Poly immobilized enzyme

FIGURE 3 Scanning electron micrograph (1200x magnification) of the surface of a porous alumina particle coated with poly(diphenoxy-phosphazene). Surface nitration, reduction, and glutaric dialdehyde coupling immobilized enzyme molecules to the surface. (From Ref. 23.)... 

Third, a poly[bis(phenoxy)phosphazene] has been coated on porous alumina particles, surface nitrated, reduced to the amino-derivative, and then coupled to the enzyme glucose-6-phosphate dehydrogenase or trypsin by means of glutaric dialdehyde. The immobilized enzymes were more stable than their counterparts in solution, and they could be used in continuous flow enzyme reactor equipment (25). 

In another approach, the alcohol moiety, formed by an enzymatic hydrolysis of an ester, can act as a nucleophile. In their synthesis of pityol (8-37a), a pheromone of the elm bark beetle, Faber and coworkers [17] used an enzyme-triggered reaction of the diastereomeric mixture of ( )-epoxy ester 8-35 employing an immobilized enzyme preparation (Novo SP 409) or whole lyophilized cells of Rhodococcus erythro-polis NCIMB 11540 (Scheme 8.9). As an intermediate, the enantiopure alcohol 8-36 is formed via kinetic resolution as a mixture ofdiastereomers, which leads to the diastereomeric THF derivatives pityol (8-37a) and 8-37b as a separable mixture with a... 

The poly(sodium acrylate) hyperbranched graft formed in a simple deprotonation was used to ionically immobilize enzymes. Studies showed that immobihzed glucose oxidase in a 3-poly(sodium acrylate)/Au film retained significant activity on immobihzation and that the enzyme in this ionic assembly remained active for more than 60 days of storage [30]. 

All the enzymes used in the work described above are quite stable at room temperature and can be used in a free form. They can also be used in an immobilized form to improve the stability and to facilitate the recovery. Many immobilization techniques are available today (25). The recent procedure developed by Whitesides et al using water-insoluble, cross-linked poly(aerylamide-acryloxysuccinimide) appears to be very useful and applicable to many enzymes (37). We have found that the non-crosslinked polymer can be used directly for immobilization in the absence of the diamine cross-linking reagent. Reaction of an enzyme with the reactive polymer produces an immobilized enzyme which is soluble in aqueous solutions but insoluble in organic solvents. Many enzymes have been immobilized by this way and the stability of each enzyme is enhanced by a factor of greater than 100. Horse liver alcohol dehydrogenase and FDP aldolase, for example, have been successfully immobilized and showed a marked increase in stability. 

In summary, the steady state and transient performance of the poly(acrylamide) hydrogel with immobilized glucose oxidase and phenol red dye (pAAm/GO/PR) demonstrates phenomena common to all polymer-based sensors and drag delivery systems. The role of the polymer in these systems is to act as a barrier to control the transport of substrates/products and this in turn controls the ultimate signal and the response time. For systems which rely upon the reaction of a substrate for example via an immobilized enzyme, the polymer controls the relative importance of the rate of substrate/analyte delivery and the rate of the reaction. In membrane systems, the thicker the polymer membrane the longer the response time due to substrate diffusion limitations as demonstrated with our pAAm/GO/PR system. However a membrane must not be so thin as to allow convective removal of the substrates before undergoing reaction, or removal of the products before detection. The steady state as well as the transient response of the pAAm/GO/ PR system was used to demonstrate these considerations with the more complicated case in which two substrates are required for the reaction. 

Havens PL, Rase HF. Reusable immobilized enzyme/poly urethane sponge for removal and detoxification of localized organophosphate pesticide spills. Ind Eng Chem Res 1993 32 2254-2258. 

Another interesting organic material usually used to immobilize enzymes is chitosan [95, 96]. Chitosan is a poly((1 4)-2-amino-2-deoxy-P-D-glucose) (Fig. 9.5), which is a product of deacetylation of chitin, the second polysaccharide ranked by its prevalence in nature, just after cellulose. 

Fernandez-Lafuente, R., Rosell, C. M., Caanan-Haden, L., Rodes, L., Guisan, J.M. (1998) Stabilization of immobilized enzymes against organic solvents complete hydrophilization of enzymes environments by solid-phase chemistry with poly-functional macromolecules, Prog. Biotechnol. 15 (Stability and Stabilization of Biocatalysis), 405-410. 

The use of electrochemically polymerized films to immobilize enzymes at electrode surfaces was reviewed by Bartlett and Cooper up to 1992 [24]. While a large variety of studies have been performed mainly using poly(pyrrole) as the conducting polymer, many problems still have to be solved, particularly with respect to the question of the speed of... 

General [13,16, 20, 22] Mediator-free [28] Various [i-di kelone mediators [25] Immobilized enzyme [38] Water in SCC02 polymerizations [19] Surfactants [21] Poly acrylamide starch graft polymers[39[. 

Howdle and coworkers [33] showed that relatively high molecular weight poly-caprolactone could be synthesized in supercritical C02 (A4n = 12-37 kDa), but with lower polydispersity (1.4-1.6) and higher yield (95-98%) than in conventional organic solvents. Furthermore, when Novozym 435 was used as catalyst in supercritical C02, the immobilized enzyme could be cleaned and recycled to allow repeated use, while unreacted monomer and low molecular weight oligomers... 

Burgmayer P and Murray R W 1984 Ion Gate Electrodes. Polypyrrole as a switchable ion conductor membrane J. Phys. Chem. 88 2515-21 Otero T F and de Larreta E 1988 Conductivity and capacity of polythiophene films impedance study J. Electroanal. Chem. 244 311-18 Sunde S, Hagen G and Odegird R 1993 Impedance analysis of the electrochemical doping of poly(3-methyl-thiophene) from aqueous nitrate solutions J. Electroanal. Chem. 345 59-82 Zaborsky O R 1973 Immobilized Enzymes (Cleveland, OH Chemical Rubber Company) (see especially pp 69,87)... 

Three kinds of silica particles with different sizes (150-250 pm, 75-150 jim and 1 pm) were selected as carriers for PPL immobilization to study the relationships between the carrier size of immobilized enzyme, the catalytic activity for ring-opening copolymerization and the polymer yield. The highest M of 26400 of poly(BTMC-co-DTC) was obtained at around 0.1 wt% of IPPL with size of 75-150 pm. Moreover, the M of poly(BTMC-co-DTC) decreased rapidly with the increasing of IPPL concentration. 

The LbL technique is undoubtedly one of the best methods to incorporate biological components into man-made devices. Therefore, sensor applications must be one of the most promising subjects for LbL assemblies of biomaterials. For example, Leblanc and coworkers used several bilayers of chitosan and poly(thiophene-3-acetic acid) as cushion layers for stable enzyme films [187]. The first five bilayers of the cushion layer allowed for better adsorption of organophosphorus hydrolase than the corresponding adsorption on a quartz slide. The immobilized enzyme becomes more stable and can be used under harsher conditions. The assembled LbL films can be used for spectroscopic detection of paraoxon, an organophosphorus compound. This cushion layer strategy provides a well-defined substrate-independent interface for enzyme immobilization, in which the bioactivity of the enzyme is not compromised. This leads to fast detection of paraoxon and quick recovery times. 

The use of polymers for the immobilization of enzymes and other bio-logically-active molecules has been discussed. The advantages of polymeric support materials and rules for their selection according to the type of use have been discussed. A review of various types of polymers which can serve as support matrices has been given. They are, e.g., polymeric carbohydrate derivatives, poly(allyl carbonate) and poly(allyl alcohol), polymers of acrylamidosalicylic acids, polyacrylamide derivatives, etc. Some examples of the use of immobilized enzymes and other biologically-active molecules were mentioned. 

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