Improvement of transglutaminase production by extending differentiation phase of Streptomyces hygroscopicus: mechanism and application.

Streptomyces transglutaminase (TGase) is an important industrial enzyme that catalyzes cross-linking of proteins. It is secreted as a zymogene and then is activated by proteases under physiological conditions. Although the activation process of TGase has been well investigated, the physiological function of TGase in Streptomyces has not been revealed. In this study, physiological function of TGase from Streptomyces hygroscopicus was found to be involved in differentiation by construction of a TGase gene interruption mutation strain (Δtg). The mutant Δtg showed an absence of differentiation compared with the parent strain. Furthermore, the production of TGase was found to be increased with the extending growth arrest phase of mycelium in submerged cultures. Thus, to enhance yield of TGase, the mycelium differentiation of Streptomyces was regulated via low temperature stress in a 3-L stirred-tank fermenter. The production of TGase increased by 39 % through extending the growth arrest phase for 4 h. This study found that TGase is involved in Streptomyces differentiation and proposed an approach to improve TGase production by regulation of mycelium differentiation in submerged cultures.

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology.

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.

Structure-based engineering of methionine residues in the catalytic cores of alkaline amylase from Alkalimonas amylolytica for improved oxidative stability.

This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H(2)O(2) at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the k(cat) values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 × 10(4) to 5.6 × 10(4) min(-1). The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.

Effect of Carbon Chain Length, Ionic Strength, and pH on the In Vitro Release Kinetics of Cationic Drugs from Fatty-Acid-Loaded Contact Lenses.

This paper explores the use of fatty acids in silicone hydrogel contact lenses for extending the release duration of cationic drugs. Drug release kinetics was dependent on the carbon chain length of the fatty acid loaded in the lens, with 12-, 14- and 18-carbon chain length fatty acids increasing the uptake and the release duration of ketotifen fumarate (KTF) and tetracaine hydrochloride (THCL). Drug release kinetics from oleic acid-loaded lenses was evaluated in phosphate buffer saline (PBS) at different ionic strengths (I = 167, 500, 1665 mM); the release duration of KTF and THCL was decreased with increasing ionic strength of the release medium. Furthermore, the release of KTF and THCL in deionized water did not show a burst and was significantly slower compared to that in PBS. The release kinetics of KTF and THCL was significantly faster when the pH of the release medium was decreased from 7.4 towards 5.5 because of the decrease in the relative amounts of oleate anions in the lens mostly populated at the polymer-pore interfaces. The use of boundary charges at the polymer-pore interfaces of a contact lens to enhance drug partition and extend its release is further confirmed by loading cationic phytosphingosine in contact lenses to attract an anionic drug.

Large-scale proteomics and phosphoproteomics of urinary exosomes.

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

Hydrogel-based ocular drug delivery systems for hydrophobic drugs.

Conventional ophthalmic dosage forms such as eye drops pose a significant challenge because physiological barriers and clearance mechanisms limit ocular bioavailability. Hydrogels are promising therapeutic materials for ocular drug delivery because of their high biocompatibility and their ability to hold and release therapeutic agents. Even though they are generally associated with the delivery of hydrophilic drugs, several approaches have been developed to integrate hydrophobic ophthalmic drugs into hydrogels. Because of the limitations associated with the traditional topical eye drop delivery of hydrophobic drugs, hydrogel-based systems represent a viable alternative for controlled ocular drug delivery. This review presents an overview on the ophthalmic applications of hydrogels for the delivery of hydrophobic drugs, with special focus on diseases occurring in the anterior segment of the eye. We summarize the key hydrogels for incorporation and delivery of hydrophobic drugs, including soft contact lenses (SCLs), stimuli-responsive hydrogels, cyclodextrin-based polymeric hydrogels, and nanoparticle-loaded hydrogels. The strategies of integrating hydrophobic drugs into hydrogels as discussed in this review provide significant potential in ocular therapeutics.

Pumpless microfluidic devices for generating healthy and diseased endothelia.

We have developed a pumpless cell culture chip that can recirculate small amounts of cell culture medium (400 µL) in a unidirectional flow pattern. When operated with the accompanying custom rotating platform, the device produces an average wall shear stress of up to 0.588 Pa ± 0.006 Pa without the use of a pump. It can be used to culture cells that are sensitive to the direction of flow-induced mechanical shear such as human umbilical vein endothelial cells (HUVECs) in a format that allows for large-scale parallel screening of drugs. Using the device we demonstrate that HUVECs produce pro-inflammatory indicators (interleukin 6, interleukin 8) under both unidirectional and bidirectional flow conditions, but that the secretion was significantly lower under unidirectional flow. Our results show that pumpless devices can simulate the endothelium under healthy and activated conditions. The developed devices can be integrated with pumpless tissues-on-chips, allowing for the addition of barrier tissues such as endothelial linings.

Effect of a Cationic Surfactant on Microemulsion Globules and Drug Release from Hydrogel Contact Lenses.

The present study evaluates the in vitro release of diclofenac sodium (DFNa) from contact lenses based on poly-2-hydroxyethyl methacrylate (pHEMA) hydrogels containing an embedded microemulsion to extend release duration. The oil (ethyl butyrate)-in-water microemulsion systems are prepared with two non-ionic surfactants, Brij 97 or Tween 80, together with a long-alkyl chain cationic surfactant, cetalkonium chloride (CKC). Without CKC, Brij 97 or Tween 80-based microemulsions showed average droplet sizes of 12 nm and 18 nm, respectively. The addition of CKC decreased the average droplet sizes to 2-5 nm for both non-ionic surfactants. Such significant reduction in the average droplet size corresponds to an increase in the DFNa release duration as revealed by the in vitro experiments. Contact lens characterization showed that important properties such as optical transparency and water content of Brij 97-based contact lenses with cationic microemulsions was excellent. However, the optical transparency of the corresponding Tween 80 based contact lenses was unsatisfactory. The results indicate that cationic microemulsion-laden contact lenses can benefit from combinatory effects of microemulsions and cationic surfactant at low CKC weight percentage, e.g., with the release of 70% of the drug in 45, 10, and 7 h for B97-CKC-0.45%, CKC-0.45%, and control lenses, respectively. However, the microemulsion effect on extending DFNa release became negligible at the highest CKC weight percentage (1.8%).

Extended delivery of non-steroidal anti-inflammatory drugs through contact lenses loaded with Vitamin E and cationic surfactants.

The purpose of this study is to extend drug release from ACUVUE Oasys® and ACUVUE TruEye® silicone hydrogel contact lenses by incorporation of vitamin E in conjunction with a cationic surfactant. In ACUVUE Oasys® and ACUVUE TruEye®, the release of ketorolac tromethamine and flurbiprofen sodium is extended from hours to several days for 11% and 21% vitamin E, (weight of vitamin E / weight of dry lens) but with a considerable reduction in the amount of drug released. Cetalkonium chloride and stearylamine increased the drug loading capacity which was otherwise compromised by the addition of vitamin E in the contact lenses. In the case of diclofenac sodium, a sustained release over 150 h for both contact lenses can be achieved. It was found that the release-time-increase factor due to vitamin E has a linear dependence with the octanol-water partition coefficient of the drug in ACUVUE Oasys®. The results in this study show that contact lenses loaded with vitamin E in conjunction with cationic surfactants achieved sustained release of non-steroidal anti-inflammatory drugs (NSAIDs) within the therapeutic window.

Formation of Drug-Participating Catanionic Aggregates for Extended Delivery of Non-Steroidal Anti-Inflammatory Drugs from Contact Lenses.

This paper focuses on extending drug release duration from contact lenses by incorporating catanionic aggregates. The aggregates consist of a long-chain cationic surfactant, i.e., cetalkonium chloride (CKC), and an oppositely charged anti-inflammatory amphiphilic drug. We studied three non-steroidal anti-inflammatory (NSAID) drugs with different octanol-water partition coefficients; diclofenac sodium (DFNa), flurbiprofen sodium (FBNa), and naproxen sodium (NPNa). Confirmation of catanionic aggregate formation in solution was determined by steady and dynamic shear rheology measurements. We observed the increased viscosity, shear thinning, and viscoelastic behavior characteristic of wormlike micelles; the rheological data are reasonably well described using a Maxwellian fluid model with a single relaxation time. In vitro release experiments demonstrated that the extension in the drug release time is dependent on the ability of a drug to form viscoelastic catanionic aggregates. Such aggregates retard the diffusive transport of drug molecules from the contact lenses. Our study revealed that the release kinetics depends on the CKC concentration and the alkyl chain length of the cationic surfactant. We demonstrated that more hydrophobic drugs such as diclofenac sodium show a more extended release than less hydrophobic drugs such as naproxen sodium.

Measurement of internal tissue optical properties at ultraviolet and visible wavelengths: Development and implementation of a fiberoptic-based system.

A novel, multi-wavelength, fiberoptic system was constructed, evaluated and implemented to determine internal tissue optical properties at ultraviolet A (UVA) and visible (VIS) wavelengths. Inverse modeling was performed with a neural network to estimate absorption and reduced scattering coefficients based on spatially-resolved reflectance distributions. The model was calibrated with simulated reflectance datasets generated using a condensed Monte Carlo approach with absorption coefficients up to 85 cm(-1) and reduced scattering coefficients up to 118 cm(-1). After theoretical and experimental evaluations of the system, optical properties of porcine bladder, colon, esophagus, oral mucosa, and liver were measured at 325, 375, 405, 445 and 532 nm. These data provide evidence that as wavelengths decrease into the UVA, the dominant tissue chromophore shifts from hemoglobin to structural proteins such as collagen. This system provides a high level of accuracy over a wide range of optical properties, and should be particularly useful for in situ characterization of highly attenuating biological tissues in the UVA-VIS.

Ultrathin-Film Titania Photocatalyst on Nanocavity for CO2 Reduction with Boosted Catalytic Efficiencies.

Photocatalytic CO2 reduction with water to hydrocarbons represents a viable and sustainable process toward greenhouse gas reduction and fuel/chemical production. Development of more efficient catalysts is the key to mitigate the limits in photocatalytic processes. Here, a novel ultrathin-film photocatalytic light absorber (UFPLA) with TiO2 films to design efficient photocatalytic CO2 conversion processes is created. The UFPLA structure conquers the intrinsic trade-off between optical absorption and charge carrier extraction efficiency, that is, a solar absorber should be thick enough to absorb majority of the light allowable by its bandgap but thin enough to allow charge carrier extraction for reactions. The as-obtained structures significantly improve TiO2 photocatalytic activity and selectivity to oxygenated hydrocarbons than the benchmark photocatalyst (Aeroxide P25). Remarkably, UFPLAs with 2-nm-thick TiO2 films result in hydrocarbon formation rates of 0.967 mmol g-1 h-1, corresponding to 1145 times higher activity than Aeroxide P25. This observation is confirmed by femtosecond transient absorption spectroscopic experiments where longer charge carrier lifetimes are recorded for the thinner films. The current work demonstrates a powerful strategy to control light absorption and catalysis in CO2 conversion and, therefore, creates new and transformative ways of converting solar energy and greenhouse gas to alcohol fuels/chemicals.

Angiogenesis inhibition and choroidal neovascularization suppression by sustained delivery of an integrin antagonist, EMD478761.

PURPOSE: To evaluate the angiogenic inhibitory effects of an alpha(v)beta(3)/alpha(v)beta(5) integrin antagonist, EMD478761, released from a polymeric implant in a chick chorioallantoic membrane (CAM) assay and laser-induced experimental choroidal neovascularization (CNV) in rats. METHODS: Polyvinyl alcohol-based reservoir implants releasing EMD478761 were designed for placement onto a CAM or intravitreally in rats. In vitro release rates of the implants were measured using HPLC. Angiogenesis was induced on 10-day-old chick embryos by basic fibroblast growth factor (bFGF), and areas of neovascularization were measured. Experimental CNV was induced in the Brown-Norway rat with a diode laser. EMD478761 or sham microimplants were placed within the vitreous chamber of Brown-Norway rats. Two weeks later, areas of CNV were determined by FITC-dextran staining of choroidal flatmounts. RESULTS: Sustained delivery of EMD478761 significantly inhibited bFGF-induced angiogenesis in CAM, as determined by a reduction in angiogenesis areas, without drug toxicity to the normal CAM vasculature. In an experimental rat model, intravitreal EMD478761 implants significantly suppressed laser-induced CNV compared with intravitreal sham implants, with the mean area reduced by 63% (P < 0.05). CONCLUSIONS: Sustained delivery of EMD478761demonstrates potent antiangiogenic properties in vivo. These results suggest that an EMD478761 implant may be beneficial in the treatment of neovascular ocular diseases.

Assessment of subconjunctival and intrascleral drug delivery to the posterior segment using dynamic contrast-enhanced magnetic resonance imaging.

PURPOSE: Sustained-release intravitreal drug implants for posterior segment diseases are associated with significant complications. As an alternative, subconjunctival infusions of drug to the episclera of the back of the eye have been performed, but results in clinical trials for macular diseases showed mixed RESULTS: To improve understanding of transscleral drug delivery to the posterior segment, the distribution and clearance of gadolinium-diethylene-triamino-penta-acetic acid (Gd-DTPA) infused in the subconjunctival or intrascleral space was investigated by means of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). METHODS: In anesthetized rabbits, catheters were placed anteriorly in the subconjunctival or intrascleral space and infused with Gd-DTPA at 1 and 10 muL/min. Distribution and clearance of Gd-DTPA were measured using DCE-MRI. Histologic examination was performed to assess ocular toxicity of the delivery system. results. Subconjunctival infusions failed to produce detectable levels of Gd-DTPA in the back of the eye. In contrast, intrascleral infusions expanded the suprachoroidal layer and delivered Gd-DTPA to the posterior segment. Suprachoroidal clearance of Gd-DTPA followed first-order kinetics with an average half-life of 5.4 and 11.8 minutes after intrascleral infusions at 1 and 10 muL/min, respectively. Histologic examination demonstrated expansion of the tissues in the suprachoroidal space that normalized after infusion termination. CONCLUSIONS: An intrascleral infusion was successful in transporting Gd-DTPA to the posterior segment from an anterior infusion site with limited anterior segment exposure. The suprachoroidal space appears to be an expandible conduit for drug transport to the posterior segment. Further studies are indicated to explore the feasibility of clinical applications.

A pharmacokinetic and safety evaluation of an episcleral cyclosporine implant for potential use in high-risk keratoplasty rejection.

PURPOSE: To determine the short and long-term pharmacokinetics and assess the toxicity of a cyclosporine (CsA) episcleral implant for the prevention of high-risk keratoplasty rejection. METHODS: CsA episcleral implants were made with a high (implant A) or low (implant B) release rate, and in vitro release rates were performed. Short-term pharmacokinetics were performed in rabbits using implant B, and the spatial and temporal spread of drug was observed by sampling from multiple corneal and conjunctival sites at 3 and 72 hours. Implant A was used in long-term pharmacokinetic studies in dogs aged more than 1 year. An ocular toxicity study was performed in dogs older than 1 year. RESULTS: A high release rate was observed with both implants over the initial 5 months followed by a steady state release. The cumulative release over the 400-day assay period from implants A and B was 3.8 +/- 0.3 and 2.3 +/- 0.3 mg, respectively. In the short-term pharmacokinetic studies, the cornea had CsA concentrations of 0.15 +/- 0.06, 0.07 +/- 0.02, and 0.05 +/- 0.02 microg/mg at sites centered 8, 13, and 18 mm away from the implant site, respectively. In the long-term pharmacokinetic studies, corneal CsA levels ranged from 0.18 +/- 0.06 to 0.009 +/- 0.004 microg/mg during the 1-year study. There were no signs of ocular toxicity at 1 year. CONCLUSIONS: Episcleral implants are safe and effective at delivering therapeutic CsA levels to the cornea to potentially prevent corneal allograft rejection. The implant can be surgically inserted at the time of penetrating keratoplasties, since the implant achieves therapeutic levels as early as 3 hours.

Transport barriers in transscleral drug delivery for retinal diseases.

Transscleral delivery has emerged as an attractive method for treating retinal disorders because it offers localized delivery of drugs as a less invasive method compared to intravitreal administration. Numerous novel transscleral drug delivery systems ranging from microparticles to implants have been reported. However, transscleral delivery is currently not as clinically effective as intravitreal delivery in the treatment of retinal diseases. Transscleral drug delivery systems require drugs to permeate through several layers of ocular tissue (sclera, Bruch's membrane-choroid, retinal pigment epithelium) to reach the neuroretina. As a result, a steep drug concentration gradient from the sclera to the retina is established, and very low concentrations of drug are detected in the retina. This steep gradient is created by the barriers to transport that hinder drug molecules from successfully reaching the retina. A review of the literature reveals 3 types of barriers hindering transscleral drug delivery: static, dynamic and metabolic. While static barriers have been examined in detail, the literature on dynamic and metabolic barriers is lacking. These barriers must be investigated further to gain a more complete understanding of the transport barriers involved in transscleral drug delivery.

Chemically surface modified gel (CSMG): an excellent enzyme-immobilization matrix for industrial processes.

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.

Synthesis of "click" alginate hydrogel capsules and comparison of their stability, water swelling, and diffusion properties with that of Ca(+2) crosslinked alginate capsules.

Ionically crosslinked alginate hydrogels have been extensively explored for encapsulation and immunoisolation of living cells/tissues to develop implantable cell therapies, such as islet encapsulation for bioartificial pancreas. Chemical instability of these hydrogels during long-term implantation hinders the development of viable cell therapy. The exchange between divalent crosslinking ions (e.g., Ca(+2) ) with monovalent ions from physiological environment causes alginate hydrogels to degrade, resulting in exposure of the donor tissue to the host's immune system and graft failure. The goal of this study was to improve stability of alginate hydrogels by utilizing covalent "click" crosslinking while preserving other biomedically viable hydrogel properties. Alginate was first functionalized to contain either pendant alkyne or azide functionalities, and subsequently reacted via "click" chemistry to form "click" gel capsules. Alginate functionalization was confirmed by NMR and gel permeation chromatography. When compared with Ca(+2) capsules, "click" capsules exhibited superior stability in ionic media, while showing higher permeability to small size diffusants and similar molecular weight cut-off and water swelling. Physicochemical properties of "click" alginate hydrogels demonstrate their potential utility for therapeutic cell encapsulation and other biomedical applications.

Immobilized thermolysin for highly efficient production of low-molecular-weight protamine--an attractive cell-penetrating peptide for macromolecular drug delivery applications.

Macromolecules present a remarkable potential as future therapeutics. However, their translation into clinical practice has been hampered by an inherently low bioavailability. Cell-penetrating peptides (CPP) have been recently shown to significantly improve on the bioavailability of macromolecules. Yet, the high cost associated with development and production of these peptides is a major factor hindering their rapid deployment beyond the laboratory. Here, we describe a facile and robust methodology for efficient and large-scale production of low-molecular-weight protamine-a potent CPP of great clinical potential. Our methodology is based on the immobilization of thermolysin, an enzyme catalyzing digestion of native protamine, on chemically surface-modified gels produced by silica sol-gel chemistry. Thermolysin was immobilized at extremely high matrix loading of 733 mg/g matrix and exhibited good thermal and pH stability, indicating robustness with respect to processing conditions. The mechanical properties of the silica matrix further allowed utilization of the immobilized thermolysin in both batch and packed-bed reactor systems to produce the LMWP peptide in high yields. Results presented here are of high significance as this efficient and cost-effective production of high purity LMWP could enable clinical translation of many potential macromolecular drugs.

Enzyme stabilization and immobilization by sol-gel entrapment.

While biocatalysts show tremendous potential for the industrial production of fine chemicals, their integration into large-scale processes has been slow. One of the main reasons for slow acceptance in industry is the inherent instability of the enzymes. Recent developments in bioengineering have shed some light on methods of improving enzyme stability. One method that has been used for many decades, successfully to varying degrees, has been the immobilization of enzymes. To this regards, silica gels have attracted much attention because of the ease of surface functionalization, high surface areas, mechanical and thermal stability, and resistance to both chemical and biological attack. We have previously shown the immobilization of invertase on silica gels with high immobilized activity and significantly improved stability. Here, we provide greater details on the methods for effecting the immobilization.