Support Material For Enzyme Immobilization Research Articles

Surfactant-based metal-organic frameworks (MOFs) in the preparation of an active biocatalysis.

Metal-organic frameworks (MOFs) are used as ideal support materials thanks to their unique properties and have become the focus of interest in enzyme immobilization studies, especially in recent years. In order to increase the catalytic activity and stability of Candida rugosa lipase (CRL), a new fluorescence-based MOF (UiO-66-Nap) derived from UiO-66 was synthesized. The structures of the materials were confirmed by spectroscopic techniques such as FTIR, 1H NMR, SEM, and PXRD. CRL was immobilized on UiO-66-NH2 and UiO-66-Nap by adsorption technique and immobilization and stability parameters of UiO-66-Nap@CRL were examined. Immobilized lipases UiO-66-Nap@CRL exhibited higher catalytic activity (204U/g) than UiO-66-NH2 @CRL (168U/g), which indicates that the immobilized lipase (UiO-66-Nap@CRL) carries sulfonate groups, this is due to strong ionic interactions between the surfactant's polar groups and certain charged locations on the protein surface. The Free CRL lost its catalytic activity completely at 60°C after 100min, while UiO-66-NH2 @CRL and UiO-66-Nap@CRL retained 45 % and 56 % of their catalytic activity at the end of 120min, respectively. After 5 cycles, the activity of UiO-66-Nap@CRL remained 50 %, while the activity of UiO-66-NH2 @CRL was about 40 %. This difference is due to the surfactant groups (Nap) in UiO-66-Nap@CRL. These results show that the newly synthesized fluorescence-based MOF derivative (UiO-66-Nap) can be an ideal support material for enzyme immobilization and can be used successfully to protect and increase the activities of enzymes.

Insight into Bioactivity of In‐situ Trapped Enzyme‐Covalent‐Organic Frameworks

AbstractSelecting a suitable support material for enzyme immobilization with excellent biocatalytic activity and stability is a critical aspect in the development of functional biosystems. The highly stable and metal‐free properties of covalent‐organic frameworks (COFs) make them ideal supports for enzyme immobilization. Herein, we constructed three kinds of COFs via a biofriendly and one‐pot synthetic strategy at room temperature in aqueous solution. Among the three developed COFs (COF‐LZU1, RT‐COF‐1 and ACOF‐1), the horseradish peroxidase (HRP)‐incorporated COF‐LZU1 is found to retain the highest activity. Structural analysis reveals that a weakest interaction between the hydrated enzyme and COF‐LZU1, an easiest accessibility by the COF‐LZU1 to the substrate, as well as an optimal conformation of enzyme together promote the bioactivity of HRP‐COF‐LZU1. Furthermore, the COF‐LZU1 is revealed to be a versatile nanoplatform for encapsulating multiple enzymes. The COF‐LZU1 also offers superior protection for the immobilized enzymes under harsh conditions and during recycling. The comprehensive understanding of interfacial interactions of COF host and enzyme guest, the substrate diffusion, as well as the enzyme conformation alteration within COF matrices represents an opportunity to design the ideal biocatalysts and opens a broad range of applications of these nanosystems.

Techniques and support materials for enzyme immobilization using Ugi multicomponent reaction: an overview

Techniques and support materials for enzyme immobilization using Ugi multicomponent reaction: an overview

Fully integrated flexible biosensor for wearable continuous glucose monitoring

Continuous physiological monitoring is a promising alternative to current chronic disease management for obtaining big data sets to help individualized therapy. Here, we present a continuous glucose monitoring platform consisting of a screen-printed electrochemical biosensor and a fully integrated wireless electrochemical analysis system. A biocompatible conjugated polymer (poly (N-phenylglycine)) was employed as the support material for enzyme immobilization. Specifically, a polyurethane outer layer was decorated onto the working electrode of the biosensor to construct a diffusion limiting membrane and improve the linear range of the glucose sensor. We optimized the fabricated glucose sensor so that it achieves a linear range of 1–30 mM and a sensitivity of 12.69 μA mM−1·cm−2 in vitro. The long-term stability is up to 30 days by storing in PBS solution at 4°C. The overall system design was very small (0.8 × 1.8 cm) and consisted of a signal conditioning part, a programmable electrochemical chip, and a wireless connection using Bluetooth Low Energy with a smartphone. Finally, we carried out biocompatibility tests and animal experiments to demonstrate the device can successfully monitor blood glucose in vivo.

Modification of zeolite with metallic ions improves the immobilization of phytase

Phytase is an enzyme applied in several fields, but its wider use is still limited due to its high cost and poor stability, which can be overcome using immobilization techniques. Zeolites are promising support materials for enzyme immobilization, due to their attractive properties, including the capacity for modification using a simple ion exchange procedure, enabling better chemical interaction with proteins. In this work, investigation was made of the immobilization of phytase onto zeolites modified with metallic ions (Ag, Cd, Zn, Cu, Ca, Mg, and Ni). The zeolite modified with nickel reached immobilization yield of 66%, while the recovered activity (150%) indicated activation of the catalytic activity due to the presence of the metallic ion. The immobilized phytase showed excellent performance under conditions of acidic pH, reaching a relative activity up to 7 times higher than observed for its free form. Furthermore, it was possible to recover and reuse effectively the zeolite-Ni/Phytase derivative in at least 4 consecutive cycles of phytate hydrolysis reaction. The phytase immobilized onto zeolite modified with nickel presented improved properties, expanding the range of potential applications of this important enzyme. These findings demonstrate an efficient and simple protocol for enzyme immobilization onto zeolites modified with metallic ions.

Nanobiochar and biochar based nanocomposites: Advances and applications

Advanced nanostructured materials like nanobiochar have come up with the sustainable solutions for a range of problems of modern era. In recent years, carbon nanomaterials have been developed as powerful tools due to their unique characteristics and number of applications in various areas like energy, materials, agriculture and the environment, specifically in phytoremediation of various organic, inorganic and heavy metal contaminants. Biochar technology and nanobiotechnology may result in production of carbon-based nanomaterials including nanobiochar and biochar nanocomposites to revolutionize the research in concerned fields. Nanobiochar is nanosized biochar material with better physical, chemical and surface properties. It has numerous advantages such as improvement in plant growth and soil properties, plant disease management, bioremediation of contaminants and pesticides, treatment of wastewater and it is also used as support material for enzyme immobilization. It may become the best alternative over conventional approaches due to its cost effectiveness, sustainability and environmental friendliness. It also can mitigate climate change by carbon sequestration function. As compared with biochar, nanobiochar has excellent capability of absorbing pollutants, nutrients and contaminants and has mobility in soil thus offering a potential waste management substitute. This review paper is an extensive compilation about production of nanobiochar and biochar based nanocomposites, their diverse applications in agriculture and environment sectors with some other novel applications and recent developments in the fields such as energy, catalysis, material science, biomedical etc. • Introduction to nanobiochar and its advantages over raw/macro biochar counterpart. • Synthesis methods of nanobiochar and biochar based nanocomposite. • Unique characteristics of nanobiochar responsible for phytoremediation through adsorption. • Applications of nanobiochar and biochar nanocomposites in the field of agriculture and the environment.

Synthesis and characterization of immobilized glucoamylase on mesocellular foams through spectroscopic techniques and catalytic activity of immobilized enzyme

Siliceous mesocellular foam (MCF) is versatile support material for enzyme immobilization. This material is attractive for its robust, well-defined pore structure with interconnected, ultra large pores that facilitate diffusion. Excellent activity and extremely low level of leaching (metal or enzyme) were attained with these robust supports. MCF was synthesized by hydrothermal procedure using tetraethylorthosilicate (TEOS), amphiphilic block co-polymers Pluronic P123 and Trimethyl benzene as auxilary chemical under acidic conditions. The amino groups have been grafted to as-synthesized mesoporous silica by 3-aminopropyl trimethoxysilane (3-APTS) and glutaraldehyde was then coupled with amino functionalized silica. The physicochemical properties of the prepared materials were characterized by Nitrogen adsorption – desorption, FTIR, XPS, CPMAS NMR techniques. Meso cellular foams having ultra large pore diameter of 241 Å. Results showed that glutaraldehyde bound silica showed highest stability for enzyme binding.

Carbonic Anhydrase Carrying Electrospun Nanofibers for Biocatalysis Applications.

Enzymes are efficient biocatalysis that catalysis a large number of reactions due to their chemical, regional, or stereo specifities and selectivity. Their usage in bioreactor or biosensor systems has great importance. Carbonic anhydrase enzyme catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. In organisms, the carbonic anhydrase enzyme has crucial roles connected with pH and CO2 homeostasis, respiration, and transport of CO2/bicarbonate, etc. So, immobilization of the enzyme is important in stabilizing the catalyst against thermal and chemical denaturation in bioreactor systems when compared to the free enzyme that is unstable at high temperatures and extreme pH values, as well as in the presence of organic solvents or toxic reagents. Nano-scale composite materials have attracted considerable attention in recent years, and electrospinning based all-nanocomposite materials have a wide range of applications. In this study, electrospun nanofibers were fabricated and used for the supporting media for carbonic anhydrase enzyme immobilization to enhance the enzyme storage and usage facilities. In this article, our motivation is to obtain attractive electrospun support for carbonic anhydrase enzyme immobilization to enhance the enzyme reusability and storage ability in biocatalysis applications. In this article, we propose electrospun nanofibers for carbonic anhydrase carrying support for achieving our aforementioned object. In the first part of the study, agar with polyacrylonitrile (PAN) nanofibers was directly fabricated from an agar-PAN mixture solution using the electrospinning method, and fabricated nanofibers were cross-linked via glutaraldehyde (GA). The morphology, chemical structure, and stability of the electrospun nanofibers were characterized. In the second part of the study, the carbonic anhydrase enzyme was immobilized onto fabricated electrospun nanofibers. Then, enzyme activity, the parameters that affect enzyme immobilization such as pH, enzyme amount, immobilization time, etc. and reusability were investigated. When the scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) analysis results are combined in the characterization process of the synthesized electrospun nanofibers, the optimum cross-linking time is found to be 8 hours using 5% glutaraldehyde cross-linking agent. Then, thermal stability measurements showed that the thermal stability of electrospun nanofibers has an excellent characteristic for biomedical applications. The optimum temperature value was found 37°C, pH 8 was determined as an optimum pH, and 100 ppm carbonic anhydrase enzyme concentration was found to be optimum enzyme concentration for the carbonic anhydrase enzyme immobilization. According to the kinetic data, carbonic anhydrase immobilized electrospun nanofibers acted as a biocatalyst in the conversion of the substrate to the product in 83.98%, and immobilized carbonic anhydrase enzyme is reusable up to 9 cycles in biocatalysis applications. After applying the framework, we get a new biocatalysis application platform for carbonic anhydrase enzyme. Electrospun nanofibers were chosen as the support material for enzyme immobilization. By using this approach, the carbonic anhydrase enzyme could easily be used in the industrial area by cost-effective advantageous aspects.

Chitin and chitosan-based support materials for enzyme immobilization and biotechnological applications

Enzymes of industrial importance are primarily employed for biotechnological applications. However, high-cost and instability issues of purified enzymes hamper their usage. Multiple reuses rather than the single use is more cost-effective. A robuster bioprocess is feasible by enzyme immobilization. Performance of immobilized enzymes depends on the nature of support materials. Chitin and its derivatives-based supports offer stability and cost-effective bioprocessing. Chitosan is biocompatible, biodegradable, non-toxic and has multiple functional groups. A variety of supports such as chitosan, chitosan film, chitosan nanoparticle and chitosan nanocomposite are employed for enzyme immobilization. Chitosan bound enzymes, as compared to free enzymes, have improved the biocatalytic performances due to exceptionally high operational stability and reusability. Here we review enzymes immobilized on chitin/chitosan supporting materials with applications ranging from agriculture to drug delivery.

Mesostructured cellular foam silica materials for laccase immobilization and tetracycline removal: A comprehensive study

Abstract Due to its exceptional textural, morphological and mechanical properties, mesostructured cellular foam (MCF) silica is a suitable support material for enzyme immobilization. However, the simple and efficient protocol of enzyme immobilization using MCF is often overlooked. Thus, in the presented study, we describe the synthesis, modification and characterization of mesostructured cellular foam silica for use in laccase immobilization. It has been established that the synthesized materials are characterized by high surface area equal to approx. 550 m2 g−1 and pores with diameters of approx. 20 nm, which makes them suitable for effective enzyme binding. Furthermore, immobilization of the laccase onto both, unmodified and Cu-modified MCF has been carried out, resulting in process yield of over 95% and 85%, respectively. Upon immobilization, irrespectively of the support used, laccase retained higher activity (over 80%) at a wider temperature (20–40 °C) and pH (4–7) range, as compared to the free enzyme, which exhibited such high activity only at pH 5 and 30 °C. In addition, the reusability of the laccase immobilized onto MCF + Cu material has also been improved as it still retained approx. 90% of its initial activity after 10 reaction cycles. Finally, the obtained biocatalytic systems have been applied for degradation of tetracycline from aqueous solutions resulting in 100% removal of the antibiotic by the MCF + Cu + lac system due to simultaneous adsorption and biocatalytic conversion.

A General Overview of Support Materials for Enzyme Immobilization: Characteristics, Properties, Practical Utility

In recent years, enzyme immobilization has been presented as a powerful tool for the improvement of enzyme properties such as stability and reusability. However, the type of support material used plays a crucial role in the immobilization process due to the strong effect of these materials on the properties of the produced catalytic system. A large variety of inorganic and organic as well as hybrid and composite materials may be used as stable and efficient supports for biocatalysts. This review provides a general overview of the characteristics and properties of the materials applied for enzyme immobilization. For the purposes of this literature study, support materials are divided into two main groups, called Classic and New materials. The review will be useful in selection of appropriate support materials with tailored properties for the production of highly effective biocatalytic systems for use in various processes.

Immobilization of horseradish peroxidase on electrospun poly(vinyl alcohol)–polyacrylamide blend nanofiber membrane and its use in the conversion of phenol

Electrospun nanofibers, with their porous structures, high surface-to-volume ratio, and good mechanical properties, are used as a support material for enzyme immobilization. In this study, the poly(vinyl alcohol) and polyacrylamide bicomponent (PVA–PAAm) nanofibers were fabricated via the electrospinning method. Synthesized PAAm was characterized with size exclusion chromatography (SEC). Nanofibers were characterized by fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscope (SEM). DSC and TGA analyses showed that the nanofibers were more durable than PVA and PAAm polymers. SEM images demonstrated that all nanofibers possessed uniform and smooth structures (average diameter about 300 nm). FTIR results have shown that PAAm successfully participates in nanofiber structure. The produced nanofibers were used as support material for covalent immobilization of horseradish peroxidase (HRP). The optimum temperature for free HRP was 45 °C, whereas it was 50 °C for the immobilized enzyme. The immobilized HRP showed better storage and thermal stability than free HRP. The kinetic parameters (K m and V max) were found to be 2.42 mM and 0.027 U for the immobilized HRP and 1.86 mM and 0.079 U for the free HRP, respectively. The immobilized enzyme could be used effectively for 25 cycles with 54% retention of the activity. The immobilized HRP was also used for the conversion of phenol. Phenol removal was found to be about 29.68% at 180 min in real wastewater. The novel PVA–PAAm nanofibrous material was successfully used as a support material for covalent immobilization of HRP. Immobilized enzymes such as oxido-reductases onto the PVA–PAAm bicomponent nanofiber could be recommended in the treatment of organic pollutants in industrial effluents.

Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk

Trypsin (EC 3.4.21.4) was successfully immobilized on the surface of Fe3O4 magnetic nanoparticles that had been pre-treated with gallic acid (GA). Measurements of protein load by using Bradford assay and the trypsin-catalyzed hydrolysis of Nα-Benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) were made for the immobilized enzyme. By using magnetic nanoparticles, which provides easy separation and decent support material for enzyme immobilization with high surface area to volume ratio, and by employing biocompatible material gallic acid, immobilized enzyme system was synthesized along with improving trypsin activity and stability. Immobilized trypsin (TR) was more stable than the free one and demonstrated higher enzymatic activity at elevated temperatures (45–55°C) and in the alkaline pH region (6–10.5). Fe3O4 NPs–GA–TR retained 92% of its initial activity after 120days of storage at 4°C in sodium phosphate buffer (0.1M, pH 7.5), whereas the free trypsin maintained about 64% of its initial activity during the same storage period. In addition, activity of the immobilized trypsin was preserved 54.5% of its initial activity after eight times successive reuse. The Michaelis–Menten kinetic constant (Km) and maximum reaction velocity (Vmax) for free trypsin were 5.1mM and 23mM/min, respectively, whereas Km and Vmax values of immobilized trypsin were 7.88mM and 18.3mM/min, respectively. The performance of the immobilized trypsin was demonstrated by carrying out the hydrolysis of bovine serum albumin (BSA) within 1h, and the assay was performed by using liquid chromatography–mass spectrometry (LC–MS/MS) technique. The hydrolysis of bovine milk as a real food was investigated by immobilized trypsin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

CTAB modified dellite: A novel support for enzyme immobilization in bio-based electrochemical detection and its in vitro antimicrobial activity

A novel support material for enzyme immobilization based on cetyltrimethylammonium bromide modified Dellite (CTAB-Del) was successfully synthesized and used to manufacture pyranose oxidase (PyOx) biosensors (CTAB-Del/PyOx). The intercalation of CTAB into Dellite was confirmed by FTIR, XRD and TGA techniques. PyOx was immobilized onto the glassy carbon electrode, via glutaraldehyde crosslinking, by using CTAB-Del as a support. In order to test the analytical performance of CTAB-Del/PyOx biosensors, chronoamperometric measurements were carried out using three electrodes configurations, at a constant potential of −0.7V in working buffer, under stirring, with successive addition of glucose. The linear response for CTAB-Del/PyOx biosensor ranged from 0.01 to 0.50mM with an equation of y=4.42x+0.004 (R2=0.998), and the limit of detection for glucose was calculated to be 0.081μM (S/N=3). In order to confirm its practical use, the CTAB-Del/PyOx biosensor was also applied for glucose measurement in various beverages. In addition, the antimicrobial activities of Del and CTAB-Del were screened in vitro by means of the disc diffusion susceptibility test, selecting a yeast, three different Gram-positive, and five different Gram-negative bacteria. The obtained results showed a moderate antibacterial activity of CTAB-Del against Gram-positive bacteria.

A comparative study of the effectiveness of β-glucosidase immobilized on CNT-nanoparticles and Ca-alginate beads

Enzymes are extensively used in various industrial, biomedical and biopharmaceutical applications. However, enzymes in their free form are unstable and expensive besides being characteristically susceptible to inhibition by high product concentrations and are highly sensitive to pH and temperature changes. Immobilization technology offers solutions to these challenges besides enhancing operational stability, longevity and ease of separation. β-glucosidase has been widely employed as model enzyme for enzymatic studies. Ca-alginate beads provide a gentle environment for immobilization, but have certain limitations such as low stability, high porosity and limitations in biocompatibility. Carbon nanotubes (CNTs) on the other hand have excellent mechanical, thermal and electrical properties, as well as dimensional and chemical compatibility with biomolecules like DNA and enzymes, suitable for biosensor design. Here, β-glucosidase was immobilized in Ca-alginate gel and multi-walled carbon nanotubes (MWCNT) using standard techniques and their activity was compared with that of free enzyme. The activity was found highest (12.53 U/mL) for the free enzyme and lowest (9.768 U/mL) for the immobilized Ca-alginate. The activity of immobilized MWCNT (12.20 U/mL) was close to the free enzyme activity. The enzyme reaction was found to follow Michaelis-Menten kinetics. The Michaelis constants, Km and Vmax, determined using Langmuir linearized plot are respectively 0.09048 μmol/mL and 0.00989 μmol/mL.min for immobilized Ca-alginate; and 0.0985 μmol/mL and 0.01237 μmol/mL.min for immobilized MWCNT. The corresponding values for the free enzyme are 0.0854 μmol/mL and 0.01263 μmol/mL.min. Thus, MWCNT appears to be a promising support material for enzyme immobilization. Keywords: β-glucosidase, immobilization, Ca-alginate, carbon-nanotube, Michaelis-Menten kinetics

Immobilization of enzyme biocatalyst on natural halloysite nanotubes

Abstract In this study, we use natural halloysite nanotubes as novel support materials to immobilize enzymes. Two typical industrial enzymes (α-amylase and urease) with different sizes were immobilized in channels of the nanotubes through simple physical adsorption. After 60 min heating, both immobilized enzymes retained more than 80% activity. Stored for 15 days, the immobilized enzymes still showed more than 90% activity. More than 55% initial activity of the enzyme was retained after 7 cycles. The immobilized enzymes exhibited thermal stability, good storage stability and reusability, which indicate that halloysite is a promising support material for enzyme immobilization.

Natural nanoclays: applications and future trends – a Chilean perspective

AbstractBecause of their large potential for agricultural, industrial and medicinal applications, nanomaterials have been the focus of much research during the past few decades. Nanoclays are natural nanomaterials that occur in the clay fraction of soil, among which montmorillonite and allophane are the most important species. Montmorillonite is a crystalline hydrous phyllosilicate (layer silicate). Organically-modified montmorillonites or ‘organoclays’, formed by intercalation of quaternary ammonium cations, have long been used as rheological modifiers and additives in paints, inks, greases and cosmetics and as carriers and delivery systems for the controlled release of drugs. Perhaps the largest single usage of organoclays over recent years has been in the manufacture of polymer-clay nanocomposites. These organic–inorganic hybrid materials show superior mechanical, thermal and gas-barrier properties. Organoclays are also useful in pollution control and water treatment. Allophane is a non-crystalline aluminosilicate derived from the weathering of volcanic ash. A large proportion of the agricultural land in Chile is covered by volcanic soils,the clay fraction of which is dominated by allophane. Consisting of nanosize (3.5–5.0 nm) hollow spherules, allophane is a suitable support material for enzyme immobilization. Allophane is also effective at adsorbing phenolic compounds and colour from kraft mill effluents and phosphate from water and wastewater.

Hollow silica nanotubes for immobilization of penicillin G acylase enzyme

In this study, porous hollow silica nanotubes (PHSNTs) synthesized via a sol–gel route using nano-sized needle-like CaCO 3 the inorganic templates were employed as a support for immobilization of penicillin G acylase (PGA) biocatalyst. The produced PHSNTs were characterized by BET and transmission electron microscopy (TEM). Effect of various factors such as loading temperature and ratio of carries to free PGA (g/mL) on the catalytic activity of the immobilized PGA was also investigated by unitary factor testing method. The results show that under optimized conditions the relative loading amount and the total activity yield of immobilized enzyme (IME) amounts to 97.20% and 88.80%, respectively. Several advantages, i.e. the rapid immobilization of PGA onto PHSNTs, the high tolerability to the pH, the less sensitivity to the temperature and the improved storage stability render PHSNTs potential support materials for enzyme immobilization.

Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis.

A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, alpha-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound alpha-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized alpha-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass-enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons

Porous zirconia: a new support material for enzyme immobilization

Four different proteases (trypsin, chymotrypsin, papain and pepsin) were covalently attached to the surface of a new type of porous zirconia, as well as a conventional porous silica, activated with 3-isothiocyanatopropyltriethoxy silane (NCS-silane). The immobilization efficiency onto the porous zirconia material was evaluated in terms of the amount of enzyme attached to the particles and from the biological activity remaining after the immobilization step. The results were compared with the corresponding experiments with a porous silica of similar surface area/g support material. In addition, the storage stability of the modified zirconia and silica biocatalysts were evaluated. These results indicated that specific immobilized enzyme biocatalysts can be achieved with this new zirconia support material which exhibits different properties to those observed with the more conventional silica-based materials. Moreover, the results with the enzyme-zirconia biocatalysts also indicate different characteristics when compared with data for the same enzymes immobilized under similar buffer conditions to organic support materials as previously described by various other investigators. The advantages of zirconia-based immobilized enzyme biocatalysts in terms of their density and chemical robustness are also described relative to other alternative support materials currently in use.