Glutaraldehyde enzyme immobilization

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Scheme 1 Covalent enzyme immobilization Aminopropyl-modified silica reacts with glutaraldehyde and the N-terminus of the enzyme.

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

Like many other useful discoveries, enzyme immobilization by cross-linking was actually an unintended by-product of another research project. In 1964, Florante Quiocho and Frederic Richards at Yale university cross-linked crystals of carboxy-peptidase-A with glutaraldehyde (pentane-1,5-dial), hoping to get stable crystals for X-ray diffraction studies. They noted that these cross-linked enzyme crystals (now... 

AOD was successfully immobilized on aminopropyl-functionalized glass beads by covalent bonding through glutaraldehyde with an average retention efficiency of 95.14% (see Table 2). The method used for enzyme immobilization showed good reproducibility with a relative SD of 2.85%. 

Acetylcholineesterase and choline oxidase Enzyme immobilized over tetra-thiafulvalene tetracyanoquinodi-methane crystals packed into a cavity at the tip of a carbon-fiber electrode. The immobilization matrix consisted of dialdehyde starch/glutaraldehyde, and the sensor was covered with an outer Nafion membrane. The ampero-metric performance of the sensor was studied with the use of FIA system. An applied potential of +100 mV versus SCE (Pt-wire auxiliary electrode) and a carrier flow rate of 1 mL/min. The Ch and ACh biosensors exhibited linear response upto 100 pM and 50 pM, respectively. Response times were 8.2 s. [97]... 

The technique is simple and fast and allows control of the physical properties and particle size of the final product. Nevertheless, care must be taken in the selection and storage of glutaraldehyde to ensure reproducible enzyme immobilizations (123). Buffers containing reactive amino groups must be avoided. Severed other bifunctional reagents are more or less successful. The most common are hexamine diisocyanate, trichloro-t-triazine, and diphenyl-4,4 -dithio-cyanate-2,2 disulfonic acid (8, 124, 125). 

The non-aqueous lipase system for flavor esters developed by our group used components and preparative techniques for enzyme immobilization, that would not only be cost effective and simple but also meet regulatory requirements. The enzyme could have been immobilized by a number of methods however for the Intended application only (i) adsorption (11) ionic bonding or (lii) glutaraldehyde cross-linking would be... 

Forccd flow mode. Invertase, an enzyme, can be chemically immobilized to the surfaces of ceramic membrane pores by the technique of covalent bonding of silane-glutaraldehyde [Nakajima et al., 1989b]. The substrate (reactant), during the sucrose conversion process, enters the membrane reactor in a crossflow mode. Under suction from the other side of the membrane, the substrate flows into the enzyme-immobilized membrane pores where the bioconversion takes place. Both the product and the unreacted substrate indiscriminately pass through the membrane pores. Thus, no permselective properties are utilized in this case. The primary purpose of the membrane is to provide a well-engineered catalytic path for the reactant, sucrose. 

Zeolite membranes are amenable by surface modification with a variety of chemical functional groups using simple silane chemistry, which may provide alternative surface chemistry pathways for enzyme immobilization. In this context, Shukla et al. [238] have recently used a chemically modified zeolite-clay composite membrane for the immobilization of porcine lipase using glutaraldehyde to provide a chemical linkage between the enzyme and the membrane. The effects of pH, temperature, and solvent on the performance of such biphasic zeohte-membrane reactors have been evaluated in the hydrolysis of olive oil to fatty acids. 

Figure 9. Schematic fabrication of LbL films comprising poly(vinylsulfonic acid) (PVS)and PAMAM-Au. The sequential deposition of LbL multilayers was carried out by immersing the substrate alternately into (a) PVS and (b) PAMAM-Au solutions for 5 min per step (c) After deposition of 3 bilayers, an ITO-PVS/PAMAM-Au)3 CoHCF electrode was prepared by potential cycling (d) The enzyme immobilization to produce ITO-PVS/PAMAM-Au)3 CoHCF-GOx was carried out in a solution containing BSA, glutaraldehyde and GOx (Adapted from Ref.[124])...

Du et al. reported a sensitive, fast and stable amperometric sensor for quantitative determination of OP insecticide, triazophos [14], Where, AChE was immobilized on MWNTs-chitosan (MC) composite matrix. Prior to enzyme immobilization, GCE surface was activated by applying a potential of +1.75 V for 300 s and scanned in the potential range +0.3 to +1.25V and +0.3 to -1.3V until a steady-state curve was obtained. This pretreated GCE surface was coated with 2.0 pi of MWCNTs, chitosan and glutaraldehyde mixture, followed by coating 4 pi of AChE solution, dried and used. CV results show that the oxidation peak of thiocholine occurs at +0.66V with much higher peak height at AChE/MC/GCE than at AChE/CS/GCE without MWCNTs. This shows that MWCNTs presence lowers the oxidation potential of thiocholine at the MC composite electrode. CV studies were also carried out to study the inhibition activity of triazophos at the composite electrode. The results show that, the peak currents decreased at the composite electrode with increase in triazophos concentration (Fig. 4). 

Fig. 5. Correlation between heat response and reaction rate of cephalosporin C transformation by immobilized D-amino acid oxidase of Trigonopsis variabilis. Enzyme immobilization techniques entrapment in polyacrylamide gel ( ), cells cross-linked with glutaraldehyde ( ), cells entrapped in polyacrylamide gel (a) [28]...

A number of methods are being tested for enzyme immobilization. The method selected depends on the operating details of the enzyme system employed and the nature of the solvent to be used, which is usually water. Enzyme, or inactivated cells, may be encapsulated in a film, or encased in a gel, which is permeable to both the substrate and product, but not to enzyme [77]. Porous glasses or insoluble polymers such as a derivatized cellulose may be used as a support onto which enzyme is adsorbed. Pendant functional groups of a polymer, such as those of the ion-exchange resins, can be used either to ionically bind the enzyme to the resin active sites or to covalently bond the enzyme to the resin [79]. The enzyme may be bonded to a polymer backbone chain using a bifunctional monomer such as glutaraldehyde to react with enzyme sites that do not affect its catalytic activity [80]. 

Mullen et al. (1986) decreased the permeability of LOD membranes in order to extend the linear range to higher lactate concentration. A polycarbonate membrane was treated with methyltrichlorosilane prior to enzyme immobilization by crosslinking with glutaraldehyde and BSA. In this way the upper limit of linearity was shifted from 0.2 to 18 mmolA. The permeation of electrochemical interferents was diminished concomitantly. On the other hand, the silanization increased the response time from 0.5-1 min to 1-3 min and reduced the sensitivity by 98-99%. 

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