Immobilization cofactor

Either the CO2 formation is followed potemiometrically (243) or the O2 consumption is measured amperometrically at an oxygen electrode (245). In the first method, the enzyme is physically immobilized with a dialysis membrane. The response is linear in the range 5-300 pg/mL of salicylate. The second technique uses chemically immobilized enzyme (GA -F BSA) attached to a pig intestine mounted on the tip of the O 2 electrode. Samples containing from 10 pM to 2 mM salicylate were analyzed. An elegant microelectrode (244) has the enzyme and the cofactor immobilized in the electrode matrix (carbon paste) and the catechol formation is monitored at -F 300 mV versus Ag/AgCl. The electrode consists of a disposable strip, allowing measurements to be made on a drop of blood within 1 min. 

Immobilization. Enzymes, as individual water-soluble molecules, are generally efficient catalysts. In biological systems they are predorninandy intracellular or associated with cell membranes, ie, in a type of immobilized state. This enables them to perform their activity in a specific environment, be stored and protected in stable form, take part in multi-enzyme reactions, acquire cofactors, etc. Unfortunately, this optimization of enzyme use and performance in nature may not be directiy transferable to the laboratory. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

The choice of a suitable immobilization method for a given enzyme and appHcation is based on a number of considerations including previous experience, new experiments, enzyme cost and productivity, process demands, chemical and physical stabiHty of the support, approval and safety issues regarding support, and chemicals used. Enzyme characteristics that greatly influence the approach include intra- or extraceUular location size surface properties, eg, charge/pl, lysine content, polarity, and carbohydrate and active site, eg, amino acids or cofactors. The size, charge, and polarity of the substrate should also be considered. 

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-... 

All soil metabolic proce.sses are driven by enzymes. The main sources of enzymes in soil are roots, animals, and microorganisms the last are considered to be the most important (49). Once enzymes are produced and excreted from microbial cells or from root cells, they face harsh conditions most may be rapidly decomposed by organisms (50), part may be adsorbed onto soil organomineral colloids and possibly protected against microbial degradation (51), and a minor portion may stand active in soil solution (52). The fraction of extracellular enzyme activity of soil, which is not denaturated and/or inactivated through interactions with soil fabric (51), is called naturally stabilized or immobilized. Moreover, it has been hypothesized that immobilized enzymes have a peculiar behavior, for they might not require cofactors for their catalysis. 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

An immobilized-enzyme continuous-flow reactor incorporating a continuous direct electrochemical regeneration of NAD + has been proposed. To retain the low molecular weight cofactor NADH/NAD+ within the reaction system, special hollow fibers (Dow ultrafilter UFb/HFU-1) with a molecular weight cut-off of 200 has been used [32],... 

Immobilized enzymes and immobilized cells can be used in principle as heterogeneous catalysts not only for the enhancement of hydrolytic reactions but also in specific synthetic reactions. Many of the enzymes involved in the synthesis of complex biological compounds -in vivo and in vitro require cofactors which have to be provided in suitable form when immobilized enzymes or cells are used. 

Adenosine triphosphate (ATP) is one of the most important cofactors involved in many of the synthetic reactions going on within the cell. Its recent large scale in vitro enzymatic synthesis from adenosine and acetylphosphate is of particular interest. Three enzymes immobilized in polyacrylamide gel were used adenosine kinase, adenylate kinase and acetate kinase (lip. ... 

When immobilized enzymes are employed in a continuous reactor, many of these limitations are avoided. Moreover, in this case the output signal is recorded at the reactor outlet, and this procedure therefore cannot affect the processes taking place in the reactor, and the signals obtained can be used in another system as actual concentrations without conversion. Yet, in this configuration the cofactor enters the reactor in the feed stream, which requires large amounts of cofactor, especially when a high flow rate is employed. 

Using this reaction fumaryl groups [24], cofactors [64] and carbohydrate derivatives [65] have been incorporated site selectively into folded four-helix bundle proteins. The reaction can also be expected to be of general use in site-selective immobilization reactions of folded proteins. 

To immobilize a protein the biologic activity of which depends on a cofactor, this cofactor has to be present in sufficient concentration during the immobilization step. Thus, for example, the lectin Concanavalin A is immobilized in the presence of a buffer containing 0.1 M glucose or a-methylmannoside, 1 mM calcium chloride and 1 mM manganese chloride. 

---