Nostoc calcicola Immobilized in Silica-coated Calcium Alginate and Silica Gel for Applications in Heavy Metal Biosorption

The present study reports the preparation and characterization of silica-based immobilization matrices for the purpose of metal accumulation using immobilized cyanobacterium Nostoc calcicola. Silica gel was prepared using aqueous sodium silicate and colloidal silica. Calcium alginate (CAG) beads were coated with silica using sodium silicate solutions. Microscopy observations and TTC tests confirmed that the immobilized cells were intact and viable. Ultrastructural studies with electron microscopy revealed a membrane thickness of approximately 10 μm around the CAG and the silica gel to be of mesoporous nature. BET surface area of silica gel-immobilized N. calcicola was 160 m² g^?1. The porous volume and average pore diameter were 0.40 cm³ g^?1 and ca. 100 Å, respectively, as calculated using the BJH model. Studies on silica-coated calcium alginate immobilized cells showed that these were superior to the uncoated CAG beads in terms of mechanical strength and metal accumulation. The silica matrices were found to be stable for repeated cycles of metal removal and with commonly used eluants for desorption processes. These matrices have potential applications in immobilization of industrially important biocatalysts.     

Facile synthesis of monodispersed silica-coated magnetic nanoparticles

Silica-coated magnetic nanoparticles (MNPs) have great potential for use in field of biotechnology owing to their unique properties, which can be manipulated by an external magnetic field gradient. Herein, we describe a method for facile synthesis of monodispersed silica-coated MNPs (MNP@SiO₂ NPs). Commercially available oleate-MNPs were successfully converted to polyvinylpyrrolidone-MNPs (PVP-MNPs), and then coated with silica by the modified Stöber method. More than 95% of MNPs were individually coated with a silica shell; non-magnetic core silica nanoparticles (NPs) were not detected. Notably, the MNP@SiO₂ NPs are highly monodispersed in size (size distribution < 2.5%) and synthesis at the scale of grams was easily obtained by a simple scale up process. Moreover, aggregation was not detected upon storage of over three months.     

Preparation and characterization of polymer-coated mesoporous silica nanoparticles and their application in Subtilisin immobilization

The preparation and characterization of polymer-coated mesoporous silica nanoparticles (MSNs) and their application in Subtilisin (Alcalase®) immobilization were investigated. For the synthesis of polymer-coated MSNs, acrylic acid (AA) and chitosan (CS) mixture were blended as poly(acrylic acid) (PAA) and CS polymer layer onto MSNs via in-situ polymerization technique. Then, both uncoated MSNs and polymer-coated mesoporous silica nanoparticles (CS-PAA/MSNs) were characterized by taking into account properties such as morphologic pattern, size distribution, surface charge of the particles as well as thermogravimetric stability with SEM, TEM, Zetasizer and TGA analyses. Subtilisin was immobilized onto polymer-coated mesoporous silica nanoparticles via adsorption technique. For optimizing the enzyme immobilization process, the percent enzyme loading depending on the matrix amount, immobilization time and pH were investigated. Then, the activity values of immobilized enzyme and free enzyme were compared at various pH and temperature values. The maximum enzyme activity was achieved at pH 9.0 for both immobilized and free enzyme. Immobilized enzyme showed more stability at higher temperatures compared with free enzyme. Furthermore, the operational and storage stability of immobilized enzyme were determined. The activity of immobilized enzyme was reduced from 100% to 45.83% after five repeated uses. The storage stability of immobilized enzyme was found to be higher than that of free enzyme. The activity of immobilized enzyme was reduced from 100% to 60% after 28 days of storage time. We concluded that the polymer-coated MSNs were a suitable matrix for Subtilisin immobilization compared to uncoated MSNs.     

Fabrication of a Chitosan‐Coated Magnetic Nanobiocatalyst for Starch Hydrolysis

The preparation of chitosan‐coated magnetic nanoparticles (MNPs) and covalent immobilization of α‐amylase for starch hydrolysis was investigated. Surface morphology, chemical composition, and structural characteristics of the MNPs were analyzed by scanning electron microscopy, energy dispersion spectrometry, and X‐ray diffractometry, respectively. Surface functional groups of MNPs, chitosan‐coated MNPs, and α‐amylase‐immobilized MNPs were characterized by Fourier transform infrared spectroscopy. Response surface methodology based on three levels was implemented to optimize three immobilization conditions and a regression model was developed. α‐Amylase‐immobilized MNPs provided better stability towards pH and temperature. The prepared thermostable nanobiocatalyst is well‐suited for industrial processes involving starch hydrolysis.     

Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

Amine functionalized cobalt ferrite (AF-CoFe₂O₄) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxy-succinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe₂O₄ were determined. TEM micrograph revealed a mean diameter of ～8 nm and showed that the AF-CoFe₂O₄ remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe₂O₄. The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe₂O₄ surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe₂O₄ MNPs can be considered as promising candidate for enzyme immobilization.     

Immobilized laccase on magnetic nanoparticles for enhanced lignin model compounds degradation

As a natural aromatic polymer, lignin has great potential but limited industrial application due to its complex chemical structure. Among strategies for lignin conversion, biodegradation has attracted promising interest recently in term of efficiency, selectivity and mild condition. In order to overcome the issues of poor stability and non-reusability of enzyme in the biodegradation of lignin, this work explored a protocol of immobilized laccase on magnetic nanoparticles(MNPs) with rough surfaces for enhanced lignin model compounds degradation.Scanning electron microscope with energy dispersive spectrometer(SEM-EDS), flourier transformation infrared spectroscopy(FTIR) and thermal gravimetric analysis(TGA) were utilized to characterize the immobilization of laccase. The results showed a maximum activity recovery of 64.7% towards laccase when it was incubated with MNPs and glutaraldehyde(GA) with concentrations of 6 mg·ml~(-1) and 7.5 mg·ml~(-1) for 5 h, respectively. The immobilized laccase showed improved thermal stability and pH tolerance compared with free laccase, and remained more than 80% of its initial activity after 20 days of storage at 4 ℃. In addition, about 40% residual activity of the laccase remained after 8 times cycles. Gas chromatography–mass spectrometry(GC–MS) was utilized to characterize the products of lignin model compound degradation and activation, and the efficiency of immobilized laccase was calculated to be 1–5 times that of free laccase. It was proposed that the synergistic effect between MNPs and laccase displays an important role in the enhancement of stability and activity in lignin model compound biodegradation.     

Preparation of Cu(II)‐chelated poly(vinyl alcohol) nanofibrous membranes for catalase immobilization

Poly(vinyl alcohol) (PVA) nanofibers were formed by electrospinning. Metal chelated nanofibrous membranes were prepared by reaction between Cu(II) solution and nanofibers, and which were used as the matrix for catalases immobilization. The constants of Cu(II) adsorption and properties of immobilized catalases were studied in this work. The Cu(II) concentration was determined by atomic absorption spectrophotometer (AAS), the immobilized enzymes were confirmed by the Fourier transform infrared spectroscopy (FTIR), and the amounts of immobilized enzymes were determined by the method of Bradford on an ultraviolet spectrophotometer (UV). Adsorption of Cu(II) onto PVA nanofibers was studied by the Langmuir isothermal adsorption model. The maximum amount of coordinated Cu(II) (q_m) was 2.1 mmol g⁻¹ (dry fiber), and the binding constant (K_l) was 0.1166 L mmol⁻¹. The immobilized catalases showed better resistance to pH and temperature inactivation than that of free form, and the thermal and storage stabilities of immobilized catalases were higher than that of free catalases. Kinetic parameters were analyzed for both immobilized and free catalases. The value of V_max (8425.8 μmol mg⁻¹) for the immobilized catalases was smaller than that of the free catalases (10153.6 μmol mg⁻¹), while the K_m for the immobilized catalases were larger. It was also found that the immobilized catalases had a high affinity with substrate, which demonstrated that the potential of PVA‐Cu(II) chelated nanofibrous membranes applied to enzyme immobilization and biosensors. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Immobilization of invertase onto crosslinked poly(p‐chloromethylstyrene) beads

Invertase was immobilized onto poly(p‐chloromethylstyrene) (PCMS) beads that were produced by a suspension polymerization with an average size of 186 μm. The beads had a nonporous but reasonably rough surface. Because of this, a reasonably large external surface area (i.e., 14.1 m²/g) could be achieved with the proposed carrier. A two‐step functionalization protocol was followed for the covalent attachment of invertase onto the bead surface. For this purpose, a polymeric ligand that carried amine groups, polyethylenimine (PEI), was covalently attached onto the bead surface by a direct chemical reaction. Next, the free amine groups of PEI were activated by glutaraldehyde. Invertase was covalently attached onto the bead surface via the direct chemical reaction between aldehyde and amine groups. The appropriate enzyme binding conditions and the batch‐reactor performance of the immobilized enzyme system were investigated. Under optimum immobilization conditions, 19 mg of invertase was immobilized onto each gram of beads with 80% retained activity after immobilization. The effects of pH and temperature on the immobilized invertase activity were determined and compared with the free enzyme. The kinetic parameters K_M and V_M were determined with the Michealis–Menten model. K_M of immobilized invertase was 1.75 folds higher than that of the free invertase. The immobilization caused a significant improvement in the thermal stability of invertase, especially in the range of 55–65°C. No significant internal diffusion limitation was detected in the immobilized enzyme system, probably due to the surface morphology of the selected carrier. This result was confirmed by the determination of the activation energies of both free and immobilized invertases. The activity half‐life of the immobilized invertase was approximately 5 times longer than that of the free enzyme. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 1268–1279, 2002     

Immobilized laccase on magnetic nanoparticles for enhanced lignin model compounds degradation

As a natural aromatic polymer, lignin has great potential but limited industrial application due to its complex chemical structure. Among strategies for lignin conversion, biodegradation has attracted promising interest recently in term of efficiency, selectivity and mild condition. In order to overcome the issues of poor stability and non-reusability of enzyme in the biodegradation of lignin, this work explored a protocol of immobilized laccase on magnetic nanoparticles (MNPs) with rough surfaces for enhanced lignin model compounds degradation. Scanning electron microscope with energy dispersive spectrometer (SEM-EDS), flourier transformation infrared spectroscopy (FTIR) and thermal gravimetric analysis (TGA) were utilized to characterize the immobilization of laccase. The results showed a maximum activity recovery of 64.7% towards laccase when it was incubated with MNPs and glutaraldehyde (GA) with concentrations of 6 mg·ml^-1 and 7.5 mg·ml^-1 for 5 h, respectively. The immobilized laccase showed improved thermal stability and pH tolerance compared with free laccase, and remained more than 80% of its initial activity after 20 days of storage at 4 ℃. In addition, about 40% residual activity of the laccase remained after 8 times cycles. Gas chromatography-mass spectrometry (GC-MS) was utilized to characterize the products of lignin model compound degradation and activation, and the efficiency of immobilized laccase was calculated to be 1-5 times that of free laccase. It was proposed that the synergistic effect between MNPs and laccase displays an important role in the enhancement of stability and activity in lignin model compound biodegradation.     

Silica coating of Fe3O4 magnetic nanoparticles with PMIDA assistance to increase the surface area and enhance peptide immobilization efficiency

The high efficiency of using N-(phosphonomethyl)iminodiacetic acid (PMIDA) as a surfactant for formation of a silica coating on Fe₃O₄ magnetic nanoparticles (MNPs) with a large surface area has been demonstrated. The coating of PMIDA-stabilized MNPs with silica and their further APS-functionalization significantly increased the specific area (up to 203 m² g^?1) and the number of amino groups (up to 1.12 mmol/g) grafted on their surface compared to nanomaterials synthesized without preliminary SiO₂-coating. The comparative study of the peptide modification efficiency, using as an example pH-low insertion peptide (pHLIP), of MNPs coated with 3-aminopropylsilane (APS) or SiO₂/APS was carried out. It has been shown that silica coating of PMIDA-stabilized MNPs leads to a significant increase in the degree of immobilization of the peptide (up to 22 μmol per g of MNPs). Comprehensive characterization of the obtained materials at each stage of the synthesis was carried out using scanning electron microscopy (SEM), energy dispersive X-ray fluorescence spectroscopy (EDX), BET analysis, ATR Fourier transformed infrared spectroscopy (FTIR), termogravimetric analysis (TGA), CHN-elemental analysis, dynamic light scattering (DLS), and vibrating sample magnetometry (VSM). The proposed approach to applying SiO₂-coating of MNPs can be useful for design of new materials for biomedical and chemical purposes.     

Sintering Behavior of Pt Metal Particles Supported on Silica-Coated Alumina Surface

The sintering behavior of Pt metal particles was studied by supporting them on silica-coated alumina. Silica coating was found to be effective for the retention of a large surface area of alumina even after calcination at elevated temperatures. Before sintering, the size of Pt metal particles on all the silica-coated aluminas, including the uncoated alumina, was identical, while the particle size was larger on silica than on alumina. After sintering the Pt catalyst at 1073 K, the particle size increased on uncoated alumina as well as on alumina coated with thicker silica layers, especially on the supports previously calcined at >1473 K. On the other hand, the size of Pt metal particles did not increase much on alumina coated with monolayer silica. The observed suppression of sintering of Pt metal particles resulted from the retention of a large surface area of alumina with a thinner silica layer. In the case of a thicker silica layer, although a large surface area was maintained after calcination at elevated temperatures, the existence of a bulk silica-like property of the support did not favor the suppression of sintering of Pt metal particles.     

Behavior of immobilized glucose oxidase on membranes from polyacrylonitrile and copolymer of methylmethacrylate‐dichlorophenylmaleimide

Two new ultrafiltration membranes were obtained from a polymer mixture, containing 60% polyacrylonitrile (PAN) and 40% copolymer of methylmethacrylate‐dichlorophenylmaleimide (MMA‐DCPMI). Membrane 1 (MB1) contains 40% DCPMI of the copolymer, and membrane 2 (MB2) contains 15% of the copolymer. The pore size, the specific surface, the water content, the water flux, and the selectivity were determined for the two membranes. The presence of dichlorophenylmaleimide in the copolymer ensures the preparation of membranes suitable for direct covalent enzyme immobilization without further modifications. These membranes were used for immobilization of glucose oxidase (GOD). High amount of bound protein was found on each of the membranes. High relative activities of the immobilized GOD were achieved, 72% for MB1 and 68% for MB2. The properties of the immobilized enzyme (GOD) were determined: optimum pH and temperature and pH, thermal, and storage stability, and then compared with the properties of the native enzyme. The kinetic parameters of the enzyme reaction, Michaelis constant (K_m) and maximum reaction rate (V_max), were also investigated. The results obtained showed that the ultrafiltration membranes prepared from the mixture of PAN and the copolymer MMA‐DCPMI were suitable for use as carriers for the immobilization of GOD. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 4334–4340, 2006     

多巴胺包埋磁性SiO2固定化漆酶催化去除4-氯酚

以磁性纳米颗粒为载体,通过多巴胺(dopamine,DA)聚合原位包埋制备了磁性SiO₂固定化漆酶(Fe₃O₄@SiO₂-PDA-Lac)。结果显示纳米颗粒尺寸均匀,并且保持较高的饱和磁性。通过最优条件制备出的固定化漆酶在50℃放置6 h后,活性保持在63%,而游离酶仅保留18%。将固定化酶用于催化降解4-氯酚(4-CP),探究了溶液pH、漆酶浓度和ABTS[2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸铵)]对4-CP去除率的影响。固定化漆酶在反应最适pH时,4-CP去除率为84.3%,而游离酶仅为65.7%。当漆酶浓度为1.2 U·ml^-1时,反应8 h后,4-CP去除率可达95%,而游离酶的4-CP去除率仅82%。ABTS可促进固定化漆酶降解4-CP,当体系中加入50 μmol·L^-1 的ABTS,反应10 min后,固定化酶对4-CP去除率可达99%。固定化漆酶在重复使用10次后,4-CP去除率仍可达67%。     

硒化镉量子点的二氧化硅包覆

将油溶性、单分散、高荧光强度的CdSe荧光纳米晶用Stber方法成功地制备了SiO2包覆的荧光纳米晶,包覆后的纳米晶在水中和醇中有良好的溶解性,具有非常好的化学稳定性。SiO2包覆后的纳米晶也具有较强的荧光特性。..     

Synthesis of poly[(5-benzyloxy-trimethylene carbonate)-co-(5,5-dimethyl-trimethylene carbonate)] catalyzed by immobilized lipase on silica particles with different size

Porcine pancreas lipase (PPL) immobilized on silica particles with different size were prepared and employed successfully for ring-opening co-polymerization of 5-benzyloxy-trimethylene carbonate (BTMC) with 5,5-dimethyl-trimethylene carbonate (DTC) for the first time. Three kinds of silica particles with different sizes (150-250, 75-150 and 1 μm) were selected as carriers for enzyme immobilization. The structure of copolymers were confirmed by ¹H and ¹³C NMR which showed no decarboxylation occurrence during the polymerization. The (M_n) of poly(BTMC-co-DTC) decreased rapidly with the increasing of immobilized PPL concentration. The carrier size of immobilized PPL affected both the catalytic activity and the polymer yield. The highest molecular weight (M_n=26,400) of poly(BTMC-co-DTC) was obtained at around 0.1% concentration of immobilized PPL on silica particles with size of 75-150 μm.     

Silver Nanoparticles Loaded Thermoresponsive Hybrid Nanofibrous Hydrogel as a Recyclable Dip‐Catalyst with Temperature‐Tunable Catalytic Activity

Silver nanoparticles (AgNPs) loaded thermoresponsive nanofibrous hydrogel is fabricated by electrospinning the aqueous solution containing the metal nanoparticles and poly((N‐isopropylacrylamide)‐co‐(N‐hydroxymethylacrylamide)) copolymer, followed by heat treatment. To avoid negative effect of the stabilizer or the residual reductant on their performances, the AgNPs of less than 5 nm size are synthesized through reducing Ag⁺ ions in the spinning solution by UV irradiation. The prepared nanofibrous hydrogel with desirable stability in aqueous medium has significant thermoresponsive property, and can reach its swelling or deswelling equilibrium state within 15 s with the medium temperature changing between 25 and 50 °C alternately. The smart nanofibrous hydrogel as a dip‐catalyst has the catalysis for the reduction of 4‐nitrothiophenol to 4‐aminothiophenol by NaBH₄, and its catalytic activity can be rapidly tuned by temperature. Moreover, it can be facilely recycled from the reaction system at least four times, without any loss of its catalytic activity.     

Immobilization of enzymes to porous-bead polymers and silica gels activated by graft polymerization of 2,3-epoxypropyl methacrylate

Three types of organic polymers and bead-shape silica gels were activated by graft polymerization of 2,3-epoxypropyl methacrylate; in some cases, epoxide groups on the support surface were modified to NH₂ groups. Eight active matrices so obtained were assessed as supports for immobilized enzymes using peroxidase, glucoamylase and urease. The immobilization yield of protein and specific activities of enzymes were better with supports containing NH₂ groups than with those containing epoxide spacer arms. Maximum enzyme immobilization and storage stabilities were obtained with silica-gel beads activated by graft polymerization of 2,3-epoxypropyl methacrylate. With all eight matrices tested, the immobilized enzymes showed good stability with not less than 82% of the original activity persisting after 28 days. The developed matrices have potential for use in process-scale biotechnological operations.     

Lipase Immobilization onto the Surface of PGMA-b-PDMAEMA-grafted Magnetic Nanoparticles Prepared via Atom Transfer Radical Polymerization

A block copolymer of 2-dimethylaminoethyl methacrylate (DMAEMA) and glycidyl methacrylate (GMA) was grafted onto the surface of magnetic nanoparticles (Fe₃O₄) via atom transfer radical polymerization. The resultant PGMA-b-PDMAEMA-grafted-Fe₃O₄ magnetic nanoparticles with amino and epoxy groups were characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction, thermo-gravimetric analysis, and scanning electron microscopy. Lipase from Burkholderia cepacia was successfully immobilized onto the magnetic nanoparticles by physical adsorption and covalent bonding. The immobilization capacity of the magnetic particles is 0.5 mg lipase per mg support, with an activity recovery of up to 43.1% under the optimum immobilization condition. Biochemical characterization shows that the immobilized lipase exhibits improved thermal stability, good tolerance to organic solvents with high lg P, and higher pH stability than the free lipase at pH 9.0. After six consecutive cycles, the residual activity of the immobilized lipase is still over 55% of its initial activity.     

Stability and magnetically induced heating behavior of lipid-coated Fe3O4 nanoparticles

Magnetic nanoparticles that are currently explored for various biomedical applications exhibit a high propensity to minimize total surface energy through aggregation. This study introduces a unique, thermoresponsive nanocomposite design demonstrating substantial colloidal stability of superparamagnetic Fe₃O₄ nanoparticles (SPIONs) due to a surface-immobilized lipid layer. Lipid coating was accomplished in different buffer systems, pH 7.4, using an equimolar mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and l-α-dipalmitoylphosphatidyl glycerol (DPPG). Particle size and zeta potential were measured by dynamic laser light scattering. Heating behavior within an alternating magnetic field was compared between the commercial MFG-1000 magnetic field generator at 7 mT (1 MHz) and an experimental, laboratory-made magnetic hyperthermia system at 16.6 mT (13.7 MHz). The results revealed that product quality of lipid-coated SPIONs was significantly dependent on the colloidal stability of uncoated SPIONs during the coating process. Greatest stability was achieved at 0.02 mg/mL in citrate buffer (mean diameter = 80.0 ± 1.7 nm; zeta potential = -47.1 ± 2.6 mV). Surface immobilization of an equimolar DPPC/DPPG layer effectively reduced the impact of buffer components on particle aggregation. Most stable suspensions of lipid-coated nanoparticles were obtained at 0.02 mg/mL in citrate buffer (mean diameter = 179.3 ± 13.9 nm; zeta potential = -19.1 ± 2.3 mV). The configuration of the magnetic field generator significantly affected the heating properties of fabricated SPIONs. Heating rates of uncoated nanoparticles were substantially dependent on buffer composition but less influenced by particle concentration. In contrast, thermal behavior of lipid-coated nanoparticles within an alternating magnetic field was less influenced by suspension vehicle but dramatically more sensitive to particle concentration. These results underline the advantages of lipid-coated SPIONs on colloidal stability without compromising magnetically induced hyperthermia properties. Since phospholipids are biocompatible, these unique lipid-coated Fe₃O₄ nanoparticles offer exciting opportunities as thermoresponsive drug delivery carriers for targeted, stimulus-induced therapeutic interventions.

PACS

7550Mw; 7575Cd; 8185Qr     

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH₂ nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V_max, k_cat and k_cat/K_m, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.