Chemically synthesized zinc finger molecules as nano-addressable probes for double-stranded DNAs

Magnetic nanoparticles (Fe₃O₄) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe₃O₄ nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.     

Carboligation Reactions Mediated by Benzoylformate Decarboxylase Immobilized on a Magnetic Solid Support

In this study, magnetic nanoparticles (Fe₃O₄, magnetite) with immobilized metal affinity ligands (MSS) were prepared and characterized by X‐ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), and vibrating‐sample magnetometer (VSM) methods for purification and immobilization of the histidine‐tagged recombinant benzoylformate decarboxylase (BFD). The MSS support was shown to be eligible for selective binding of HIS‐tagged BFD by SDS‐page analysis. Loading capacity of the MSS support was determined as 43.6 ± 1.1 mg/g. The regeneration ability for protein binding was also studied. An immobilized BFD was tested to catalyze benzoin condensation and representative cross acyloin reaction. Conversion and enantiomeric excess values were comparable with that of free enzyme catalyzed reactions. Chirality 25:415–421, 2013. © 2013 Wiley Periodicals, Inc.     

Preparation and characterization of YADH-bound magnetic nanoparticles

The covalently binding of yeast alcohol dehydrogenase (YADH) to magnetic nanoparticles via carbodiimide activation was studied. The magnetic nanoparticles Fe₃O₄ with a mean diameter of 10.6 nm were prepared by co-precipitating Fe²⁺ and Fe³⁺ ions in an ammonia solution and treating under hydrothermal conditions. Transmission electron microscopy (TEM) micrographs showed that the magnetic nanoparticles remained discrete and had no significant change in size after binding YADH. X-ray diffraction (XRD) patterns indicated both the magnetic nanoparticles before and after binding YADH were pure Fe₃O₄. Magnetic measurement revealed the resultant magnetic nanoparticles were superparamagnetic characteristics, and their saturation magnetization was reduced only slightly after enzyme binding. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed the binding of YADH to magnetic nanoparticles and suggested a possible binding mechanism. In addition, the measurement of protein content revealed that the maximum weight ratio of YADH bound to magnetic nanoparticles was 0.125, below which the binding efficiency of YADH was almost 100%. The kinetic measurements indicated the bound YADH retained 62% of its original activity and exhibited a 10-fold improved stability than did the free enzyme. The maximum specific activities and Michaelis constants were also determined.     

Preparation and characterization of amino and carboxyl functionalized core-shell Fe3O4/SiO2 for L-asparaginase immobilization: A comparison study

Abstract

Magnetic nanoparticles are well known as facile and effective support for enzyme immobilization since they have a high surface area, large surface-to-volume ratio, easy separation, a fast and high enzyme loading. This study aims to provide insights on whether acidic or basic modified particles are more effective for L-asparaginase (ASNase) immobilization. Therefore, amino (Fe₃O₄/SiO₂/NH₂) and carboxyl-functionalized (Fe₃O₄/SiO₂/COOH) particles were prepared. The functional groups, crystalline structure, magnetic properties, morphology, chemical composition and thermal behaviour of the prepared modified nanoparticles were examined via Fourier-transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), vibrating-sample magnetometer (VSM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDAX). Under the optimum conditions, the immobilized enzymes were more stable within a certain range of temperatures and pH values in comparison to free enzyme. On the other hand, the immobilized enzymes showed greater stability after incubation for 3?h at 50?°C. The free enzyme maintained only 30% of its initial activity for 4?weeks at 4?°C, while Fe₃O₄/SiO₂/NH₂/ASNase and Fe₃O₄/SiO₂/COOH/ASNase retained more than 78.9% and 56.5% of initial activities under the same conditions, respectively. Moreover, Fe₃O₄/SiO₂/NH₂/ASNase (77.2%) and Fe₃O₄/SiO₂/COOH/ASNase (57.4%) displayed excellent operational stability after 17 repeated cycles. These findings suggested that the Fe₃O₄/SiO₂/NH₂ and Fe₃O₄/SiO₂/COOH may be utilized as efficient and sustainable supports to developed immobilized ASNase in several biotechnological applications.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Cellulases immobilization on chitosan-coated magnetic nanoparticles: application for <Emphasis Type="Italic">Agave Atrovirens</Emphasis> lignocellulosic biomass hydrolysis

In the present study, Trichoderma reesei cellulase was covalently immobilized on chitosan-coated magnetic nanoparticles using glutaraldehyde as a coupling agent. The average diameter of magnetic nanoparticles before and after enzyme immobilization was about 8 and 10 nm, respectively. The immobilized enzyme retained about 37 % of its initial activity, and also showed better thermal and storage stability than free enzyme. Immobilized cellulase retained about 80 % of its activity after 15 cycles of carboxymethylcellulose hydrolysis and was easily separated with the application of an external magnetic field. However, in this reaction, K _m was increased eight times. The immobilized enzyme was able to hydrolyze lignocellulosic material from Agave atrovirens leaves with yield close to the amount detected with free enzyme and it was re-used in vegetal material conversion up to four cycles with 50 % of activity decrease. This provides an opportunity to reduce the enzyme consumption during lignocellulosic material saccharification for bioethanol production.     

Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability

To obtain regenerable magnetic nanoparticles, triethoxy(3-isocyanatopropyl)silane and iminodiacetic acid (IZ) were used as the starting material and immobilized on Fe₃O₄ nanoparticles. Copper ions (Cu²⁺ ions) were loaded on the Fe-IZ nanoparticles and used for cellulase immobilization. The support was characterized by spectroscopic methods (FTIR, NMR) and thermogravimetric analysis, transmission electron microscopy, scanning electron microscope, X-ray diffraction, energy dispersive X-ray analysis, and vibrating sample magnetometer techniques. As a result of experiments, the amount of protein bound to immobilized cellulase (Fe-IZ-Cu-E) and cellulase activity was found to be 33.1 mg/g and 154 U/g at pH 5, 50°C, for 3 h. The results indicated that the free cellulase had kept only 50% of its activity after 2 h, while the Fe-IZ-Cu-E was observed to be around 77%, at 60°C. It was found that the immobilized cellulase maintained 93% of its initial catalytic activity after its sixth use. Furthermore, the Fe-IZ-Cu-E retained about 75% of its initial activity after 28 days of storage. To reuse the support material (Fe-IZ-Cu), it was regenerated by thorough washing with ammonia or imidazole.     

Direct binding and characterization of lipase onto magnetic nanoparticles

Lipase was covalently bound onto Fe(3)O(4) magnetic nanoparticles (12.7 nm) via carbodiimide activation. The Fe(3)O(4) magnetic nanoparticles were prepared by coprecipitating Fe(2+) and Fe(3+) ions in an ammonia solution and treating under hydrothermal conditions. The analyses of transmission electron microscopy (TEM) and X-ray diffraction (XRD) showed that the size and structure of magnetic nanoparticles had no significant changes after enzyme binding. Magnetic measurement revealed the resultant lipase-bound magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu/g (only slightly lower than that of the naked ones (64 emu/g)), a remanent magnetization of 1.0 emu/g, and a coercivity of 7.5 Oe. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed the binding of lipase onto magnetic nanoparticles. The binding efficiency of lipase was 100% when the weight ratio of lipase bound to Fe(3)O(4) nanoparticles was below 0.033. Compared to the free enzyme, the bound lipase exhibited a 1.41-fold enhanced activity, a 31-fold improved stability, and better tolerance to the variation of solution pH. For the hydrolysis of pNPP by bound lipase at pH 8, the activation energy within 20-35 degrees C was 6.4 kJ/mol, and the maximum specific activity and Michaelis constant at 25 degrees C were 1.07 micromol/min mg and 0.4 mM, respectively. It revealed that the available active sites of lipase and their affinity to substrate increased after being bound onto magnetic nanoparticles.     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

A surfactant-coated lipase immobilized in magnetic nanoparticles for multicycle ethyl isovalerate enzymatic production

Gum arabic coated magnetic Fe₃O₄ nanoparticles (GAMNP) were prepared by chemical co-precipitation method and their surface morphology, particle size and presence of polymer-coating was confirmed by various measurements, including transmission electron microscopy (TEM), X-ray diffraction (XRD), thermo gravimetric analysis (TGA), and Fourier transform infra red (FTIR) analysis. Magnetic particles were employed for their potential application as a support material for lipase immobilization. Glutaraldehyde was used as a coupling agent for efficient binding of lipase onto the magnetic carrier. For this purpose, the surface of a Candida rugosa lipase was initially coated with various surfactants, to stabilize enzyme in its open form, and then immobilized on to the support. This immobilized system was used as a biocatalyst for ethyl isovalerate, a flavor ester, production. The influence of various factors such as type of surfactant, optimum temperature and pH requirement, organic solvent used, amount of surfactant in coating lipase and effect of enzyme loadings on the esterification reaction were systematically studied. Different surfactants were used amongst which non-ionic surfactant performed better, showing about 80% esterification yield in 48 h as compared to cationic/anionic surfactants. Enhanced activity due to interfacial activation was observed for immobilized non-ionic surfactant–lipase complex. The immobilized surfactant coated lipase activity was retained after reusing seven times.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

Magnetic single-enzyme nanoparticles with high activity and stability

Magnetic single-enzyme nanoparticles (SENs) encapsulated within a composite inorganic/organic polymer network were fabricated via the surface modification and in situ aqueous polymerization of separate enzyme molecule. The resultant nanoparticles were characterized by transmission electron microscope (TEM), Fourier transform infrared (FTIR) spectrometer and X-ray diffraction (XRD). These particles are almost spherical in shape and have a unique size of about 50 nm in diameter. Electrical and magnetic measurements reveal that the magnetic SENs have a conductivity of 2.7 × 10⁻³ S cm⁻¹, and are superparamagnetic with a saturation magnetization of 14.5 emu g⁻¹ and a coercive force of 60 Oe. Compared with free enzyme, encapsulated enzyme exhibits a strong tolerance to the variation of solution pH, high temperature, organic solvent and long-term storage, thus showing significantly enhanced enzyme performance and stability.     

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe₃O₄/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe₃O₄/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.     

Conjugation of enzyme on superparamagnetic nanogels covered with carboxyl groups

Alpha-chymotrypsin (CT) as model enzyme was conjugated onto the novel carboxyl-functionalized superparamagnetic nanogels, prepared via facile photochemical in situ polymerization, by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) as coupling reagent. The obtained magnetic immobilized enzyme was characterized by use of photo correlation spectroscopy (PCS), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) measurement, thermogravimetric (TG) analysis and vibrating sample magnetometer (VSM) measurement. PCS result showed that the immobilized enzyme was 68 nm in diameter while the magnetic nanogels with carboxyl groups were only 38 nm; enzyme immobilization led to pronounced change in size. Superparamagnetic properties were retained for Fe3O4 after enzyme immobilization while slightly reducing its value of saturation magnetization. Immobilization and surface coating did not induce phase change of Fe3O4 by XRD analysis. The binding capacity was 30 mg enzyme/g and 37.5 mg enzyme/g nanogel determined by TG analysis and BCA protein assay, respectively. Specific activity of the immobilized CT was calculated to be 0.77 U/(mg min), 82.7% as that of the free form.     

Biodesulfurization of Dibenzothiophene by Microbial Cells Coated with Magnetite Nanoparticles

Microbial cells of Pseudomonas delafieldii were coated with magnetic Fe₃O₄ nanoparticles and then immobilized by external application of a magnetic field. Magnetic Fe₃O₄ nanoparticles were synthesized by a coprecipitation method followed by modification with ammonium oleate. The surface-modified Fe₃O₄ nanoparticles were monodispersed in an aqueous solution and did not precipitate in over 18 months. Using transmission electron microscopy (TEM), the average size of the magnetic particles was found to be in the range from 10 to 15 nm. TEM cross section analysis of the cells showed further that the Fe₃O₄ nanoparticles were for the most part strongly absorbed by the surfaces of the cells and coated the cells. The coated cells had distinct superparamagnetic properties. The magnetization (δ_s) was 8.39 emu · g⁻¹. The coated cells not only had the same desulfurizing activity as free cells but could also be reused more than five times. Compared to cells immobilized on Celite, the cells coated with Fe₃O₄ nanoparticles had greater desulfurizing activity and operational stability.     

Immobilization of porcine pancreatic <Emphasis Type="Italic">α</Emphasis>-amylase on magnetic Fe<Subscript>2</Subscript>O<Subscript>3</Subscript> nanoparticles: Applications to the hydrolysis of starch

Enzymes play a pivotal role in catalyzing diverse reactions. However, their instability upon repetitive/prolonged use, as well as their inhibition by high substrates and product concentration, remains an area of concern. In this study, porcine pancreatic α-amylase was immobilized on magnetic Fe₂O₃ nanoparticles (Fe₂O₃-NPs) in order to hydrolyze starch. The magnetic nanoparticle bound enzymes retained 94% of their initial enzyme activity. X-ray diffraction and atomic force microscopy analyses showed that the prepared matrix had advantageous microenvironment and a large surface area for binding significant amounts of protein. Functional groups present in enzyme and support were monitored by Fourier transform infrared spectroscopy. Immobilized enzyme exhibited lowered pH optimum (pH 6.0) to a greater degree than its soluble counterpart (pH 7.0). Optimum temperature for the immobilized enzyme shifted towards higher temperatures. The immobilized enzyme was significantly more resistant to inactivation caused by various metal ions and chemical denaturants. Immobilized α-amylase hydrolyzed 92% starch in a batch process, after 8 h at 40°C; while the free enzyme could hydrolyze only 73% starch under similar experimental conditions. A reusability experiment demonstrated that the immobilized enzyme retained 83% of its original activity even after its 8^th repeated use.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

DNA binding and cytotoxicity studies of magnetic nanofluid containing antiviral drug oseltamivir

In this work, the possibility of preparing a nanoparticle with improved treatment properties was investigated. In this regard, synthesis, characterization, in vitro cytotoxicity and DNA binding of Fe₃O₄@oleate/oseltamivir magnetic nanoparticles (MNPs) were investigated. Fe₃O₄ nanoparticles were synthesized via chemical co-precipitation and coated by oleate bilayers. Then, Fe₃O₄@OA MNPs were functionalized with an antiviral drug (oseltamivir), for better biological applications. The MNPs were subsequently characterized by zeta sizer and Zeta potential measurements, Fourier transform infrared (FT-IR) spectroscopy, vibrating sample magnetometer (VSM) and transmission electron microscopy (TEM) analyses. The TEM image demonstrated that average sizes of Fe₃O₄@OA/oseltamivir MNPs were about 8?nm. The in vitro cytotoxicity of Fe₃O₄@OA/oseltamivir MNPs was studied against cancer cell lines (MCF-7 and MDA-MB-231) and compared with oseltamivir drug. The results illustrated that Fe₃O₄@OA/oseltamivir magnetic nanoparticles have better antiproliferative effects on the mentioned cell lines as compared with oseltamivir. Also, in vitro DNA binding studies were done by UV–Vis, circular dichroism, and Fluorescence spectroscopy. The results indicated that Fe₃O₄@OA/oseltamivir MNPs bound to DNA via groove binding. Moreover, this magnetic nanofluid has potential for magnetic hyperthermia therapy due to magnetic core of its nanoparticles.

Communicated by Ramaswamy H. Sarma     

Preparation of NiO nanoparticles from metal-organic frameworks via a solid-state decomposition route

This study focuses on the preparation and characterization of single phase NiO nanoparticles. At first, nickel-o-phthalate complexes as precursor were synthesized through semisolid phase reaction method and then NiO nanoparticles were obtained via a solid-state decomposition procedure of layered coordination nickle-o-phthalate complexes formulated as [Ni(pht)(H₂O)₂] and [Ni(pht)₂]. In addition, the effects of calcination temperature and metal-to-ligand ratio on the particle size and morphology of NiO were investigated. Thermogravimetric analysis (TGA) was applied to determine the thermal behavior of complexes. The crystalline structure of products by X-ray diffractometer (XRD), morphology of particles by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were studied. The composition of as-prepared sample was studied by X-ray photoelectron spectroscopy (XPS) spectra. Analysis of FT-IR spectra confirmed the composition of products. The magnetic property was studied with vibrating sample magnetometer (VSM).     

Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability

Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe₃O₄ magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g^–1, only slightly lower than that of the naked ones (63 emu g^–1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol^–1, 0.23 mol min^–1 mg^–1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.