High Activity Recovery Research Articles

Hybrid Catalysts for CO2 Conversion into Cyclic Carbonates

The conversion of carbon dioxide into valuable chemicals such as cyclic carbonates is an appealing topic for the scientific community due to the possibility of valorizing waste into an inexpensive, available, nontoxic, and renewable carbon feedstock. In this regard, last-generation heterogeneous catalysts are of great interest owing to their high catalytic activity, robustness, and easy recovery and recycling. In the present review, recent advances on CO2 cycloaddition to epoxide mediated by hybrid catalysts through organometallic or organo-catalytic species supported onto silica-, nanocarbon-, and metal–organic framework (MOF)-based heterogeneous materials, are highlighted and discussed.

Multi-Functional Laccase Immobilized Hydrogel Microparticles for Efficient Removal of Bisphenol A.

Hghly stable, reusable, and multi-functional biocatalytic microparticles with Laccase (Lac) enzyme (Lac/particles) were synthesized for bisphenol A (BPA) removal from aqueous solution. The Lac/particles were prepared by encapsulating Lac enzymes into poly ethylene glycol (PEG) hydrogel via the UV assisted emulsion polymerization method followed by cross linking with glutaraldehyde (GA). The obtained Lac/particles were spherical and micron sized (137–535 μm), presenting high enzyme entrapment efficiency of 100%, high activity recovery of 18.9%, and great stability at various pHs (3–7) than the free Lac. The Lac/particles could adsorb the BPA into the catalytic particles in a short time, promoting contact between BPA and enzyme, and further enzymatically degrade them without the shaking process and independent surrounding buffer solution. The Lac/particles could be reused for another round BPA adsorption and biotranformation by maintaining over 90% of BPA removal efficiency after seven times reuse. The synergistic effects of adsorption and biocatalytical reaction of Lac/particles have significant values in high efficient and cost-effective BPA removal.

Hard template-induced internal solidification synthesis of Cu NPs- supported glutaraldehyde-crosslinked polyethyleneimine-modified calcium alginate beads with enhanced catalytic activity

The development of a sustainable catalyst with high catalytic activity and easy recovery property is an important subject in the field of heterogeneous catalysis. In this work, we developed a hard template-induced internal solidification procedure for preparation of ultrafine metal nanoparticles (NPs) supported on GLA-PEI-CA beads. During the fabrication process, CaCO3 NPs had a dual function, not only serviced as a hard template to adjust the structure of the as-prepared catalyst, but also as an indirect crosslinking agent to obtain hydrogel beads. The resulting beads were characterized using FT-IR, XRD, BET, SEM, TEM, EDS and XPS. The results revealed that Cu NPs with a narrow size distribution in 2–3 nm were uniformly immobilized on the surface of GLA-PEI-CA-1 beads with high specific surface area and well-developed hierarchical pore structure. As a result, the Cu@GLA-PEI-CA-1 beads exhibited remarkably catalytic activity and high stability, with more than 90% conversion within 10 successive cycles, towards the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) by NaBH4 in aqueous solution. The result showed that the Cu@GLA-PEI-CA-1 catalyst displayed high kinetic responses with a kinetic reaction rate constant of 1.60 × 10−2 s−1, which was higher than that of Cu@GLA-PEI-CA-0 (without CaCO3 template) and Cu@GLA-CA-1 (without PEI coating). The enhanced catalytic performance was resulted from homogeneous distribution of Cu NPs along with the tailored microstructure and the hydrophilic surface of the support. Additionally, the dynamic catalytic experiment was conducted to investigate the catalytic performance of Cu@GLA-PEI-CA-1 beads as the catalyst. We expected that this work could provide a new and effective strategy to design highly efficient catalysts for various catalytic reactions.

Self-Cleaning Interfaces of Polydimethylsiloxane Grafted with pH-Responsive Zwitterionic Copolymers.

Self-cleaning surfaces allow the reversible attachment and detachment of microorganisms which show great promise in regards to their reusability as smart biomaterials. However, a widely used biomaterial such as polydimethylsiloxane (PDMS) suffers from high biofouling activity and hydrophobic recovery that results in decreased efficiency and stability. A current challenge is to modify and fabricate self-cleaning PDMS surfaces by incorporating antifouling and pH-sensitive properties. To address this, we synthesized a zwitterionic and pH-sensitive random poly(glycidyl methacrylate- co-sulfobetaine methacrylate- co-2-(dimethylamino)ethyl methacrylate) polymer, poly(GMA- co-SBMA- co-DMAEMA). In this work, chemical modification of PDMS was done by grafting onto poly(GMA- co-SBMA- co-DMAEMA) after surface activation via UV and ozone for 90 min to ensure the formation of covalent bonds necessary for stable grafting. The PDMS grafted with G20-S40-D40 exhibit antifouling and pH-sensitive properties by mitigating fibrinogen adsorption, blood cell adhesion, and releasing 98% adhered E. coli bacteria after immersion at basic pH. The grafting of poly(GMA- co-SBMA- co-DMAEMA) presented in this work shows attractive potential for biomedical and industrial applications as a simple, smart, and effective method for the modification of PDMS interfaces.

Design and synthesis of multipotent 3-aminomethylindoles and 7-azaindoles with enhanced protein phosphatase 2A-activating profile and neuroprotection

We report the synthesis and pharmacological evaluation of new 3-aminomethylindoles derivatives with neuroprotective properties designed to present multi-target activity centered on reducing the neuronal Ca2+ overload and preventing phosphatase 2A (PP2A) inhibition, which are two important early physiophathological events observed in neurodegenerative scenarios. Chemical syntheses of proposed compounds were achieved in two straightforward reaction steps with high yields. Most of the compounds mitigated the okadaic acid-provoked inhibition of PP2A and protected SH-SY5Y cells against toxic stimuli related to Tau-hyperphosphorylation and oxidative stress, similarly to the observed in Alzheimer's disease (AD). In addition, some of them mitigated the Ca2+ overload induced by depolarization. The derivative 1-(1-benzyl-5-chloro-1H-indol-3-yl)-N,N-dimethylmethanamine (19) outstood by its high recovery of the PP2A activity and blockade of voltage-gated Ca2+ channels, accompanied by good neuroprotective profile. These findings make this compound eligible for further preclinical assays with the goal of positioning new innovative drugs for the treatment of AD.

Immobilization of cellulase on a core-shell structured metal-organic framework composites: Better inhibitors tolerance and easier recycling

For the first time, cellulase was successfully immobilized on a magnetic core-shell metal-organic framework (MOF) material, UIO-66-NH2. The as-prepared immobilized cellulase demonstrated a high protein loading efficiency of 126.2 g/g support and a high enzyme activity recovery of 78.4%. Cellulase immobilized on magnetic UIO-66-NH2 exhibited a superior performance in terms of pH stability, thermal stability and catalytic efficiency compared to its free form. Notably, immobilized cellulase could be recycled for up to 5 consecutive runs. Furthermore, compared to free cellulase, immobilized cellulase showed better tolerance to formic acid and vanillin, two typical inhibitors found in lignocellulosic prehydrolysates. In the presence of 5 g/L of formic acid and vanillin, immobilized cellulase demonstrated 16.8% and 21.5% higher activity than free enzyme, respectively, and its improvement in hydrolysis yield was 18.7% and 19.6% respectively. This is firstly confirmed that immobilization can alleviate the inhibitory effects of certain pretreatment inhibitors on cellulase.

Silica encapsulated catalase@metal-organic framework composite: A highly stable and recyclable biocatalyst

Metal-organic frameworks (MOFs) have recently emerged as a promising candidate for the immobilization of enzymes due to their diversified structures and porosity. However, it is difficult to handle and fully recover the enzyme-MOF composites from the reaction medium due to their nanometer size and good dispersity in solvents, which limits their practical applications. Here, a novel enzyme-MOF composite with both highly stable and easily reusable features was prepared via encapsulating catalase/ZIF-8 nanocrystals into large mesoporous silica layer (silica@CAT/ZIF-8). This immobilized system exhibited high activity recovery (81%). The silica layer around the catalase/ZIF-8 particles provided a “shield” to protect from biological and chemical degradation for enzyme. As a result, the silica@CAT/ZIF-8 composites exhibited higher stability against proteolytic agent and extreme conditions (such as low pH) than that of conventional catalase/ZIF-8 composites. More importantly, the micrometer-sized silica@CAT/ZIF-8 can be easily repeatedly used without obvious activity loss. The silica@CAT/ZIF-8 composites still remained 50% of their original activity after 10 cycles, whereas the conventional catalase/ZIF-8 composites only retained 7% of their original activity after 5 cycles. These results demonstrated that this approach could be an efficient strategy to prepare enzyme-ZIF-8 composite with both high stability and excellent recyclability.

Cross-linked cytochrome P450 BM3 aggregates promoted by Ru(II)-diimine complexes bearing aldehyde groups

Cross-linked enzyme aggregate (CLEA) methodology has been applied to immobilize cytochrome P450 BM3 variants (F87A and 21B3) with peroxygenase activity. Several Ru(II)-diimine complexes were found to be suitable cross-linking agents, surpassing the traditional glutaraldehyde and dextran aldehyde. They offer modular numbers of aldehyde functionalities and a more rigid framework than their organic counterparts. The F87A CLEAs display significant activity loss compared to the protein in solution. Meanwhile, for the 21B3 CLEAs, high activity recovery (up to 95%) is obtained. In order to minimize enzyme leaching from the CLEA, sodium cyanoborohydride was used to reduce the CLEAs imine bonds. The reduced CLEAs were active for several rounds of reactions leading to an overall increase in protein activity of 170% compared to the free protein in solution.

Preparation and Evaluation of Coal Fly Ash/Chitosan Composites as Magnetic Supports for Highly Efficient Cellulase Immobilization and Cellulose Bioconversion.

Two magnetic supports with different morphologies and particle sizes were designed and prepared for cellulase immobilization based on chitosan and industrial by-product magnetic coal fly ash (MCFA). One was prepared by coating chitosan onto spherical MCFA particles to form non-porous MCFA@chitosan gel microcomposites (Support I) with a size of several micrometers, and the other was prepared using the suspension method to form porous MCFA/chitosan gel beads (Support II) with a size of several hundred micrometers. Cellulase was covalent binding to the support by glutaraldehyde activation method. The morphology, structure and magnetic property of immobilized cellulase were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and a vibrating-sample magnetometer. The cellulase loading on Support I was 85.8 mg/g with a relatlvely high activity recovery of 76.6%, but the immobilized cellulase exhibited low thermal stability. The cellulase loading on Support II was 76.8 mg/g with a relative low activity recovery of 51.9%, but the immobilized cellulase showed high thermal stability. Cellulase immobilized on Support I had a glucose productivity of 219.8 mg glucose/g CMC and remained 69.9% of the original after 10 cycles; whereas the glucose productivity was 246.4 mg glucose/g CMC and kept 75.5% of its initial value after 10 repeated uses for Support II immobilized cellulase. The results indicate that the two supports can be used as cheap and effective supports to immobilize enzymes.

Preparation of a novel nanobiocatalyst by immobilizing penicillin acylase onto magnetic nanocrystalline cellulose and its use for efficient synthesis of cefaclor

Magnetic nanocrystalline cellulose (MNCC) was prepared and used as an enzyme support for the immobilization of penicillin acylase (PA). A novel coupling agent, tri(hydroxymethyl)phosphine(THP) instead of the conventional glutaraldehyde(GA), was used as a crosslinker in this study. The obtained results showed that the immobilized PA with THP (PA-THP-MNCC) had high enzyme loading (172.3 mg/g) and activity recovery (77.6%) in the optimal preparation conditions, which were remarkably superior to those of the counterpart using GA (PA-GA-MNCC, 148.4 mg/g and 48.7%, respectively). Compared with free PA and PA-GA-MNCC, PA-THP-MNCC displayed a higher optimum pH and temperature, and manifested relatively higher enzyme-substrate affinity and catalytic efficiency. In addition, PA-THP-MNCC exhibited significantly enhanced stability. Furthermore, PA-THP-MNCC was successfully employed for synthesis of cefaclor, an important second-generation cephalosporin antibiotic, affording a significantly higher yield of 84% than that reported previously.

Biotransformation of emerging pollutants in groundwater by laccase from P. sanguineus CS43 immobilized onto titania nanoparticles

Biotransformation of organic compounds by using biocatalysts such as laccases provides an environmentally attractive alternative to supplement conventional wastewater treatment. However, loss and inactivation of enzymes are challenges to solve for their potential use in water treatment applications. Titania presents a high chemical stability and easy functionalization, which are important characteristics in a support used for immobilization processes. Therefore, in this study, the immobilization of laccase enzymes produced by Pycnoporus sanguineus CS43 onto titania nanoparticles (∼21 nm) was optimized. Surface modification of the support was carried out by using different weight% (wt%) of 3-aminopropyltriethoxysilane (APTES) as a coupling agent and the cross-linker glutaraldehyde (GLU) as a laccase binder. Free and immobilized enzymatic activity were measured based on 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays and were compared regarding apparent activity and activity recovery. In addition, the effect of ionic strength on the immobilization process was studied, finding that low ionic strength (25 mM phosphate buffer) promote a high activity recovery, greater than 90% and even higher than previous reports. Immobilized laccases showed high thermostability at 50 and 60 °C (half-lives of 45.7 and 3.7 h, respectively), and high stability at low pH values of 2 and 3 (half-lives of 31.8 and 107.1 h respectively). Furthermore, the biocatalyst was assessed in the biotransformation of emerging pollutants such as acetaminophen and diclofenac by using 100 UL−1 of immobilized laccase at pH 4; the maximum biotransformation percentage of DCF was 68% after 8 h, and more than 90% biotransformation of ACE was reached after 2 h of treatment.

Immobilization of laccase onto porous polyvinyl alcohol/halloysite hybrid beads for dye removal.

Laccase was immobilized in polyvinyl alcohol beads containing halloysite nanotubes (PVA/HNTs) to improve the stability and reusability of enzyme. The porous structure of PVA/HNTs beads facilitates the entrapment of enzyme and prevents the leaching of immobilized laccase as well. Halloysite nanotubes act as bridge to connect the adjacent pores, facilitating the electron transfer and enhancing the mechanical properties. PVA/HNTs beads have high laccase immobilization capacity (237.02 mg/g) and activity recovery yield (79.15%), indicating it can be used as potential support for laccase immobilization. Compared with free laccase, the immobilized laccase on hybrid beads exhibits enhanced pH tolerance (even at pH 8.0), good thermal stability (57.5% of the initial activity can be maintained at 75 °C), and excellent storage stability (81.17% of enzyme activity could be retained after storage at 4 °C for 5 weeks compared with that for free enzyme of 60%). Also, the removal efficiency for reactive blue can reach as high as 93.41% in the presence of redox mediator 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate), in which adsorption and degradation exist simultaneously. The remarkable pH tolerance, thermal and storage stability, and reuse ability imply potential application of porous PVA/HNTs immobilized enzyme in environmental fields.

Cryopreservation of Proteins Using Ionic Liquids: A Case Study of Cytochrome c.

Aqueous ionic liquid (IL) solutions form a glassy state at 77 K over a wide concentration of ILs. They have potential as novel cryopreservation/refolding solvents for proteins. However, even if proteins in glass-forming concentrations of ILs are preserved at 77 K, the recovery of activity and the structure of the proteins after cryopreservation are still unclear. To achieve high recovery of protein activity and structure by removal of ILs after cryopreservation at 77 K, we studied the recovery of activity and structural stability after cryopreservation of bovine heart cytochrome c in aqueous solutions with ILs, including ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazolium thiocyanate ([bmim][SCN]) over wide IL concentrations using UV-vis, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy. On the whole, although the addition of both ILs induced a decrease of activity and unfolding of the secondary structure of cytochrome c before and after cooling to 77 K, EAN, a weak denaturant, showed a reduction in protein damage (decrease of activity and unfolding of secondary structure) during the reheating process from 77 K (protection ability). In contrast, [bmim][SCN], a strong denaturant, did not have this protective ability. A remarkable result is that although the addition of both ILs caused cytochrome c denaturation, > 90% of activity and structure after cryopreservation (X > 10 mol %IL) was recovered after the removal of both ILs by dialysis. These recoveries after the removal of ILs are slightly higher than the results for dimethyl disulfide (DMSO), another cryoprotectant. The present results indicate that concentrated aqueous IL solutions have potential as one-pot (i.e., solubilization/preservation/refolding) solvents for proteins, which easily aggregate after purification, with comparable results to DMSO.

Laccase immobilized on magnetic nanoparticles by dopamine polymerization for 4-chlorophenol removal

In this work, a new immobilization method based on dopamine (DA) self-polymerization was developed for laccase immobilization on magnetic nanoparticles (Fe3O4 NPs). To optimize the immobilization condition including reaction pH, DA concentration and enzyme concentration, a central composite response surface method was applied. The optimal condition was determined as pH value of 5.92, laccase concentration of 0.25 mg mL−1 and DA concentration of 12.74 mg mL−1, under which a high enzyme activity recovery of 88.17% was obtained. By comparing with free laccase, the stabilities of immobilized laccase towards pH, thermostability, storage were enhanced significantly. Approximately 60% of relative activity for immobilized laccase was remained after being incubated for 6 h at 50 °C, but the free laccase only remained 25%. After 40 days of storage at 4 °C, the laccase immobilized by DA kept about 89% of its original activity, but the free laccase only retained 48%. After recycled 10 times, the relative activity of immobilized laccase still retained 70%. The immobilized laccase was then applied to catalyze the degradation of 4-chlorophenol (4-CP), 86% percentage of 4-CP was removed within 2 h. After degraded 10 times, the relative activity of immobilized laccase still remained 64% of its initial activity, which exhibits an excellent reusability and operational stability.

High activity and selectivity immobilized lipase on Fe 3 O 4 nanoparticles for banana flavour synthesis

Abstract Lipase (E.C.3.1.1.3) from Thermomyces lanuginosus (TL) was directly bonded, through multiple physical interactions, on citric acid functionalized monodispersed Fe 3 O 4 nanoparticles (NPs) in presence of a small amount of hydrophobic functionalities. A very promising scalable synthetic approach ensuring high control and reproducibility of the results, and an easy and green immobilization procedure was chosen for NPs synthesis and lipase anchoring. The size and structure of magnetic nanoparticles were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The samples at different degree of functionalization were analysed through thermogravimetric measurements. Lipase immobilization was further confirmed by enzymatic assay and Fourier transform infrared (FT-IR) spectra. Immobilized lipase showed a very high activity recovery up to 144% at pH = 7 and 323% at pH = 7.5 (activity of the immobilized enzyme compared to that of its free form). The enzyme, anchored to the Fe 3 O 4 nanoparticles, to be easy recovered and reused, resulted more stable than the native counterpart and useful to produce banana flavour. The immobilized lipase results less sensitive to the temperature and pH, with the optimum temperature higher of 5 °C and optimum pH up shifted to 7.5 (free lipase optimum pH = 7.0). After 120 days, free and immobilized lipases retained 64% and 51% of their initial activity, respectively. Ester yield at 40 °C for immobilized lipase reached 88% and 100% selectivity.

Improving Pullulanase Catalysis via Reversible Immobilization on Modified Fe3O4@Polydopamine Nanoparticles.

To improve the catalysis of pullulanase from Anoxybacillus sp.WB42, Fe3O4@polydopamine nanoparticles (Fe3O4@PDA) were prepared and modified with functional groups for immobilization of pullulanases via covalent binding or ionic adsorption. Immobilized pullulanases had lower thermal stability than that of free pullulanase, whereas their catalysis depended on the surface characteristics of nanoparticles. As for covalent immobilization of pullulanases onto Fe3O4@PDA derivatives, the spacer grafted onto Fe3O4@PDA made the catalytic efficiency of pullulanase increase up to the equivalence of free enzyme but dramatically reduced the pullulanase thermostability. In contrast, pullulanases bounded ionically to Fe3O4@PDA derivatives had higher activity recovery and catalytic efficiency, and their catalytic behaviors varied with the modifier grafted onto Fe3O4@PDA. Among these immobilized pullulanases, ionic adsorption of pullulanase on Fe3O4@PDA-polyethyleneimine-glycidyltrimethylammonium gave a high-performance and durable catalyst, which displayed not only 1.5-fold increase in catalytic efficiency compared to free enzyme but also a significant improvement in operation stability with a half of initial activity after 27 consecutive cycles with a total reaction time of 13.5h, and was reversible, making this nanoparticle reusable for immobilization.

One-step purification of α1-antitrypsin by regulating polyelectrolyte ligands on mussel-inspired membrane adsorber

One-step purification of biomolecules from complex feed is challenging with commercial membrane adsorbers since it is difficult to screen and regulate the ligands on the membrane with traditional techniques for removal of various impurities. By readily grafting cationic polyelectrolyte ligands on a polydopamine (PDA)-coated membrane, a membrane adsorber with suitable ligand (750kDa polyethylenimine, PEI) was selected to achieve one-step purification of α1-antitrypsin (AAT) from pretreated human plasma fraction IV. It was found that ligand structure and molecular weight showed a significant effect on the properties and separation performance of membrane adsorbers. The attached polyelectrolytes increased membrane hydrophilicity but caused an enhancement of filtration resistance, and quaternization of the ligands (PEI and polyallylamine) further improved the hydrophilicity but decreased the charge quantity. By further screening elution salt concentration, flow rate, buffer pH, loading volume as well as molecular weight of PEI ligands, a high activity recovery of 96.6% and a high purity of 94.6% of AAT were obtained by one-step membrane chromatography, which was even better than those by multiple-step method. This could be owing to the special multilayer structure of PEI chains, which could suppress the impurity protein binding on the adsorption sites by exclusion effect. The present work not only provides new insights into the purification of biomolecules from complex streams with polyelectrolyte-coupled membrane adsorbers, but also offers a new approach to design the ligands on membrane adsorber for specific application.

Preparation of spherical cross-linked lipase aggregates with improved activity, stability and reusability characteristic in water-in-ionic liquid microemulsion

BACKGROUND Cross-linking enzyme aggregates (CLEAs) seem to be a promising immobilization method due to mild processing conditions and short preparation time. However, the CLEAs prepared in aqueous solution exhibit amorphous large clusters that cause significant mass-transfer limitations, leading to low catalytic efficiency. In this work, novel spherical CLEAs of lipase from bovine pancreas (spherical CLEAs) were prepared within droplets dispersed in a water-in-hydrophobic ionic liquid microemulsion rather than a conventional aqueous solution. The properties of the spherical CLEAs were determined. RESULTS The resultant CLEAs exhibited spherical structure with good monodispersity instead of the amorphous clusters of conventional CLEAs, and showed higher activity recovery (84.6%) than that of conventional CLEAs (52.8%). The high activity recovery of spherical CLEAs could be attributed to both interfacial activation of the microemulsion system and the well-defined structure of the spherical CLEAs, which not only activated lipases in an active form, but also decreased mass-transfer limitations. Moreover, the thermostability, storage stability, tolerance against hydrophilic solvents, and mechanical stability of the spherical CLEAs were enhanced significantly. The spherical CLEAs evidenced excellent reusability in comparison with conventional CLEAs. CONCLUSIONS The novel strategy in this study might open an attractive way towards effectively improving the catalytic properties of traditional CLEAs. © 2016 Society of Chemical Industry

Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS)

ABSTRACT Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na2SO4 at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE.

Catalase immobilization on amino-activated Fe3O4@SiO2 nanoparticles: Loading density affected activity recovery of catalase

A blackberry-shaped Fe3O4@SiO2 nanoparticles were prepared, characterized and applied in covalently binding catalase. The enzyme loading decreased with the increase of pH, however, the activity recovery increased simultaneously. To elucidate the influence factor of the activity recovery, the enzyme loading was further regulated by changing the initial free enzyme content. The relationship between enzyme loading and activity recovery showed the consistent trend, whether the variation of enzyme loading was incurred by pH or by initial enzyme content. The simulated parameters showed the similar values according to the experiment at different conditions. It was concluded that activity recovery was dominated by protein density on surface, not by the orientation of the enzyme on surface, due to the negligible diffusion limit for H2O2 as the substrate of catalase. The immobilized catalase at pH 7.0 has a high activity recovery of 100% at 14.4 enzyme μg/mg nanoparticles. The Km and Vmax of the immobilized enzyme above are 0.215mol and 0.797mol/min, similar to 0.167mol and 0.727mol/min for the free enzyme, respectively.