Covalent linkage of proteins to surface-modified poly(organophosphazenes): immobilization of glucose-6-phosphate dehydrogenase and trypsin

Abstract

Glucose-bphosphate dehydrogenase (G-6-PDH) and trypsin have been linked covalently to a surface-modified poly[bis(aryloxy)phosphazene] supported on porous alumina particles. Poly(diphenoxyphosphazene) was surface-nitrated and then reduced to the aminophenoxy derivative. The aminophenoxy sites were then activated by reaction with cyanogen bromide, nitrous acid, or glutaric dialdehyde, followed by treatment with the enzymes in aqueous buffer solutions. The activities of the immobilized enzymes were monitored spectrophotometrically with the use of glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (for G-6-PDH) and N-a-benzoyl-L-arginine-p-nitmudide (for trypsin). Thew data were supplemented by an assessment of the total yield of immobilized enzyme by means of a Lowry protein measurement. The maximum immobilization yields (&lo% for G-6-PDH and 50-60% for trypsin) were achieved when the glutaric dialdehyde activated system was used. For both proteins, the immobilized enzyme retained its activity and remained linked to the support through numerous cycles. By contrast, the same enzymes adsorbed on uncoated alumina particles were displaced readily by washing. The storage stabilities of both immobilized enzymes were much greater than those of the free enzymes in solution. A continuous-flow assaying system showed that immobilized trypsin generated a constant activity at three different flow rates. For the G-6-PDH system, enzyme activity and scanning electron microscopy were used to examine the relationship between the substrate topography/surface area and the concentration of immobilized protein molecules.

Immobilized redox enzymatic catalysts: Baeyer–Villiger monooxygenases supported on polyphosphazenes

“…in the case of 1 to 2.5 U. Therefore, for subsequent experiments a PAMO/G6PDH proportion 1 to 10 U was employed to prove that G6PDH was able to work when supported on a polyphosphazene carrier, as previously shown for other similar supports [15].…”

“…Due to their high chemical resistance, these derivatives posses a great potential for biotechnological applications and, in fact, novel materials derived from this type of polymer have recently been used in the field of medicine [13,14]. Functionalized polyphosphazenes have proven to be an efficient material to covalently attach different types of enzymes like trypsin or 3 glucose-6-phosphate dehydrogenase on a support of [NP(OPh) 2 ] n on an alumina carrier [15], or an invertase on spherical particles of [NP(OCH 2 CF 3 ) 2 ] n [16]. In another contribution, an urease was encapsulated on a hydrogel derived from poly[bis(methoxyethoxy-ethoxy)phosphazene] through irradiation with γ-rays to cross-link the polymer and trap the biocatalyst [17].…”

Surface‐modified polysulfone hollow fibers. IV. Chloromethylated fibers and their derivatives

“…This research involves investigation of chloromethylated polysulfone membranes by surface reaction and their derivatives. The enzyme and affinity ligands will be easily immobilized from glutaraldehyde, [12][13][14] succinimide, l5,I6 or ~arbodiimide'~ reactions on the surfaces of the membranes. Ultrafiltration experiments for the modified membranes are also shown and discussed in this study.…”

Polyphosphazenes as Tunable and Recyclable Supports To Immobilize Alcohol Dehydrogenases and Lipases: Synthesis, Catalytic Activity, and Recycling Efficiency

“…2b In a first article, Allcock and Kwon covalently immobilized tripsine and glucose-6-phosphate dehydrogenase on a support of modified [NP(OPh) 2 ] n on an alumina carrier. 8 The process consisted on a nitration-reduction protocol to obtain the corresponding polyaminophosphazene derivative which was subsequently activated with glutaraldehyde and then reacted with the enzyme solution. Later, an invertase was supported on spherical particles of [NP(OCH 2 CF 3 ) 2 ] n , first displacing the trifluoroethoxy moieties with NaOCH 2 CH 2 NH 2 and then anchoring the enzyme, obtaining good activities for this preparation.…”