Lactose hydrolysis in packed-and fluidized-bed reactors using a recombinant ß-galactosidase immobilized on magnetic core-shell capsules.

The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core-shell enzymatic system featuring ß-galactosidase fused to a Cellulose Binding Domain (CBD) tag (ß-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing ß-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability.

Enhanced Immobilization of Enzymes on Plasma Micro-Nanotextured Surfaces and Microfluidics: Application to HRP.

The enhanced and direct immobilization of the enzyme horseradish peroxidase on poly(methyl methacrylate) (PMMA) microchannel surfaces to create a miniaturized enzymatic reactor for the biocatalytic oxidation of phenols is demonstrated. Enzyme immobilization occurs by physical adsorption after oxygen plasma treatment, which micro-nanotextures the PMMA surfaces. A five-fold enhancement in immobilized enzyme activity was observed, attributed to the increased surface area and, therefore, to a higher quantity of immobilized enzymes compared to an untreated PMMA surface. The enzymatic reaction yield reached 75% using a flow rate of 2.0 µL/min for the reaction mixture. Additionally, the developed microreactor was reused more than 16 times without affecting the enzymatic conversion yield. These results demonstrate the potential of microchannels with plasma micro/nanotextured surfaces for the rapid and facile fabrication of microfluidic enzymatic microreactors with enhanced catalytic activity and stability.

Recent trends using natural polymeric nanofibers as supports for enzyme immobilization and catalysis.

All the disciplines of science, especially biotechnology, have given continuous attention to the area of enzyme immobilization. However, the structural support made by material science intervention determines the performance of immobilized enzymes. Studies have proven that nanostructured supports can maintain better catalytic performance and improve immobilization efficiency. The recent trends in the application of nanofibers using natural polymers for enzyme immobilization have been addressed in this review article. A comprehensive survey about the immobilization strategies and their characteristics are highlighted. The natural polymers, e.g., chitin, chitosan, silk fibroin, gelatin, cellulose, and their blends with other synthetic polymers capable of immobilizing enzymes in their 1D nanofibrous form, are discussed. The multiple applications of enzymes immobilized on nanofibers in biocatalysis, biosensors, biofuels, antifouling, regenerative medicine, biomolecule degradation, etc.; some of these are discussed in this review article.

Nanotechnology and enzyme immobilization: a review.

The synergy between enzymes and nanotechnology (nano-biocatalysts) has created some of the most promising biomaterials fabricated by synergistically incorporating advanced nano-biotechnology. The incorporation of enzymes into nanotechnology is of great significance for making nanomaterials that are rarely harmful to the environment. However, the unique/specific physicochemical characteristics and supramolecular nature ascribed to functional nanostructures (nanomaterials), have made them novel, interesting, and exceptional matrices for the creation of nano-biocatalysts. These have a lot of potential for improving the enzyme stability, function, efficiency, kinetic characteristics, vulnerability to diffusional constraints, and engineering performance in bioprocessing. Hence, the nano-biocatalysts developed contain exceptional properties with many potential applications in diverse fields. This review covers a wide range of the nanotechnology and enzyme technology involved in producing nano-biocatalysts, including different mechanisms, strategies in nanomaterial enzyme immobilization, and various nanocarriers, as well as recent developments in controlling enzyme activity. The vast range of potential applications of nano-biocatalysts in various fields, including food, pharmaceuticals, biofuels, and bioremediation, has been discussed.

Metal-Organic Framework-Based Artificial Organelle Corrects Microenvironment Interference for Accurate Intratumoral Glucose Analysis.

Enzymatic catalysis with high efficiency allows them a great prospect in metabolite monitoring in living cells. However, complex tumor microenvironments, such as acidity, H2 O2 , and hypoxia, are bound to disturb catalytic reactions for misleading results. Here, we report a spatially compartmentalized artificial organelle to correct intratumoral glucose analysis, where the zeolitic imidazolate framework-8 immobilized glucose oxidase-horseradish peroxidase cascade core and catalase-directed shell act as signal transduction and guarding rooms respectively. The acid-digested core and stable shell provide appropriate spaces to boost biocatalytic efficiency with good tolerability. Notably, the endogenous H2 O2 is in situ decomposed to O2 by catalase, which not only overcomes the interference in signal output but also alleviates the hypoxic states to maximize glucose oxidation. The marked protective effect and biocompatibility render artificial organelles to correct the signal transduction for dynamic monitoring glucose in vitro and in vivo, achieving our goal of accurate intratumoral metabolite analysis.

Overview on immobilization of enzymes on synthetic polymeric nanofibers fabricated by electrospinning.

The arrangement and type of support has a significant impact on the efficiency of immobilized enzymes. 1-dimensional fibrous materials can be one of the most desirable supports for enzyme immobilization. This is due to their high surface area to volume ratio, internal porosity, ease of handling, and high mechanical stability, all of which allow a higher enzyme loading, release and finally lead to better catalytic efficiency. Fortunately, the enzymes can reside inside individual nanofibers to remain encapsulated and retain their three-dimensional structure. These properties can protect the enzyme's tolerance against harsh conditions such as pH variations and high temperature, and this can probably enhance the enzyme's stability. This review article will discuss the immobilization of enzymes on synthetic polymers, which are fabricated into nanofibers by electrospinning. This technique is rapidly gaining popularity as one of the most practical ways to fibricate polymer, metal oxide, and composite micro or nanofibers. As a result, there is interest in using nanofibers to immobilize enzymes. Furthermore, present research on electrospun nanofibers for enzyme immobilization is primarily limited to the lab scale and industrial scale is still challanging. The primary future research objectives of this paper is to investigate the use of electrospun nanofibers for enzyme immobilization, which includes increasing yield to transfer biological products into commercial applications.

Janus Surface Micelles on Silica Particles: Synthesis and Application in Enzyme Immobilization.

In these years, synthesis and applications of Janus structures have aroused great interest for large-scale applications in chemistry and materials science. Up to now, Janus particles with different morphologies and different functionalities have been synthesized in solutions, but the synthesis of Janus particles on solid surfaces has not been touched. In this research, Janus surface micelles (JSMs) are fabricated on the surfaces of silica particles by polymerization induced surface self-assembly (PISSA) approach, and the JSMs are used for enzyme immobilization. Usually, enzyme immobilization should be able to optimize the performance of the immobilized enzymes, and an ideal immobilization system must offer protection to the immobilized enzyme with retained bioactivity. Herein, it is demonstrated that JSMs on silica particles can be used as an ideal platform for the immobilization of enzymes. To prepare JSMs, poly(2-(dimethylamino) ethyl methacrylate) macro chain transfer agent (PDMAEMA-CTA) brushes on silica particles and poly(di(ethylene glycol) methyl ether methacrylate) macro CTA (PDEGMA-CTA) are employed in reversible addition-fragmentation chain transfer dispersion polymerization of styrene. After polymerization, JSMs with polystyrene cores and PDMAEMA/PDEGMA patches on the surfaces are prepared on silica particles. After quaternization reaction, the quaternized PDMAEMA patches are used for the immobilization of enzymes. Experimental results turn out that enhanced bioactivities of the immobilized enzymes are achieved and the enzyme molecules are well protected by surface Janus structures.

Tailor-made novel electrospun polystyrene/poly(d,l-lactide-co-glycolide) for oxidoreductases immobilization: Improvement of catalytic properties under extreme reaction conditions.

Immobilized enzymes find applications in many areas such as pharmacy, medicine, food production and environmental protection. However, protecting these biocatalysts against harsh reaction conditions and retaining their enzymatic activity even after several biocatalytic cycles are major challenges. Properly selected supports and type of surface modifier therefore seem to be crucial for achieving high retention of catalytic activity of immobilized biomolecules. Here we propose production of novel composite electrospun fibers from polystyrene/poly(d,l-lactide-co-glycolide) (PS/PDLG) and its application as a support for immobilization of oxidoreductases such as alcohol dehydrogenase (ADH) and laccase (LAC). Two strategies of covalent binding, (i) (3-aminopropyl)triethoxysilane (APTES) with glutaraldehyde (GA) and (ii) polydopamine (PDA), were applied to attach oxidoreductases to PS/PDLG. The average fiber diameter was shown to increase from 1.252 µm to even 3.367 µm after enzyme immobilization. Effective production of PS/PDLG fibers and biomolecule attachment were confirmed by Fourier transform infrared spectroscopy analysis. The highest substrate conversion efficiency was observed at pH 6.5 and 5 for ADH and LAC, respectively, and at 25 °C for enzymes attached using the APTES + GA approach. Improvement of enzyme stabilization at high temperatures was confirmed in that relative activities of enzymes immobilized onto PS/PDLG fibers were over 20% higher than those of the free biomolecules, and enzyme leaching from the support using acetate and MES buffers was below 10 mg/g.

A Crosstalk- and Interferent-Free Dual Electrode Amperometric Biosensor for the Simultaneous Determination of Choline and Phosphocholine.

Choline (Ch) and phosphocholine (PCh) levels in tissues are associated to tissue growth and so to carcinogenesis. Till now, only highly sophisticated and expensive techniques like those based on NMR spectroscopy or GC/LC- high resolution mass spectrometry permitted Ch and PCh analysis but very few of them were capable of a simultaneous determination of these analytes. Thus, a never reported before amperometric biosensor for PCh analysis based on choline oxidase and alkaline phosphatase co-immobilized onto a Pt electrode by co-crosslinking has been developed. Coupling the developed biosensor with a parallel sensor but specific to Ch, a crosstalk-free dual electrode biosensor was also developed, permitting the simultaneous determination of Ch and PCh in flow injection analysis. This novel sensing device performed remarkably in terms of sensitivity, linear range, and limit of detection so to exceed in most cases the more complex analytical instrumentations. Further, electrode modification by overoxidized polypyrrole permitted the development of a fouling- and interferent-free dual electrode biosensor which appeared promising for the simultaneous determination of Ch and PCh in a real sample.

Removal of Persistent Sulfamethoxazole and Carbamazepine from Water by Horseradish Peroxidase Encapsulated into Poly(Vinyl Chloride) Electrospun Fibers.

Enzymatic conversion of pharmaceutically active ingredients (API), using immobilized enzymes should be considered as a promising industrial tool due to improved reusability and stability of the biocatalysts at harsh process conditions. Therefore, in this study horseradish peroxidase was immobilized into sodium alginate capsules and then trapped into poly(vinyl chloride) electrospun fibers to provide additional enzyme stabilization and protection against the negative effect of harsh process conditions. Due to encapsulation immobilization, 100% of immobilization yield was achieved leading to loading of 25 µg of enzyme in 1 mg of the support. Immobilized in such a way, enzyme showed over 80% activity retention. Further, only slight changes in kinetic parameters of free (Km = 1.54 mM) and immobilized horseradish peroxidase (Km = 1.83 mM) were noticed, indicating retention of high catalytic properties and high substrate affinity by encapsulated biocatalyst. Encapsulated horseradish peroxidase was tested in biodegradation of two frequently occurring in wastewater API, sulfamethoxazole (antibiotic) and carbamazepine (anticonvulsant). Over 80% of both pharmaceutics was removed by immobilized enzyme after 24 h of the process from the solution at a concentration of 1 mg/L, under optimal conditions, which were found to be pH 7, temperature 25 °C and 2 mM of H2O2. However, even from 10 mg/L solutions, it was possible to remove over 40% of both pharmaceuticals. Finally, the reusability and storage stability study of immobilized horseradish peroxidase showed retention of over 60% of initial activity after 20 days of storage at 4 °C and after 10 repeated catalytic cycles, indicating great practical application potential. By contrast, the free enzyme showed less than 20% of its initial activity after 20 days of storage and exhibited no recycling potential.

Evaluation of Enzyme Inhibitory Activity of Flavonoids by Polydopamine-Modified Hollow Fiber-Immobilized Xanthine Oxidase.

In this study, a polydopamine (PDA)-modified hollow fiber-immobilized xanthine oxidase (XOD) was prepared for screening potential XOD inhibitors from flavonoids. Several parameters for the preparation of PDA-modified hollow fiber-immobilized XOD, including the dopamine concentration, modification time, XOD concentration and immobilization time, were optimized. The results show that the optimal conditions for immobilized XOD activity were a dopamine concentration of 2.0 mg/mL in 10.0 mM Tris-HCl buffer (pH 8.5), a modification time of 3.0 h, an XOD concentration of 1000 µg/mL in 10.0 mM phosphate buffer (pH 7.5) and an immobilization time of 3.0 h. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were investigated, and the enzyme kinetics and inhibition parameters were determined. The results indicate that the optimal pH value (7.5) and temperature (37 °C) of the PDA-modified hollow fiber-immobilized XOD were consistent with the free enzyme. Moreover, the PDA-modified hollow fiber-immobilized XOD could still maintain above 50% of its initial immobilized enzyme activity after seven consecutive cycles. The Michaelis-Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of allopurinol on the immobilized XOD were determined as 0.25 mM and 23.2 µM, respectively. Furthermore, the PDA-modified hollow fiber-immobilized XOD was successfully applied to evaluate the inhibitory activity of eight flavonoids. Quercetin, apigenin, puerarin and epigallocatechin showed a good inhibition effect, and their percentages of inhibition were (79.86 ± 3.50)%, (80.98 ± 0.64)%, (61.15 ± 6.26)% and (54.92 ± 0.41)%, respectively. Finally, molecular docking analysis further verified that these four active compounds could bind to the amino acid residues in the XOD active site. In summary, the PDA-modified hollow fiber-immobilized XOD is an efficient method for the primary screening of XOD inhibitors from natural products.

High-level production in a plant system of a thermostable carbonic anhydrase and its immobilization on microcrystalline cellulose beads for CO2 capture.

KEY MESSAGE: Plant-produced SazCA and its application to CO2 capture. Technologies that rely on chemical absorption or physical adsorption have been developed to capture CO2 from industrial flue gases and sequester it at storage sites. Carbonic anhydrases (CAs), metalloenzymes, that catalyze the reversible hydration of CO2 have recently received attention as biocatalysts in the capture of CO2 from flue gases, but their cost presents a major obstacle for use at an industrial scale. This cost, however, can be reduced either by producing a long-lasting enzyme suitable for CO2 capture or by lowering production costs. High-level expression, easy purification, and immobilization of CAs from Sulfurihydrogenibium azorense (SazCA) were investigated in a plant system. Fusion of the 60-amino acid-long ectodomain (M-domain) of the human receptor-type tyrosine-protein phosphatase C increased the levels of SazCA accumulation. Fusion of the cellulose-binding module (CBM3) from Clostridium thermocellum resulted in tight binding of recombinant protein to microcrystalline cellulose beads, enabling easy purification. The chimeric fusion protein, BMC-SazCA, which consisted of SazCA with the M and CBM3 domains, was expressed in tobacco (Nicotiana benthamiana), giving a recombinant protein yield in leaf extracts of 350 mg/kg fresh weight. BMC-SazCA produced in planta was active in the presence of various chemicals used in CO2 capture. Immobilization of BMC-SazCA on the surface of microcrystalline cellulose beads extended its heat stability, allowing its reuse in multiple rounds of the CO2 hydration reaction. These results suggest that production of SazCA in plants has great potential for CA-based CO2 sequestration and mineralization.

Enzymatic degradation of ginkgolic acid by laccase immobilized on novel electrospun nanofiber mat.

BACKGROUND: Ginkgo biloba leaf extract contains many active ingredients that are beneficial for health. However, ginkgolic acid, one of the major components found in G. biloba extract, may cause serious allergic and toxic side effects. The purpose of this study is to immobilize the laccase system on the electrospun nylon fiber mat (NFM) to hydrolyze the ginkgolic acid in G. biloba leaf extract efficiently. RESULTS: Novel electrospinning technology successfully produced high-quality nanoscopic fiber mats made of a mixture of multi-walled carbon nanotube and nylon 6,6. Laccase that was immobilized onto the NFM exhibited much higher efficiency in the catalyzation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) than nylon 6,6 pellets. After being immobilized onto the NFM, the pH and temperature stability of laccase were significantly improved. The NFM-immobilized laccase could maintain more than 50% of its original activity even after 40 days of storage or 10 operational cycles. The kinetic parameters, including rate constant (K), the time (τ50) in which 50% of ginkgolic acid hydrolysis was reached, the time (τcomplete) required to achieve complete ginkgolic acid hydrolysis, Km and Vmax were determined, and were 0.07 ± 0.01 min-1 , 8.97 ± 0.55 min, 45.45 ± 2.79 min, 0.51 ± 0.09 mM and 0.49 ± 0.03 mM min-1 mg-1 , respectively. CONCLUSION: The result successfully demonstrated the strong potential of using novel electrospun nanofiber mats as enzyme immobilization platforms, which could significantly enhance enzyme activity and stability. © 2020 Society of Chemical Industry.

Polydopamine-Capped Bimetallic AuPt Hydrogels Enable Robust Biosensor for Organophosphorus Pesticide Detection.

Noble metal hydrogels/aerogels with macroscopic nanoassemblies characterized by ultralow density, profuse continuous porosity, and extremely large surface area have gained abundant interest due to not only their tunable physicochemical properties, but also promising applications in catalysis and sensing. Coupling the increased reaction temperature with dopamine-induced effect, herein, a one-step synthetic approach with accelerated gelation kinetics is reported for the synthesis of polydopamine-capped bimetallic AuPt hydrogels. 3D porous nanowire networks with surface functionalization of polydopamine make them a promising biocompatible microenvironment for immobilizing acetylcholinesterase (AChE) and constructing enzyme-based biosensors for sensitive detection of organophosphorus compounds. Taking advantage of their favorable structure and composition, the optimized product exhibits superior electrochemical activity toward thiocholine produced by AChE-catalyzed hydrolysis of acetylthiocholine. Based on the inhibition of organophosphorus pesticide on the enzymatic activity of AChE, the inhibition mode for the detection of paraoxon-ethyl is established, displaying linear regions over the range of 0.5-1000 ng L-1 with a low detection limit of 0.185 ng L-1 .

Immobilized enzyme on pulp fiber through layer-by-layer technique using cationic polyacrylamide for whitewater treatment from papermaking.

Anionic pectic substances in whitewater from papermaking are detrimental to machine operation and product quality. Pectinase was immobilized on pulp fiber using cationic polyacrylamide with layer-by-layer method to obtain bound enzyme with tunable activity and good performance for wastewater treatment. It was revealed that high charge density and low molecular weight for cationic polyacrylamide were advantageous for enzymatic activity. During the layer-by-layer adsorption process, the enzymatic activity of the immobilized enzyme increased nearly linearly with the layer number from 983 to 3074 U/g until the fourth layer. The stability of the four-layer immobilized enzyme was improved. The multilayer immobilized enzyme exhibited good reusability and storage stability compared with monolayer enzyme. At dosage of 10 U/mL, the cationic demand of the whitewater samples was reduced by 15% using four-layer immobilized enzyme. The results indicated a potential route to prepare immobilized enzyme with good performance for wastewater treatment in papermaking industry.

Non-Covalent Functionalization of Carbon Nanotubes for Electrochemical Biosensor Development.

Carbon nanotubes (CNTs) have been widely studied and used for the construction of electrochemical biosensors owing to their small size, cylindrical shape, large surface-to-volume ratio, high conductivity and good biocompatibility. In electrochemical biosensors, CNTs serve a dual purpose: they act as immobilization support for biomolecules as well as provide the necessary electrical conductivity for electrochemical transduction. The ability of a recognition molecule to detect the analyte is highly dependent on the type of immobilization used for the attachment of the biomolecule to the CNT surface, a process also known as biofunctionalization. A variety of biofunctionalization methods have been studied and reported including physical adsorption, covalent cross-linking, polymer encapsulation etc. Each method carries its own advantages and limitations. In this review we provide a comprehensive review of non-covalent functionalization of carbon nanotubes with a variety of biomolecules for the development of electrochemical biosensors. This method of immobilization is increasingly being used in bioelectrode development using enzymes for biosensor and biofuel cell applications.

Ionic Liquid-Polypyrrole-Gold Composites as Enhanced Enzyme Immobilization Platforms for Hydrogen Peroxide Sensing.

In this work, three different aqueous solutions containing imidazole-based ILs with different alkyl chain lengths ([Cnmim]Br, n = 2, 6, 12) were adopted as the medium for the synthesis of ionic liquid-polypyrrole (IL-PPy) composites. Herein, the ILs undertook the roles of the pyrrole solvent, the media for emulsion polymerization of PPy and PPy dopants, respectively. The electrochemical performances of the three IL-PPy composites on a glassy carbon electrode (GCE) were investigated by electrochemical experiments, which indicated that [C12mim]Br-PPy (C12-PPy) composites displayed better electrochemical performance due to their larger surface area and firmer immobilization on the GCE. Further, C12-PPy/GCE were decorated with Au microparticles by electrodeposition that can not only increase the conductivity, but also immobilize sufficient biomolecules on the electrode. Then, the obtained C12-PPy-Au/GCE with outstanding electrochemical performance was employed as a horseradish peroxidase (HRP) immobilization platform to fabricate a novel C12-PPy-Au-HRP/GCE biosensor for H2O2 detection. The results showed that the prepared C12-PPy-Au-HRP/GCE biosensor exhibited high sensitivity, fast response, and a wide detection range as well as low detection limit towards H2O2. This work not only provides an outstanding biomolecule immobilization matrix for the fabrication of highly sensitive biosensors, but also advances the understanding of the roles of ILs in improving the electrochemical performance of biosensors.

Simple Preparation of Thiol-Ene Particles in Glycerol and Surface Functionalization by Thiol-Ene Chemistry (TEC) and Surface Chain Transfer Free Radical Polymerization (SCT-FRP).

Thiol-ene (TE)-based polymer particles are traditionally prepared via emulsion polymerization in water (using surfactants, stabilizers, and cosolvents). Here, a green and simple alternative is presented with excellent control over particle size, while avoiding the addition of stabilizers. Glycerol is applied as a dispersing medium for the preparation of off-stoichiometric TE microparticles, where sizes in the range of 40-400 µm are obtained solely by changing the mixing speed of the emulsions prior to crosslinking. Control over surface chemistry is achieved by surface functionalization of excess thiol groups via photochemical thiol-ene chemistry resulting in a functional monolayer. In addition, surface chain transfer free radical polymerization is used for the first time to introduce a thicker polymer layer on the particle surface. The application potential of the system is demonstrated by using functional particles as adsorbent for metal ions and as a support for immobilized enzymes.

Improved enzymatic performance of graphene-immobilized ß-glucosidase A in the presence of glucose-6-phosphate.

Optimization of cellulose enzymatic hydrolysis is crucial for cost-effective bioethanol production from lignocellulosic biomass. Enzyme immobilization in solid support allows enzyme recycling for reuse, lowering hydrolysis costs. Graphene is a nanomaterial isolated in 2004, which possesses exceptional properties for biomolecule immobilization. This study evaluates the potential for ß-glucosidase recycling by immobilization on graphene nanosheets. Data reported here demonstrated that graphene-immobilized ß-glucosidase can be recycled for at least eight cycles. Immobilization did not change the optimal temperature of catalysis and improved enzymatic stability upon storage. The role of glucose-6-phosphate on immobilized enzyme was also investigated, demonstrating that glucose-6-phosphate acts as a mixed-type activator and improves storage stability of immobilized enzyme. Complete cellulose hydrolysis using graphene-immobilized ß-glucosidase in the presence of glucose-6-phosphate resulted in greatly improved hydrolysis rates, demonstrating the potential of this strategy for biomass hydrolysis.

Synthesis of pH-responsive ß-CD-based star polymer and impact of its self-assembly behavior on pectinase activity.

A novel type of pH-responsive star polymer based on ß-cyclodextrin (ß-CD) was synthesized and further covalently conjugated with enzyme. The impact of its self-assembly behavior on enzyme activity was investigated. In our design, azide containing the polymer (N3 )7 -ß-CD-(PtBA)14 was synthesized via atom transfer radical polymerization of tert-butyl acrylate using (N3 )7 -ß-CD-(Br)14 as the multifunctional initiator. The final product (N3 )7 -ß-CD-(PAA)14 was obtained via hydrolysis and covalently conjugating pectinase onto pH-responsive polyacrylic acid (PAA) arms. PAA can change its conformation with the self-assembly by altered pH, leading its nanostructure into micellar nanoparticles in aqueous solution and further affecting the activity of immobilized pectinase. The results were proved by fluorescence spectroscopy and dynamic light scattering. This system proves that the activity of immobilized enzyme can be tailored predictably, and this pH-responsive polymer holds great potential for controllable delivery of enzymes.