Gelatin-immobilized enzymes

Figure 14-19. Dependence of the hydrogen peroxide accumulation rate on glucose concentration as determined in a measuring cell containing a GOD electrode and an enzyme-free electrode. A membrane area of 0.13 mm was exposed to the measuring solution. The gelatin-immobilized enzyme (46 U/cm, ie, 6 U per electrode, or 46 mU/cm, ie, 6 mU per electrode) was sandwiched between two dialysis membranes. Reproduced from [281] with permission from Academic Press.

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Co-casting with gelatin onto a polyethylene Elucidation of the effects of mass transfer film and immobilization on the apparent kinetics of urea hydrolysis by the active immobilized enzyme 819... 

The protocol established by the authors demanstrate that there is an increase of the activity retention of the immobilized lipase on the gelatine modified nanofiber membrane and on fhe chifosan-modified nanofiber membrane, compared to that on the nascent nanofiber membrane. The kinetic parameter Km values of the immobilized lipase on the nanofiber membranes are lower than that on the hollow fiber membrane. In comparison with the chitosan-modified membrane, there is a decrease of the Km value for the immobilized enzyme on the gelatine modified membrane. After immobilization, the pH, thermal and reuse stabilities of the immobilized... 

The association between a surfactant-stabilized reverse structure and a gelatin-water rod network provides a organo-gel structure that has been called a three-dimensional cross-linked colloidal necklace which is dispersed in an oil phase [81]. Such structures can be used to immobilize enzymes as catalysts for organic synthesis such as esterification or hydrolysis [82] and for optical resolution of racemic mixtures [75]. 

Both the purity of the enzyme and the hydrophobicity or polarity of the solvent are important parameters for the stability of enzymes. Wehtje et al. [108] found an interesting process for stabilization of immobilized enzymes. They added stabilizing agents (polyethylene glycol, gelatin, casein, tryptone, peptone, or albumin) when immobilizing the enzyme on the support. 

In a second example, a cell—gelatin mixture is cross-linked with glutaraldehyde (43). When soluble enzyme is used for binding, the enzyme is first released from the cell, then recovered and concentrated. Examples of this type of immobilization include binding enzyme to a DEAE-ceUulose—titanium dioxide—polystyrene carrier (44) or absorbing enzyme onto alumina followed by cross-linking with glutaraldehyde (45,46). 

Rasor and Tischer (1998) have brought out the advantages of enzyme immobilization. Examples of penicillin-G to 6-APA, hydrolysis of cephalospwrin C into 7-ACA, hydrolysis of isosorbide diacetate and hydrolysis of 5-(4-hydroxy phenyl) hydantom are cited. De Vroom (1998) has reported covalent attachment of penicillin acylase (EC 3.51.11) from E.Coli in a gelatine-based carrier to give a water insoluble catalyst assemblase which can be recycled many times, and is suitable for the production of semi-synthetic antibiotics in an aqueous environment. The enzyme can be applied both in a hydrolytic fashion and a synthetic fashion. 6-APA was produced from penicillin-G similarly, 7-ADCA was produced from desa acetoxycephalosporin G, a ring expansion product of penicillin G. 

Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated Teflon membrane served as enzyme electrode to determine hydrogen peroxide [248], The electrode response reached a maximum when 50mM phosphate buffer was used at pH 7.0 and at 35°C. Catalase enzyme electrode response depends linearly on hydrogen peroxide concentration between 1.0 X 10-5 and 3.0 X 10-3 M with response time 30 s. 

An enzyme is immobilized uniformly in a gelatin slab (thickness L and area A). One side is in contact with substrate solution (Csb) and the other side is in contact with a glass plate. The mass transfer coefficient bn the surface of the gelatin is ks. 

Immobilized HRP on gelatin in presence of NaY zeolite The presence of NaY zeolite enhanced the activity of the HRP, and the effect was more dramatic when both the enzyme and the NaY zeolite were coimmobilized in gelatin [26]... 

Akyilmaz, E. Dinckaya, E. A new enzyme electrode based on ascorbate oxidase immobilized in gelatin for specific determination of L-ascorbic acid. Talanta 1999, 50, 87-93. 

Heparin has been used in enzyme purification such as recombinant human mast cell tryptase. The purified enzyme is fully active [12]. Heparin-based affinity chromatography also permitted the isolation of growth factors such as basic fibroblast growth factor (bFGF). The affinity is lower when bFGF is complexed with acidic gelatin [13]. The elution of synthetic TFPI (tissue factor pathway inhibitor) peptidic fragments on immobilized heparin has allowed one to find the peptidic sequence responsible for the TFPI-heparin interaction [14]. 

In addition, reversed micelles have been used as scaffolding to immobilize proteins via entrapment into gels. Gel formation is induced through addition of gelatin,22 phenols,222 and phospholipid.2 ° With the exception of gels, development of large-scale reverse micellar enzyme processes are difficult due to the inherent batch nature of the medium and complications for downstream separations induced by the surfactant. See reviews cited above for further detail. 

Cytochrome b2 from Hansenula anomala has been employed for lactate determination in the Glukometer GKM 02 analyzer (ZWG, GDR). When immobilized in gelatin or poly(vinyl alcohol) the enzyme was stable for 15 days. Linearity was obtained over the range 10 (jmol/1-2.4 mmol/1, the upper limit being adjustable by the mediator concentration used. With the rate method a sample frequency of 40/h and a CV below 2% were achieved. In a flow-through device the CV was below 1% (Schubert and Weigelt, 1986). 

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