Enzyme Adsorption Research Articles

Designing mixed cationic/anionic supports to covalently immobilize/stabilize enzymes with high isoelectric point by enzyme adsorption and support-enzyme glutaraldehyde crosslinking

Ficin fully immobilized on Asp-agarose beads at pH 7 but not on an aminated support. This made enzyme adsorption plus glutaraldehyde modification non-viable for this enzyme. Modifying glyoxyl-agarose beads with mixtures of Asp and 1,6-hexamethylenediamine (HA) at different ratios, mixed anion/cation exchanger supports were built. Only if HA greatly exceed Asp in the support, immobilization did not work. While only using the Asp-agarose support immobilized enzyme molecules were only ionically adsorbed after glutaraldehyde treatment (visualized in SDS-PAGE analysis), the mixed supports gave covalent immobilization. The glutaraldehyde modification of these biocatalysts permitted to establish covalent bonds with the support, and this was more effective when using higher amounts of HA in the support. When around 60 % of the groups in the support were HA, the treatment with glutaraldehyde fully suppressed enzyme release from the support after boiling in SDS. The glutaraldehyde treated biocatalysts were more stable than just the adsorbed enzymes or the enzyme adsorbed only on Asp supports and then treated with glutaraldehyde (the optimal biocatalyst retained 90 % of the initial activity while the just adsorbed ficin retained 50 % of the initial activity). This strategy can be utilized to immobilize other proteins with high isoelectric points following this immobilization strategy.

Effect of surfactants on enzymatic hydrolysis of PET

Enzyme is difficult to adsorb on the surface of high-crystallinity poly(ethylene terephthalate) fibers (PET，crystallinity of 48.56 %) due to the high hydrophobicity of PET fibers. Surfactants can effectively reduce the PET/water interface tension and improve the adsorption of enzyme on PET fibers, increasing the degradation of PET fibers promoted by enzyme. In this paper, different nonionic surfactants (Triton X-100, Triton X-114, Triton X-405, PEG 200, PEG 400, PEG 600, Tween 20, Tween 80) were selected to investigate the effect of surfactant on the degradation of PET fibers by enzyme. It was found that the addition of Triton X-405 can improve the concentration of degradation products. However, other surfactants selected in this paper, such as Tween-80 caused the decrease of the degradation yields. The results indicated that the structure of surfactant plays a very important roll on the degradation of PET catalyzed by enzyme. This is probably due to the transformation of enzyme conformation induced by surfactant, and affects on the activity of enzyme. The hydrophilic surface is beneficial to the adsorption of enzyme onto the surface of PET fibers, and further improve the degradation of PET fibers.

A customized self-assembled synergistic biocatalyst for plastic depolymerization

The enzymatic degradation of plastic offers a green, sustainable strategy and scalable circular carbon route for solving polyester waste. Among the earlies discovered plastic-degrading enzymes are PET hydrolase (PETase) and MHET hydrolase (MHETase), which act synergistically. To promote the adsorption of enzymes on PET surfaces, increase their robustness, and enable directly depolymerization, we designed hydrophobin HFBI fused-PETase and MHETase. A customized self-assembled synergistic biocatalyst (MC@CaZn-MOF) was further developed to promote the two-step depolymerization process. The tailored catalysts showed better adhesion to the PET surface and desirable durability, retaining over 70% relative activity after incubation at pH 8.0 and 60 °C for 120 h. Importantly, MC@CaZn-MOF could directly decompose untreated AGf-PET to generate 9.5 mM TPA with weight loss over 90%. The successful implementation of a bifunctional customized catalyst makes the large-scale biocatalytic degradation of PET feasible, contributing to polymer upcycling and environmental sustainability.

Fabrication of functional diatomaceous earth with enhanced papain enzyme adsorption

Diatomaceous earth (DE) is a sedimentary rock comprising fossilized shells of diatoms, which are a type of algae. The shells of the diatoms are made of silicon dioxide, which is also the main component of DE. DE has excellent moisture absorption and desorption properties and is widely used as an adsorbent, abrasive, insulator, catalyst, and carrier in the stationary phase. It has also been widely studied for its application as a new silicon material in emerging growth fields, such as biosensors. However, because organic matter is adsorbed on the surface of DE, the organic matter must be removed to exploit the properties of mesoporous silica. In this study, the organic matter on the DE surface was removed by alkali treatment to expose the pores and increase the adsorption capacity. Additionally, papain was physisorbed onto DE using a rotator, and l-cysteine was added for papain activation. Under these optimised conditions, the activation of the papain enzyme was effective, resulting in improved DE performance. The adsorption power of DE was correlated with the amount of papain adsorbed, which, in turn, was correlated with its absorbance. Trichloroacetic acid was added to stop the reaction, and the absorbance was measured. The prepared samples were designated as papain/DE (P/DE) complexes. The final concentration of the P/DE was 1 mg/mL. In addition, cross-linked and non-cross-linked P/DE samples were prepared using N-hydroxysuccinimide and water-soluble carbodiimide in a 5:3 ratio and compared. The results showed that surface treatment and functionalisation of DE improved its functionality (enzyme activity), indicating its potential as a new DE material.

Urea Biosensing through Integration of Urease to the PEDOT-Polyamine Conducting Channels of Organic Electrochemical Transistors: pH-Change-Based Mechanism and Urine Sensing

We present the construction of an organic electrochemical transistor (OECT) based on poly(3,4-ethylendioxythiophene, PEDOT) and polyallylamine (PAH) and its evaluation as a bioelectronic platform for urease integration and urea sensing. The OECT channel was fabricated in a one-step procedure using chemical polymerization. Then, urease was immobilized on the surface by electrostatic interaction of the negatively charged enzyme at neutral pH with the positively charged surface of PEDOH-PAH channels. The real-time monitoring of the urease adsorption process was achieved by registering the changes on the drain–source current of the OECT upon continuous scan of the gate potential during enzyme deposition with high sensitivity. On the other hand, integrating urease enabled urea sensing through the transistor response changes resulting from local pH variation as a consequence of enzymatic catalysis. The response of direct enzyme adsorption is compared with layer-by-layer integration using polyethylenimine. Integrating a polyelectrolyte over the adsorbed enzyme resulted in a more stable response, allowing for the sensing of urine even from diluted urine samples. These results demonstrate the potential of integrating enzymes into the active channels of OECTs for the development of biosensors based on local pH changes.

Immobilization of Thiol-Modified Horseradish Peroxidase on Gold Nanoparticles Enhances Enzyme Stability and Prevents Proteolytic Digestion.

The specificity and efficiency of enzyme-mediated reactions have the potential to positively impact many biotechnologies; however, many enzymes are easily degraded. Immobilization on a solid support has recently been explored to improve enzyme stability. This study aims to gain insights and facilitate enzyme adsorption onto gold nanoparticles (AuNPs) to form a stable bioconjugate through the installation of thiol functional groups that alter the protein chemistry. In specific, the model enzyme, horseradish peroxidase (HRP), is thiolated via Traut's reagent to increase the robustness and enzymatic activity of the bioconjugate. This study compares HRP and its thiolated analog (THRP) to deduce the impact of thiolation and AuNP-immobilization on the enzyme activity and stability. HRP, THRP, and their corresponding bioconjugates, HRP-AuNP and THRP-AuNP, were analyzed via UV-vis spectrophotometry, circular dichroism, zeta potential, and enzyme-substrate kinetics assays. Our data show a 5-fold greater adsorption for THRP on the AuNP, in comparison to HRP, that translated to a 5-fold increase in the THRP-AuNP bioconjugate activity. The thiolated and immobilized HRP exhibited a substantial improvement in stability at elevated temperatures (50 °C) and storage times (1 month) relative to the native enzyme in solution. Moreover, HRP, THRP, and their bioconjugates were incubated with trypsin to assess the susceptibility to proteolytic digestion. Our results demonstrate that THRP-AuNP bioconjugates maintain full enzymatic activity after 18 h of incubation with trypsin, whereas free HRP, free THRP, and HRP-AuNP conjugates are rendered inactive by trypsin treatment. These results highlight the potential for protein modification and immobilization to substantially extend enzyme shelf life, resist protease digestion, and enhance biological function to realize enzyme-enabled biotechnologies.

Co-production of fermentable sugars and highly active lignin from eucalyptus via a mild preprocessing with diethylene glycol and chromic chloride

Eucalyptus was pretreated with diethylene glycol catalyzed by 0.02 mol/L CrCl3 for 10 min, resulting in 91 % delignification and 98 % cellulose recovery, with trace fermentation inhibitors generated. After the mild pretreatment, the accessibility and affinity of cellulase to eucalyptus was enhanced, especially since enzyme adsorption rate increased by 1.6-fold. Therefore, glucose yield of pretreated eucalyptus was 7.9-fold higher than that of untreated eucalyptus after hydrolyzed 48 h, in which the maximum glucose concentration reached 62 g/L from eucalyptus by adding Tween 80. According to the characterization analysis, the structure of the eucalyptus lignin-carbohydrate complexes structure was destroyed during the pretreatment, while lignin fragments was likely reacted with diethylene glycol to form the stabilized aromatic ethers. Moreover, the extracted Deg-lignin exhibited better performances than commercial alkali lignin such as higher fluorescence intensity, less negative surface charge, and lower particle size. The mild pretreatment method with diethylene glycol and CrCl3 provided a promising approach for co-production of fermentable sugars and high activity lignin from lignocellulosic biomass.

Study on the mechanism of lignin non-productive adsorption on cellobiohydrolase

Enzymatic hydrolysis is important for lignocellulosic biomass conversion into fermentable sugars. However, the nonproductive adsorption of enzyme on lignin was major hinderance for the enzymatic hydrolysis efficiency. In this study, non-productive adsorption mechanism of cellulase component cellobiohydrolase (CBH) onto lignin was specific investigated. Research revealed that the adsorption behavior of CBH on eucalyptus alkali lignin (EuA) was affected by reaction conditions. As study on the adsorption kinetic, it was indicated that the adsorption cellulose binding domain (CBD) of CBH onto EuA well fitted with Langmuir adsorption model and pseudo second-order adsorption kinetics model. And the tyrosine site related to the adsorption of CBD onto lignin was proved by the fluorescence and UV spectra analysis. The results of this work provide a theoretical guidance to understanding the nonproductive adsorption mechanism and building method to reduce the adsorption of cellulase on the lignin.

Surface-associated residues in subtilisins contribute to poly-L-lactic acid depolymerization via enzyme adsorption.

Poly-L-lactic acid (PLLA) is currently the most abundant bioplastic; however, limited environmental biodegradability and few recycling options diminish its value as a biodegradable commodity. Enzymatic recycling is one strategy for ensuring circularity of PLLA, but this approach requires a thorough understanding of enzymatic mechanisms and protein engineering strategies to enhance activity. In this study, we engineer PLLA depolymerizing subtilisin enzymes originating from Bacillus species to elucidate the molecular mechanisms dictating their PLLA depolymerization activity and to improve their function. The surface-associated amino acids of two closely related subtilisin homologues originating from Bacillus subtilis (BsAprE) and Bacillus pumilus (BpAprE) were compared, as they were previously engineered to have nearly identical active sites, but still varied greatly in PLLA depolymerizing activity. Further analysis identified several surface-associated amino acids in BpAprE that lead to enhanced PLLA depolymerization activity when engineered into BsAprE. In silico protein modelling demonstrated increased enzyme surface hydrophobicity in engineered BsAprE variants and revealed a structural motif favoured for PLLA depolymerization. Experimental evidence suggests that increases in activity are associated with enhanced polymer binding as opposed to substrate specificity. These data highlight enzyme adsorption as a key factor in PLLA depolymerization by subtilisins.

Immobilization of Horseradish Peroxidase onto Montmorillonite/Glucosamine-Chitosan Composite for Electrochemical Biosensing of Polyphenols.

Glucosamine-chitosan synthesized by the Maillard reaction was combined with montmorillonite to obtain a nanohybrid composite to immobilize horseradish peroxidase. The material combines the advantageous properties of clay with those of the chitosan derivative; has improved water solubility and reduced molecular weight and viscosity; involves an eco-friendly synthesis; and exhibits ion exchange capacity, good adhesiveness, and a large specific surface area for enzyme adsorption. The physicochemical characteristics of the composite were analyzed by infrared spectroscopy and X-ray diffraction to determine clay-polycation interactions. The electrochemical response of the different polyphenols to glassy carbon electrodes modified with the composite was evaluated by cyclic voltammetry. The sensitivity and detection limit values obtained with the biosensor toward hydroquinone, chlorogenic acid, catechol, and resorcinol are (1.6 ± 0.2) × 102 µA mM-1 and (74 ± 8) nM; (1.2 ± 0.1) × 102 µA mM-1 and (26 ± 3) nM; (16 ± 2) µA mM-1 and (0.74 ± 0.09) μM; and (3.7± 0.3) µA mM-1 and (3.3 ± 0.2) μM, respectively. The biosensor was applied to quantify polyphenols in pennyroyal and lemon verbena extracts.

Towards a mechanistic model of oxidase deactivation in a bubble column

Despite the many advantages of biocatalysis, enzyme stability remains an issue. This work investigates the long-term kinetic stability of a water-forming NAD(P)H-oxidase (NOX2), an enzyme of potential industrial interest for NAD(P)H regeneration, in a bubble column sparged with air, which enables reasonable oxygen transfer but without drastic enzyme deactivation. Experiments have been performed with the particular goal of explaining the observed two-stage deactivation trend. We show evidence supporting the hypothesis that the first stage is related to the adsorption of enzymes to the interface, followed by a subsequent deactivation stage at the interface.The characterization of NOX deactivation in the bubble column setup, complemented by additional’quiescent’ experiments, has enabled the development of a first principles model as the first step towards a complete mechanistic model of oxidase deactivation at gas–liquid interfaces.

Functionalized electrospun nanofibers to enhance β-Galactosidase immobilization and catalytic activity for efficient galactooligosaccharide synthesis

This study aimed to immobilize β-galactosidase (β-GAL) into enhanced polystyrene (PS) electrospun nanofiber membranes (ENMs) with functionalized graphene oxide (GO). Initially, GO sheets were functionalized by salinization with 3-aminopropyl triethoxysilane (APTES). Then the ENMs (PS, PS/GO, and PS/GO-APTES) were prepared and characterized. Then, the β-GAL was immobilized in the different ENMs to produce the β-GAL-bound nanocomposites (PS-GAL, PS/GO-GAL, and PS/GO-APTES-GAL). Immobilization of β-GAL into PS/GO-APTES significantly improved enzyme adsorption by up to 87 %. Also, PS/GO-APTES-GAL improved the enzyme activity, where the highest enzyme activity was obtained at enzyme concentrations of 4 mg/L, 50 °C, and pH 4.5. Likewise, the storage stability and reusability of immobilized β-GAL were improved. Furthermore, this process led to enhanced catalytic behavior and transgalactosylation efficiency, where GOS synthesis (72 %) and lactose conversion (81 %) increased significantly compared to the free enzyme. Overall, the immobilized β-GAL produced in this study showed potential as an effective biocatalyst in the food industry.

Probing Enzymatic PET Degradation: Molecular Dynamics Analysis of Cutinase Adsorption and Stability.

Understanding the mechanisms influencing poly(ethylene terephthalate) (PET) biodegradation is crucial for developing innovative strategies to accelerate the breakdown of this persistent plastic. In this study, we employed all-atom molecular dynamics simulation to investigate the adsorption process of the LCC-ICCG cutinase enzyme onto the PET surface. Our results revealed that hydrophobic, π-π, and H bond interactions, specifically involving aliphatic, aromatic, and polar uncharged amino acids, were the primary driving forces for the adsorption of the cutinase enzyme onto PET. Additionally, we observed a negligible change in the enzyme's tertiary structure during the interaction with PET (RMSD = 1.35 Å), while its secondary structures remained remarkably stable. Quantitative analysis further demonstrated that there is about a 24% decrease in the number of enzyme-water hydrogen bonds upon adsorption onto the PET surface. The significance of this study lies in unraveling the molecular intricacies of the adsorption process, providing valuable insights into the initial steps of enzymatic PET degradation.

Kinetic Model for the Heterogeneous Biocatalytic Reactions Using Tethered Cofactors.

Understanding the mechanism of interfacial enzyme kinetics is critical to the development of synthetic biological systems for the production of value-added chemicals. Here, the interfacial kinetics of the catalysis of β-nicotinamide adenine dinucleotide (NAD+)-dependent enzymes acting on NAD+ tethered to the surface of silica nanoparticles (SiNPs) has been investigated using two complementary and supporting kinetic approaches: enzyme excess and reactant (NAD+) excess. Kinetic models developed for these two approaches characterize several critical reaction steps including reversible enzyme adsorption, complexation, decomplexation, and catalysis of the surface-bound enzyme/NAD+ complex. The analysis reveals a concentrating effect resulting in a very high local concentration of enzyme and cofactor on the particle surface, in which the enzyme is saturated by surface-bound NAD, facilitating a rate enhancement of enzyme/NAD+ complexation and catalysis. This resulted in high enzyme efficiency within the tethered NAD+ system compared to that of the free enzyme/NAD+ system, which increases with decreasing enzyme concentration. The role of enzyme adsorption onto solid substrates with a tethered catalyst (such as NAD+) has potential for creating highly efficient flow biocatalytic systems.

Advancement in Sustainable Wastewater Treatment: A Multifaceted Approach to Textile Dye Removal through Physical, Biological and Chemical Techniques

AbstractThe global manufacturing sector heavily relies on the textile industry, yet the widespread utilization of synthetic dyes within this sector has raised serious environmental apprehensions, notably regarding wastewater contamination. The release of untreated wastewater containing residual dyes poses a significant risk to both aquatic ecosystems and human well‐being. In reaction to this issue, considerable efforts have been made to develop efficient and sustainable methods for the removal of textile dyes from wastewater. This comprehensive review paper furnishes in‐depth literature information covering various aspects of dyes, including their classification, toxicity effects, and evaluation of recent advancements in dye removal technology, emphasizing their advantages, limitations, and future potential. Various strategies are covered, including physical, chemical, and biological methods. Physical techniques like adsorption, filtration, and membrane technologies are contrasted with chemical approaches such as coagulation, precipitation, and advanced oxidation processes. Biological methods include the utilization of micro‐organisms, fungi, and enzymes to break down or metabolize dye compounds. Key considerations in the critical analysis include the efficiency, cost‐effectiveness, and scalability of each method. This review focuses on enzyme breakdown (biological) and adsorption (physical) color removal procedures, which are currently considered the most effective. It also proposes the utilization of a composite adsorbent, anticipating enhanced efficiency and faster removal of dyes through this technique.

Сенсорное покрытие на основе липидного ленгмюровского монослоя с молекулами фермента глюкозооксидазы

A sensor coating was created based on a Langmuir monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with immobilized molecules of the enzyme glucose oxidase and its sensor properties were studied. The process of incorporation of glucose oxidase enzyme molecules into a Langmuir monolayer of phospholipid DPPE molecules was studied using the compression isotherm method. Adsorption of glucose oxidase molecules has an expanding effect on the Langmuir monolayer of DPPE and leads to an increase in the area per molecule from 32.5 Å2 to 49 Å2 and a decrease in the compression modulus of the monolayer from 133 mN/m to 83 mN/m. Also, the adsorption of glucose oxidase leads to an increase in the desorption coefficient of the monolayer into the kd subphase from 0.5·10-3 to 1.2·10-3. The morphology of films with immobilized enzyme molecules at different adsorption times was characterized by atomic force microscopy. Increasing the enzyme adsorption time leads to a decrease in the surface roughness of the formed film from 3.6 nm at 5 minutes to 2.4 nm at 60 minutes. The formed DPPE monolayers with immobilized enzyme molecules were transferred to graphite electrodes. Their sensory properties were studied using cyclic voltammetry and impedance measurements. The resulting coatings were sensitive to glucose in solutions with concentrations from 0 to 1 mg/ml. The presence of a linear concentration dependence of the maximum and minimum currents and resistances in the specified concentration range makes the created films promising for use as touch coatings.

The Effect of Stereocomplexation and Crystallinity on the Degradation of Polylactide Nanoparticles.

Polymeric nanoparticles (PNPs) are frequently researched and used in drug delivery. The degradation of PNPs is highly dependent on various properties, such as polymer chemical structure, size, crystallinity, and melting temperature. Hence, a precise understanding of PNP degradation behavior is essential for optimizing the system. This study focused on enzymatic hydrolysis as a degradation mechanism by investigation of the degradation of PNP with various crystallinities. The aliphatic polyester polylactide ([C3H4O2]n, PLA) was used as two chiral forms, poly l-lactide (PlLA) and poly d-lactide (PdLA), and formed a unique crystalline stereocomplex (SC). PNPs were prepared via a nanoprecipitation method. In order to further control the crystallinity and melting temperatures of the SC, the polymer poly(3-ethylglycolide) [C6H8O4]n (PEtGly) was synthesized. Our investigation shows that the PNP degradation can be controlled by various chemical structures, crystallinity and stereocomplexation. The influence of proteinase K on PNP degradation was also discussed in this research. AFM did not reveal any changes within the first 24 h but indicated accelerated degradation after 7 days when higher EtGly content was present, implying that lower crystallinity renders the particles more susceptible to hydrolysis. QCM-D exhibited reduced enzyme adsorption and a slower degradation rate in SC-PNPs with lower EtGly contents and higher crystallinities. A more in-depth analysis of the degradation process unveiled that QCM-D detected rapid degradation from the outset, whereas AFM exhibited delayed changes of degradation. The knowledge gained in this work is useful for the design and creation of advanced PNPs with enhanced structures and properties.

Adsorption features of reduced aminated supports modified with glutaraldehyde: Understanding the heterofunctional features of these supports

Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.

Carbamoylase-based impedimetric electronic tongue for rapid detection of paralytic shellfish toxins

Phytotoxins produced by marine microalgae, such as paralytic shellfish toxins (PSTs), can accumulate in bivalve molluscs, representing a human health concern due to the life-threatening symptoms they cause. To avoid the commercialization of contaminated bivalves, monitoring programs were established in the EU. The purpose of this work is the implementation of a PST transforming enzyme—carbamoylase—in an impedimetric test for rapid simultaneous detection of several carbamate and N-sulfocarbamoyl PSTs. Carbamoylase hydrolyses carbamate and sulfocarbamoyl toxins, which may account for up to 90% of bivalve toxicity related to PSTs. Conformational changes of carbamoylase accompanying enzymatic reactions were probed by Fourier transform mid-infrared spectroscopy (FT-MIR) and electrochemical impedance spectroscopy (EIS). Furthermore, a combination of EIS with a metal electrode and a carbamoylase-based assay was employed to harness changes in the enzyme conformation and adsorption on the electrode surface during the enzymatic reaction as an analytical signal. After optimization of the working conditions, the developed impedimetric e-tongue could quantify N-sulfocarbamoyl toxins with a detection limit of 0.1 µM. The developed e-tongue allows the detection of these toxins at concentration levels observed in bivalves with PST toxicity close to the regulatory limit. The quantification of a sum of N-sulfocarbamoyl PSTs in naturally contaminated mussel extracts using the developed impedimetric e-tongue has been demonstrated.

Seed‐Induced Living Two‐Dimensional (2D) Supramolecular Polymerization in Water: Implications on Protein Adsorption and Enzyme Inhibition

AbstractIn biological systems, programmable supramolecular frameworks characterized by coordinated directional non‐covalent interactions are widespread. However, only a small number of reports involve pure water‐based dynamic supramolecular assembly of artificial π‐amphiphiles, primarily due to the formidable challenge of counteracting the strong hydrophobic dominance of the π‐surface in water, leading to undesired kinetic traps. This study reveals the pathway complexity in hydrogen‐bonding‐mediated supramolecular polymerization of an amide‐functionalized naphthalene monoimide (NMI) building block with a hydrophilic oligo‐oxyethylene (OE) wedge. O‐NMI‐2 initially produced entropically driven, collapsed spherical particles in water (Agg‐1); however, over a span of 72 h, these metastable Agg‐1 gradually transformed into two‐dimensional (2D) nanosheets (Agg‐2), favoured by both entropy and enthalpy contributions. The intricate self‐assembly pathways in O‐NMI‐2 enable us to explore seed‐induced living supramolecular polymerization (LSP) in water for controlled synthesis of monolayered 2D assemblies. Furthermore, we demonstrated the nonspecific surface adsorption of a model enzyme, serine protease α‐Chymotrypsin (α‐ChT), and consequently the enzyme activity, which could be regulated by controlling the morphological transformation of O‐NMI‐2 from Agg‐1 to Agg‐2. We delve into the thermodynamic aspects of such shape‐dependent protein‐surface interactions and unravel the impact of seed‐induced LSP on temporally controlling the catalytic activity of α‐ChT.