Polyvinyl alcohol-glutaraldehyde network as a support for protein immobilisation

Polyvinyl alcohol-glutaraldehyde (PVA-glut) network was synthesised in bead and disc forms and used for protein immobilisation. Xanthine oxidase, a-amylase and amyloglucosidase were covalently fixed on the beads yielding preparations with specific activities retention of 72.3%, 1.6% and 1.4%, respectively. Km of xanthine oxidase PVA-glut beads (24 ± 4 µM) was slightly higher than that estimated for the soluble enzyme (16 ± 2 mM). Antigens from Schistosoma mansoni were covalently fixed onto PVA-glut discs and ELISA was carried out. The relationship between the amount of fixed antigen and the results obtained by ELISA showed a hyperbolic curve and the saturation of antigen-antibody complex was achieved at the plateau.This revised version was published online in October 2005 with corrections to the Cover Date.     

Protein-alginate gel for enzyme immobilisation

Summary Propylene glycol alginate forms strong, covalently bonded gel when mixed with certain proteins in alkaline conditions. Enzymes can be immobilised onto the alginate ester before gelating with protein to form the above gel. Some data on the immobilisation of -glucosidase are presented.     

Cellulosic exopolysaccharide produced by Zoogloea sp. as a film support for trypsin immobilisation

Trypsin (E.C. 3.4.21.4) was covalently immobilised onto a membrane of a cellulosic exopolysaccharide produced by Zoogloea sp. in sugarcane molasses. Carbonyl groups were introduced into the matrix by sodium metaperiodate oxidation and the enzyme was immobilised either directly or through bovine serum albumin (BSA) as a spacer. The trypsin-membrane and trypsin–BSA-membrane retained, respectively, 37.2% and 9.16% of the specific activity of the native enzyme acting on N-benzoil-dl-arginine-p-nitroanilide (BAPNA). No activity decrease was observed in both preparations after seven reutilisations as well as they showed to be more thermal stable than the native enzyme. The trypsin–BSA-membrane presented the same initial activity (99%) after 54 days stored in 0.1 M Tris–HCl buffer, pH 8.0, at 4 °C but the trypsin-membrane lost 15% of activity. Furthermore, the trypsin–BSA-membrane lost 31% of activity after reuse at 9 days interval during 54 days of storage at 4 °C whereas the trypsin-membrane lost 69% of activity under the same conditions. These results showed an additional application for this biofilm, namely, to act as a reusable matrix for trypsin immobilisation and the presence of BSA improved the derivative performance.     

Advances in enzyme immobilisation

Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.     

Spent grains – a new support for brewing yeast immobilisation

A novel carrier obtained from spent grains, a brewing by-product, was used for brewing yeast immobilisation in a continuous bubble-column reactor. The multiple-layer cell adhesion to the carrier particles resulted in a maximum cell load of 430 mg dry cell g^–1 dry carrier (d.c.). After 120 h of reactor operation, the cell load of DEAE-modified carrier was below 40 mg dry cell g^–1 d.c. while the values for non-modified carrier reached at least 100 mg dry cell g^–1 d.c. The changes in substrate composition on the rate of yeast attachment and on its stability were also studied.     

Curdlan as a support matrix for immobilization of enzyme

Curdlan, a high molecular weight extracellular β(1→3) glucan produced by pure culture fermentation by Agrobacterium radiobacter NCIM 2443 contains large number of free hydroxyl groups. The reaction of hydroxyl containing supports with epichlorohydrin results in activated epoxy groups that can covalently link with available amino, hydroxyl, or sulfhydryl groups of enzymes, thereby immobilizing it. The present work reports on preparation of epoxy-activated matrix for immobilization of a model enzyme, porcine pancreatic lipase. The binding capacity of the matrix prepared by extraction of epoxy-activated curdlan by isopropyl alcohol was found to be 58.7% with about 0.6% loss of the enzyme activity during immobilization. Further, the specific activity of the enzyme increased marginally from 9.37 to 10.2. The corresponding value was 10.15 for a commercial sample of curdlan, epoxy-activated as for laboratory-isolated curdlan. Sepharose, the most widely used support matrix for the immobilization of enzymes was used for comparison in this study.     

Natural silk fibroin as a support for enzyme immobilization

Silk fibroin derived from Bombyx mori cocoon is being developed and utilized for purposes besides traditional textile material. Fibroin can be easily made up into various forms, several of which can serve as enzyme-immobilized supports. There are numerous reports on immobilized enzymes using these forms of silk fibroin as supports in which the enzyme-immobilized fibroin membranes were characterized in detail by means of spectrophotometry, infrared spectra, NMR, ESR. Enzyme-immobilized fibroin membranes have been successfully used in several biosensors for the determinations of glucose, hydrogen peroxide and uric acid in which glucose and urate biosensors in a flow injection system were able rapidly to analyze various biosamples including human whole blood or serum.     

Application of advanced materials as support for immobilisation of lipase from Candida rugosa

Ni/Al-layered double hydroxides (Ni-LDHs) and Ni/Al-sodium dodecyl sulfonate layered double hydroxide nanocomposites (Ni-SDS-LDHs) with a molar ratio of Ni:Al (4:1) have been prepared by a co-precipitation (or salt-base) method. Their structures were determined using Powder X-Ray Diffractometer (PXRD) and the spectra showed that basal spacings for Ni-LDHs and Ni-SDS-LDHs synthesised were around 8.1?Å and 34.8?Å, respectively. Lipase from Candida rugosa was immobilised onto these advanced materials, by physical adsorption. The activity of immobilised lipase was investigated through esterification of palmitic acid and isopropyl alcohol in hexane. The effects of reaction temperature, thermostability, stability in organic solvent, operational stability, leaching and storage studies of the immobilised lipase were investigated. These biocatalysts exhibited higher activities than the native lipase with an optimum temperature of 40°C. Immobilised lipases showed higher storage stability than native lipase (up to 60 days) and during operational studies at 30°C for 5?h, more than 50% of its activity was retained. Leaching studies showed that physical adsorption is suitable for the attachment of enzymes onto LDHs.     

Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production

Nanobiotechnology is emerging as a new frontier of biotechnology. The potential applications of nanobiotechnology in bioenergy and biosensors have encouraged researchers in recent years to investigate new novel nanoscaffolds to build robust nanobiocatalytic systems. Enzymes, mainly hydrolytic class of enzyme, have been extensively immobilised on nanoscaffold support for long-term stabilisation by enhancing thermal, operational and storage catalytic potential. In the present report, novel nanoscaffold variants employed in the recent past for enzyme immobilisation, namely nanoparticles, nanofibres, nanotubes, nanopores, nanosheets and nanocomposites, are discussed in the context of lipase-mediated nanobiocatalysis. These nanocarriers have an inherently large surface area that leads to high enzyme loading and consequently high volumetric enzyme activity. Due to their high tensile strengths, nanoscale materials are often robust and resistant to breakage through mechanical shear in the running reactor making them suitable for multiple reuses. The optimisation of various nanosupports process parameters, such as the enzyme type and selection of suitable immobilisation method may help lead to the development of an efficient enzyme reactor. This might in turn offer a potential platform for exploring other enzymes for the development of stable nanobiocatalytic systems, which could help to address global environmental issues by facilitating the production of green energy. The successful validation of the feasibility of nanobiocatalysis for biodiesel production represents the beginning of a new field of research. The economic hurdles inherent in viably scaling nanobiocatalysts from a lab-scale to industrial biodiesel production are also discussed.     

Different phyllosilicates as supports for lipase immobilisation

The aim of this work was to determine the enzymatic activities resulting from the adsorption of Rhizomucor miehei lipase (RML) and Candida cylindracea lipase (CCL) onto three different phyllosilicates (sepiolite, palygorskite and montmorillonite), comparing the resultant activities with those obtained following similar immobilisation technique on a widely used resin (Duolite A-568). Due to the different adsorption mechanisms produced, different derivatives with higher hydrolytic activities can be obtained. Comparing the clays tested, the results showed that, in comparison with the laminar silicate (montmorillonite sample) and Duolite A-568 (spherical particles), fibrous materials (palygorskite and sepiolite) resulted in derivatives with higher hydrolytic activities in the hydrolysis of different ethyl esters. Moreover, according to the data obtained with the electrophoresis, the selectivity of immobilisation for RML in the case of fibrous silicates was optimal. As a conclusion, and according to the activities and selectivities measured, at least two out of the four studied materials (sepiolite and palygorskite) would be useful as supports for immobilisation for proteins of relatively low molecular weight (such as RML) for further use in biotransformations, while for C. cylindracea the immobilisation onto duolite rendered a derivative specially active in the hydrolysis of ethyl formiate (esterasic activity).     

Activation of nylon by alkaline glutaraldehyde solution for enzyme immobilisation

Summary Nylon tube was directly activated by alkaline glutaraldehyde solution. PEI was utilised as a spacer molecule. Glucose oxidase was immobilised to the nylon tube after reactivating the spacer molecules with glutaraldehyde. On immobilising glucose oxidase there was more protein binding and higher immobilised enzyme activity when compared to immobilised enzyme tube activated by triethyloxonium salt. The optimal condition for direct glutaraldehyde activation of nylon was incubation with 18.5% (w/v) glutaraldehyde in 0.12M borate pH 9.0 for 15 min at 90 °.     

Reducing enzyme conformational flexibility by multi-point covalent immobilisation

Summary A thermostable esterase was immobilised to glyoxyl-agarose under conditions designed to generate limited-linkage and multi-point covalent derivatives. The multi-point derivative was 830-fold more thermostable than the limited-linkage derivative and retained more activity in the presence of sodium chloride and organic solvents. Medium chain (C8) aliphatic p-nitrophenyl ester substrates, which inactivate the soluble enzyme, were shown to be more readily hydrolysed. Together these data support the contention that multi-point covalent immobilisation results in a more rigid, less conformationally flexible protein structure.     

Partially deacetylated chitin as an acid-stable support for enzyme immobilization

Partially deacetylated chitin (PDAC) obtained by boiling chitin in 28.6% (w/w) sodium hydroxide was not dissolved when it was suspended in 2% acetic acid (pH 2.6) at 60°C for 12 h or autoclaved in acetate buffer (pH 5.0) for 20 min. The enzyme binding ability of the PDAC with glutaraldehyde was similar to that of chitosan. Immobilized pullulanase had low enzyme activity for high-molecular-weight material such as pullulan, but its activity for maltosyl β-cyclodextrin was almost the same as that of the free enzyme. The immobilized enzyme produced branched cyclodextrin through a reverse reaction in acetate buffer of pH 3.75 at 53°C.     

An evaluation of a ceramic monolith as an enzyme support material

Catalase was immobilized on commercially available monolithic catalyst supports and also on participate support obtained by crushing the monolith. The kinetics of the monolith- and particulate-supported enzymes were analyzed in a continuous tubular reactor system and pressure drop was also monitored. Analysis of the results indicates that the monolith-supported system presents very little resistance to flow which results in a considerably smaller pressure drop than is obtained in flow through particulate-supported systems under comparable conversion conditions. Ceramic monoliths thus appear to be very suitable for use as enzyme supports in continuous tubular reactor applications, particularly where high pressure drops might be expected.     

Hydrophobised sawdust as a carrier for immobilisation of the hydrocarbon-oxidizing bacterium Rhodococcus ruber

Pine sawdust treated by a series of hydrophobising agents (drying oil, organosilicon emulsion, n-hexadecane and paraffin) was examined as carrier for adsorption immobilisation of hydrocarbon-oxidizing bacterial cells Rhodococcus ruber. It was shown that hydrophobising agents based on drying oil turned out to be optimal (among the other modifiers examined) for the preparation of sawdust carriers suitable for the efficient immobilisation. The results obtained demonstrate promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity.     

An experiment illustrating the effect of immobilisation on enzyme properties

The experiment described in this paper introduce to students to immobilisation of β-glucosidase, induced in Aspergillus niger cultures, by entrapment method in calcium alginate gel. Additionally, the determination of apparent kinetic parameters, the pH/enzyme activity curves and the study of the thermal stability permits students to check and understand the effect of the immobilisation on the properties of the enzyme.     

Dual polarisation interferometry characterisation of DNA immobilisation and hybridisation detection on a silanised support

Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterise the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19 mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was approximately 35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was approximately 85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.     

Direct immobilisation of antibodies on a bioinspired architecture as a sensing platform

A sensitive and selective immunosensor for the nonlabeled detection of sulfate-reducing bacteria (SRB) is constructed using a self-polymerised polydopamine film as the immobilisation platform. Self-polymerisation of dopamine is used as a powerful approach for applying multifunctional coatings onto the surface of a gold electrode. The polydopamine film is used not only as the immobilisation platform, but also as a cross-linker reagent for the immobilisation of the anti-SRB antibody. The polydopamine film is loaded with a high density of anti-SRB antibodies linked to the substrate to obtain high response signals. The formation and fabrication of the biosensor and the quantification of antibody anchoring are monitored, and SRB detection is performed by either quartz crystal microbalance (QCM) or electrochemical impedance spectroscopy (EIS). After modeling the impedance Nyquist plots of the SRB/anti-SRB/polydopamine/gold electrode for increasing concentrations of SRB, the electron transfer resistance (R(ct)) is used as a measure of immunocomplex binding. The R(ct) is correlated with the concentration of bacterial cells in the range of 1.8×10(2) to 1.8×10(6) CFU mL(-1); the detection limit is 50 CFU mL(-1). This work demonstrates a new immobilisation platform for the development of a sensitive and label-less impedimetric and piezoelectric immunosensor. This immunosensor may be broadly applied in clinical diagnoses and the monitoring of water environmental pollution. The method proposed is distinct in its ease of application, use of a simple protocol, and mild reaction conditions. These allow it to be applied to a wide variety of materials.