutectogels as innovative scaffolds that support biocatalytic reactions and discuss different applications where these systems could show plain benefits compared to traditional materials. Future directions for this newly developed technology are highlighted.

Monitoring Enzyme Activity Using Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

Enzymes serve as pivotal biological catalysts that accelerate essential chemical reactions, thereby influencing a variety of physiological processes. Consequently, the monitoring of enzyme activity and inhibition not only yields crucial insights into health and disease conditions but also forms the basis of research in drug discovery, toxicology, and the understanding of disease mechanisms. In this context, near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) have emerged as effective tools for tracking enzyme activity and inhibition through diverse strategies. This perspective explores the physicochemical attributes of SWCNTs that render them well-suited for such monitoring. Additionally, we delve into the various strategies developed so far for successfully monitoring enzyme activity and inhibition, emphasizing the distinctive features of each principle. Furthermore, we contrast the benefits of SWCNT-based NIR probes with conventional gold standards in monitoring enzyme activity. Lastly, we highlight the current challenges faced in this field and suggest potential solutions to propel it forward. This perspective aims to contribute to the ongoing progress in biodiagnostics and seeks to engage the wider community in developing and applying enzymatic assays using SWCNTs.

The Rhodium Analogue of Coenzyme B12 as an Anti‐Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors

AbstractCoenzyme B12 (AdoCbl; 5′‐deoxy‐5′‐adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light‐responsive cobalt‐corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA‐binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl‐bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non‐natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound “base‐off/His‐on” to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti‐photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl‐based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non‐radical syn‐1,2‐elimination mechanism, an aspect not previously considered for this photoreceptor.

The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

A Potentially Versatile Enzyme Sensor Platform: Enzyme-Loaded, Tagged, Porous Polymeric Nanocapsules.

Enzymes are essential to life and indispensable in a wide range of industries (food, pharmaceutical, medical, biosensing, etc.); however, a significant shortcoming of these fragile biological catalysts is their poor stability. To address this challenge, a variety of immobilization methods have been described to enhance the enzyme's stability. These immobilization methods generally are specific to an individual enzyme or optimal for a particular application. The aim of this study is to explore the utility of porous, indicator moiety-tagged, polymeric nanocapsules (NCs) for the encapsulation of enzymes and measurement of the enzyme's substrate. As a model enzyme, glucose oxidase (GOx) is used. The GOx enzyme-loaded, fluorophore-tagged NCs were synthesized by using self-assembled surfactant vesicle templates. To show that the biological activity of GOx is preserved during entrapment, the rate of the GOx enzyme catalyzed reaction was measured. To evaluate the protective features of the porous NCs, the encapsulated GOx enzyme activity was followed in the presence of hydrolytic enzymes. During the encapsulation of GOx and the purification of the GOx-loaded NCs, the GOx activity decayed less than 10%, and up to 30% of the encapsulated GOx activity could be retained for 3-5 days in the presence of hydrolytic enzymes. In support of the potentially unique advantages of the enzyme-loaded NCs, as a proof-of-concept example, the fluorophore-tagged, GOx-loaded NCs were used for the determination of glucose in the concentration range between 18 and 162 mg/dL and for imaging the distribution of glucose concentration in imaging experiments.

Traveling wave solution and the stability of critical points of an enzyme-inhibitor system under diffusion effects: with special reference to dimer molecule

Enzymes are absolutely essential biological catalysts in human body that catalyze all cellular processes in physiological network. However, there are certain low molecular weight chemical compounds known as inhibitors, that reduce or completely inhibit the enzyme catalytic activity. Mathematical modeling plays a key role in the control and stability of metabolic enzyme inhibition. Enzyme stability is an important issue for protein engineers, because of its great importance and impact on optimal utility of material in biological tissues. In this outlook, we have first determined the existence of traveling wave solution for the enzyme-inhibitor system and then emphasized the stability of critical points that arise in the reactions. The study of traveling wave solution of an enzyme-inhibitor system with reaction diffusion equations involve quite complex mathematical analysis. The results obtained in this model indicate that the traveling wave solution may give a well explained method for improving enzyme kinetic stability. The present study will be helpful in understanding the stability of critical points of an enzyme-inhibitor system to give an idea about the inhibition of less stable enzymes. Moreover, the role of diffusion on the enzyme activity has been exhaustively discussed using mathematical tools related to eigen values and eigen function analysis.

Biosensors Precision in Healthcare: A Scientific Odyssey

Biosensors have been the beacon of precision in the ever-evolving landscape of health care and offer a profound impact on diagnostics and patient care. These devices are designed to detect specific biological markers and convert then into measurable signals, seamlessly blending biology with cutting-edge technology. The diverse tapestry of biosensors encompass enzyme based sensors, immuno-assays, photo-biosensors, tissue-based sensors, DNA biosensors and nano-biosensors. Enzyme-based biosensors showcase unparalleled catalytic precision exemplified by glucose biosensors. Glucose oxidase serves as the biological catalyst and converts glucose into gluconic acid and hydrogen peroxide. Electrodes of the sensor then transduce this reaction into measurable signals which enables the precise quantification of glucose levels in diabetes management. Similarly, immunoassays rely on the specificity of antigen-antibody interactions. Most widely used immunoassay, Enzyme-linked immunosorbent assay (ELISA) exemplify this category, which finds applications in a number of diagnostics and provide a window into immunological responses and diseases such as HIV. Advancing the field of biosensing are the photobiosensors which utilize diverse technologies such as fluorescence resonance energy transfer (FRET) to illuminate biological processes. Genetically encoded biosensors that employ proteins like green fluorescent protein (GFP), enable non-invasive monitoring of cellular dynamics and offer real-time insights. Another cateogory, tissue-based biosensors converge the technology with the physiological environments. For instance, implantable glucose sensors that continuously monitor blood glucose concentrations and provide real-time data for personalized treatment approaches. At the core of precision diagnostics lie DNA biosensors such as DNA microarrays. They offer parallel analysis of gen expression, influencing decisions in cancer diagnostics and pharmacogenomics. Nanobiosensors, represented by nanoparticle-based sensors, navigate the nano-frontiers with exceptional sensitivity. For example, quantum dots exhibit unique optical properties for sensitive detection. Likewise, gold nanoparticles based biosensors enable real-time monitoring of drug release in drug delivery systems and emphasize their role in precision medicine. As biosensors advance, ethical considerations come to the forefront. Issues such as data privacy, consent, and equitable access to these technologies demand careful attention. It becomes imperative to ensure that biosensors are developed and deployed responsibly with a keen awareness of potential societal implications in navigating the ethical landscape. In the symphony of precision that biosensors bring to healthcare, their types and applications reflect not only technological prowess but also the ethical responsibility inherent in their development and deployment. As these devices continue to evolve, the healthcare landscape stands on the brink of a new era, where precision and ethics intertwine for the betterment of patient outcomes.

The use of biological catalyst (enzyme) for enhanced oil recovery in Niger Delta

This study is aimed at using bio-catalyst (enzymes) for enhanced oil recovery in Niger Delta. Three different enzymes produced from microbial isolates with code ID; LPE1, LPE2 and AME were use in analyzing enzymes effect on the rock-fluid and fluid-fluid interaction test to simulate the conditions in the reservoir. The tests carried out include; wettability, interfacial tension (IFT) and adhesion test. The results from these tests show that addition of enzymes to rock-fluid interface has the potential of enhancing recovery of crude oil. From the result, the use of enzyme caused an alteration in the wettability of the rock from oil-wet to water-wet with a 33° difference. The selected enzyme LPE1 showed a significant difference in reduction of interfacial tension by a factor of 4, while LPE2 and AME did not show any significant difference. In conclusion, the wettability and IFT tests results were the basis for choosing LPE1 for core flooding process. Enzyme (LPE1) flooding after waterflood was able to give an additional 11.5 % oil recovery. From the results, the percentage recovery is a pointer to the fact that when the primary and secondary oil recovery methods are no longer viable in terms of economics, the Enyme enhanced oil recovery is the best method of mobilizing trapped crude oil in the Secondly, there is no negative effect on the crude oil quality using the enzyme-EOR because the enzymes only increase the rate of the chemical reaction on the rock media without undergoing any permanent chemical change.

Non-statistical assembly of multicomponent [Pd2ABCD] cages.

Self-assembled hosts, inspired by biological receptors and catalysts, show application potential in sustainable synthesis, energy conversion and medicine. Implementing multiple functionalities in the form of distinguishable building blocks, however, is difficult without risking narcissistic self-sorting or a statistical mess. Here we report a systematic series of integratively self-assembled heteroleptic cages in which two square-planar PdII cations are bridged by four different bis-pyridyl ligands, A, B, C and D, via synergistic effects to exclusively form a single isomer-the lantern-shaped cage [Pd2ABCD]. This self-sorting goal-forming just one out of 55 possible structures-is reached under full thermodynamic control and can be realized progressively (by combining progenitors, such as [Pd2A2C2] with [Pd2B2D2]), directly from ligands and PdII cations or by mixing all four corresponding homoleptic cages. The rational design of complex multicomponent assemblies that enables the modular incorporation of diverse chemical moieties will advance their applicability in functional nanosystems.

Influence of acetate concentration on acetone production by a modified Acetobacterium woodii

Global warming is the driving force for developing production processes of chemical compounds based on CO2 reduction technologies. Bacteria can act as biological catalysts that reduce this gaseous substrate in added-value compounds. Acetobacterium woodii is one of the best-performing strains on H2–CO2 blends and naturally produces acetate. Acetone is a raw material deeply used in the chemical industry, and its global demand is increasing. Acetone-butanol-ethanol (ABE) fermentation is the oldest microbial production platform for acetone synthesis from organic substrates, and Clostridium acetobutylicum is the model strain for its production. In various wild-type acetogens and ABE-producing Clostridium species, acetate positively influences the synthesis of reduced products. In this work, a modified A. woodii strain expressing the enzymes of the acetone pathway from C. acetobutylicum was used to convert H2–CO2 streams into acetone. This study aims to assess the impact of acetate on acetone production catalyzed by such a modified A. woodii. Tests were carried out in serum bottles and a continuous stirred tank reactor up to a pressure of 10 bar, in gas-batch or in continuous gassing, providing different gas mixes. Outcomes indicated that acetone synthesis was stimulated when acetate concentration in the medium exceeded the threshold of 100–120 mM. Thus, acetic acid can affect acetone productivity in the modified A. woodii strain. This outcome should be considered in the design of fermentation processes, especially in setting up fermentations with the liquid continuous operative mode.

Enzyme stability in polymer hydrogel-enzyme hybrid nanocarrier containing phosphorylcholine group.

Enzymes are biological catalysts with good biocompatibility and high efficiency and have been widely used in many fields, such as wastewater treatment, biosensors, and the medical industry. However, their inherently low stability under conditions of practical use limits further applications. Zwitterionic polymers possessing a pair of oppositely charged groups in their repeating units can increase protein stability because of their good biocompatibility and high water content. In this study, zwitterionic copolymer nanogels comprising poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-methacrylic acid-N-hydroxy succinimide ester (MNHS)) (PMS) were synthesized via reversible addition-fragmentation chain-transfer polymerization (RAFT). β-Galactosidase (β-gal) was post-modified within zwitterionic polymer nanogels with a covalently-bound spacer and the activity was compared with that of directly immobilized β-gal and free β-gal. Compared with direct immobilization, covalent immobilization with a spacer could reduce the structural change of β-gal, as confirmed by the circular dichroism spectra. Although the activity of β-gal decreased after immobilization, the hybrids of the β-gal immobilized nanogels, termed hybrid nanogel-enzymes, demonstrated superior stability compared to the free enzymes. The hybrid nanogel-enzymes maintained their function against inactivation by organic solvents and proteinases owing to their high water content, anti-biofouling properties, and limited mass transfer. They can also withstand protein aggregation at high temperatures and maintain their activity. Compared to direct immobilization, immobilization with a spacer resulted in a dramatic increase in the enzyme activity and a slight decrease in the stability. These results indicate that polymer nanogels containing phosphorylcholine units are promising materials for enzyme immobilization, expanding the scope of enzyme applications.