Immobilization of α-glucosidase on polystyrene plates: A practical application to α-amylase detection.

Chromogenic and fluorigenic substrates for sulfurtransferases

Stable and efficient immobilization technique of aldolase under consecutive microwave irradiation at low temperature

Porous materials are widely employed as supports in the immobilisation of enzymes. Traditionally macroporous materials with pore diameters >50 nm were believed to be the most suitable support material, ensuring no spatial restrictions upon enzyme molecules entering such large pores. In recent years however, there has been growing emphasis in the use of mesoporous supports with pore diameters ranging between 2 and 50 nm. It is thought this smaller pore range may offer enhanced conformational stability to immobilised enzymes while not being so small as to restrict enzyme access. Despite their increasing popularity, many argue that mesoporous materials have not yet proven superior to traditional macroporous supports for enzyme immobilisation. Through the design and application of a unique confidence rating system we were able to accurately compare data and establish trends between pore characteristics and protein loading. By analysing published data (182 experiments in total) and extracting pore characteristics and protein loading values, we have described three categories of pore diameters in which correlations between pore characteristics and protein loading are noted. With pore diameters less than 10 nm we see a general decrease in protein loading as the enzymes find physical restrictions in accessing the high surface offered in this pore diameter range. At pore sizes greater than 100 nm, protein loading generally decreases due to a concomitant reduction in available surface area. In the pore range of 10-100 nm there it is expected to see a decrease in protein loading level with increasing pore diameter. In fact protein loading in this range remains largely constant, suggesting some degree of protein-protein interaction blocking pores and restricting access to the increasing surface area available at decreasing pore diameters. No trends were established between pore characteristics and retention of activity.

This paper presents a strategy for preparing an efficient immobilised alcohol dehydrogenase preparation for a gas-phase reaction. The effects of additives such as buffers and sucrose on the immobilisation efficiency (residual activity and protein loading) and on the gas-phase reaction efficiency (initial reaction rate and half-life) of Thermoanaerobacter sp. alcohol dehydrogenase were studied. The reduction of acetophenone to 1-phenylethanol under in situ cofactor regeneration using isopropanol as co-substrate was used as a model reaction at fixed reaction conditions (temperature and thermodynamic activities). A strongly enhanced thermostability of the enzyme in the gas-phase reaction was achieved when the enzyme was immobilised with 50 mM phosphate buffer (pH 7) containing sucrose five times the protein amount (on weight/weight basis). This resulted in a remarkable productivity of 200 g L(-1) day(-1) even at non-optimised reaction conditions. The interaction of additives with the enzyme and water affects the immobilisation and gas-phase efficiencies of the enzyme. However, it was not possible to predict the effect of additives on the gas-phase reaction efficiency even after knowing their effect on the immobilisation efficiency.

Immobilization of L-methionine γ-lyase on different cellulosic materials and its potential application in green-selective synthesis of volatile sulfur compounds

A rapid, convenient and accurate method, based upon an enzyme linked immunosorbent assay (ELISA), is described for the determination of paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit anti-mouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of paraquat.

Mesocellular foam silica (MCF) was synthesized using Pluronic P-123 triblock copolymer as a template. Calcination and solvent extraction were employed to remove templates and compared. Through physical, chemical and functional characterization interesting results have been observed. Microstructures of the two MCFs were very different. Pore structure of the calcined MCF was found to be more ordered than that of the solvent extracted MCF. However, pores of the solvent extracted MCF seemed more defined and spongy under SEM observations. Surface functional groups and their concentrations characterized by ToF-SIMS were also found to be different. Surface silanol groups were found to be more concentrated in the calcined MCF. Each type of MCFs was subsequently grafted by an amine functional group, adsorbed by Au ion precursors, and reduced to form Au nanoparticles (AuNPs) on the MCF surfaces. These decorated MCF surfaces were immobilized with enzyme acetylcholinesterase (AChE), modified on screen-printed carbon electrodes and tested for electrochemical responses with acetylthiocholine. AuNPs were successfully distributed within the pores for both types of MCFs. Before the functional biosensor electrochemical tests, calcination seemed more promising than solvent extraction due to periodic structure of pores, high surface area, less contamination and higher concentration of surface silanol groups available for grafting of the amine functional group. However, after immobilization of the AuNPs for electron transport improvement and enzyme immobilization, a few observations of biosensing performances were noteworthy. Firstly, having AuNPs distributed throughout the MCF structure did not help the electron transport. Results of reduced response currents from both types of MCFs compared to MCFs without AuNPs indicated that MCF walls were too thick to allow electron transport. Secondly, the higher concentration of surface silanol groups in calcined MCFs resulted in Ostwald’s ripening of small AuNPs giving bigger AuNPs than the case of solvent extraction. Therefore, although the AuNPs in enzyme biosensors are expected to help electron transport and enzyme immobilization, this study showed that although the former did not occur, the latter was well demonstrated. The smaller AuNPs in the solvent extraction case resulted in higher surface area than the bigger AuNPs in the calcination case. This was the reason why the response current was higher for the solvent extracted MCFs compared to the calcined MCFs.

Enzyme immobilization has been demonstrated to be a favorable protocol for promoting the industrialization of bioactive molecules, but still with formidable challenge. Addressing this challenge, we create a dynamic defect generation strategy for enzyme immobilization by using the dissociation equilibrium of metal-organic frameworks (MOFs) mediated by enzymes. Enzymes can act as "macro ligands" to generate competitive coordination against original ligands, along with the release of metal clusters of MOFs to generate defects, hence promoting the gradual transport of enzymes from the surface to inside. Various enzymes can be efficiently immobilized in MOFs to afford composites with good enzymatic activities, protective performances and exceptional reusabilities. Moreover, multienzyme bioreactors capable of efficient cascade reactions can also be generated. This study provides new opportunities to construct highly efficient biocatalysts incorporating different types of enzymes.

The enzyme stability is a key parameter for its analytical applications. In this backdrop, the current chapter gives an insight about enzyme immobilization using various strategies highlighting its pros and cons. The chapter provides an overview about industrial application of immobilized α-amylase on various matrices with major focus on emerging technologies. The nanotechnology coupled with biotechnological advances provides new avenue in the area of enzyme immobilization. The chapter reveals the excellent properties of nanomaterials as efficient matrices for enzyme immobilization with enhanced catalytic activity, stability and reusability leading the process economically viable and environmentally feasible. The immobilized α-amylase on nanomatrices possess excellent properties contrary to their soluble forms are more preferred for industrial processes. Hence, the present chapter gives an idea about the ongoing research on industrially important α-amylase highlighting new approaches of nanotechnology for enzyme immobilization, which suggests its efficient role for various analytical applications.

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Immobilization of chymotrypsin on hierarchical nylon 6,6 nanofiber improves enzyme performance

Hen egg white as a feeder protein for lipase immobilization

In recent years, researchers have focused on developing simple and efficient methods based on electrochemical biosensors to determine hydroxycinnamic acids from various real samples (wine, beer, propolis, tea, and coffee). Enzymatic biosensors represent a promising, low-cost technology for the direct monitoring of these biologically important compounds, which implies a fast response and simple sample processing procedures. The present review aims at highlighting the structural features of this class of compounds and the importance of hydroxycinnamic acids for the human body, as well as presenting a series of enzymatic biosensors commonly used to quantify these phenolic compounds. Enzyme immobilization techniques on support electrodes are very important for their stability and for obtaining adequate results. The following sections of this review will briefly describe some of the laccase (Lac) and tyrosinase (Tyr) biosensors used for determining the main hydroxycinnamic acids of interest in the food or cosmetics industry. Considering relevant studies in the field, the fact has been noticed that there is a greater number of studies on laccase-based biosensors as compared to those based on tyrosinase for the detection of hydroxycinnamic acids. Significant progress has been made in relation to using the synergy of nanomaterials and nanocomposites for more stable and efficient enzyme immobilization. These nanomaterials are mainly carbon- and/or polymer-based nanostructures and metallic nanoparticles which provide a suitable environment for maintaining the biocatalytic activity of the enzyme and for increasing the rate of electron transport.

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead-Glu) or carboxyl groups through acid solution (Immobead-Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β-galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead-Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10-500 mg g-1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g-1 support. Gal immobilized on Immobead-Glu and Immobead-Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half-lifes than the soluble enzyme, where the half-lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934-943, 2018.

Review: 229 refs.