Hydrazide-activated immobilized

Covalent immobilization of lipase on nylon fibers has been done, using the enzymes carbohydrate groups as chemical link. Oxidation of the lipases carbohydrates with periodate provides aldehyde groups for the binding to hydrazide activated nylon (Lopez, Braun Klein, 1996). 

While the batch process is the dominant one in current use, researchers and companies have attempted to create continuous bioreactor systems. Lopez et al. immobilized Candida rugosa in polymethacrylamide hydrazide beads and polyurethane foam 3 with the intent to achieve the continuous production of lipase enzymes. Despite flow problems with the polyurethane foam, it showed high lipolytic activity. Biomass buildup was problematic. Feijoo et al. immobilized Phanerochaete chry-sosporium on polyurethane foam in packed bed bioreactors under near-plug flow conditions. Continuous lignin peroxidase production was accomplished, the rate of which was studied as a function of recycle ratio. 

Arenkov et al. prepared poly(acrylamide) gel pads for use in protein microarrays [199], The gels were prepared by photopolymerization of acrylamide and crosslinkers. Capture probes were immobilized, either by use of glutaraldehyde or by converting some of the acrylamide groups into hydrazides and subsequent coupling of aldehyde-modified antibodies to the pending hydrazide groups. Then, immunoassays were performed on the pads, either assays with directly labeled analytes or sandwich assays. Furthermore, the gel pads were used for enzyme activity studies. 

Periodate Oxidation mainly of polysaccharide supports has become a popular activation technique for protein immobilization [149]. Sodium periodate (Nal04) can react with vicinal cw-hydroxyl groups on cellulose, dextran, or any other diols to produce aldehyde groups. These aldehyde groups can easily be transformed into secondary amines by reductive oxidation or to hydrazides by reaction with dihydrazine. Further attachment of ligands or spacer molecules can be performed via primary amino groups. 

For the reversible immobilization of amines to such linkers, the most frequently used strategy is conversion of the hydroxymethyl linkers into activated carbonic esters (Figure 14.7) [8, 39, 40]. These react with primary and secondary amines or hydrazines to form immobilized carbamates or carbazates analogous to the classical benzyloxycarbonyl protecting group. Amines and hydrazides attached by this strategy to Wang resin can be cleaved with TFA [8, 40]. 

Many different chemistries can be used to immobilize antibodies onto chromatographic media and only a few will be discussed. In most cases, the chromatographic media is coated with the active chemistry, which will then react with the antibody. These include amine reactive chemistries such as epoxide-, aldehyde-, and cyanogen bromide (CNBr)-activated media, carboxyl reactive chemistries such as carbodiimides, aldehyde-reactive chemistries such as amino and hydrazide, and thiol-reactive chemistries such as iodoacetyl and reduce thiol media. Although there are several antibody isotypes (IgA, IgE, IgG, IgM), the most... 

Enzymes have also been immobilized on collagen membrane after its stepwise modification to esters, hydrazides and azides [171]. Another method of enzyme electrode preparation consists of enzyme immobilization on polyacryl acid modified with p-nitroaniline and by a subsequent reduction of N02-groups with titanous chloride and following diazota-tion of resulting aromatic amines [150]. An enzyme electrode has also been prepared by the direct immobilization of an enzyme on the surface of a Pt-electrode which was formerly modified first with 3-aminopropyl triethoxysilane and secondly with glutaraldehyde and bovine serum albumin [172]. Enzymes can also be immobilized on p-benzoquinone-carbon paste[173] or on the graphite electrode after its activation with cyanuric chloride [174]. In a similar way an enzyme electrode has been prepared by using iridium diiodide electrode as a support [175]. 

Neuraminidase has been immobilized to give a water-insoluble derivative with retention of enzymic activity by covalent reaction with agarose cyclic imidocarbonate, a hydrazide derivative of agarose cyclic imidocarbo-nate, and periodate-oxidized controlled-pore glass (CPG)-glycophase. ... 

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