Enzymic methods poly gels

This process involves the suspension of the biocatalyst in a monomer solution which is polymerized, and the enzymes are entrapped within the polymer lattice during the crosslinking process. This method differs from the covalent binding that the enzyme itself does not bind to the gel matrix. Due to the size of the biomolecule it will not diffuse out of the polymer network but small substrate or product molecules can transfer across or within it to ensure the continuous transformation. For sensing purposes, the polymer matrix can be formed directly on the surface of the fiber, or polymerized onto a transparent support (for instance, glass) that is then coupled to the fiber. The most popular matrices include polyacrylamide (Figure 5), silicone rubber, poly(vinyl alcohol), starch and polyurethane. 

The same methods are also used to immobilize whole cells. Immobilization of cells avoids the tedious isolation and puriHcation of enzymes. The activity of the immobilized cells can be reduced by toxicity during immobilization and by hindered diffusion of the substrate. But the immobilized cells may also be more active than the free cells as well, since complete or incomplete destruction of the cell walls removes certain proteases from the cell which would otherwise degrade and thereby deactivate enzymes. For example, E. coli bacteria encapsulated in poly(acrylamide) gels are used commercially to convert sodium fumarate to L-aspartic acid. 

The main steps of the activity gel assay are outlined in Table 1. The in situ detection of poly(ADP-ribose) polymerase activities includes gel electrophoresis in SDS, renat-uration of proteins with appropriate buffers, incubation of the intact gel with [ P]-NAD, removal of nonincorporated precursor by TCA, washing, and autoradiography. The method was developed by using extracts and partially purified fractions of the poly(ADP-ribose) polymerase. The catalytic peptides of the enzyme are identified as activity bands and their Mj. determined by referring to protein markers [1]. 

Poly(ADP-ribose) was purified almost to homogeneity fi om bull testes by the method of Agemori et al. (5). Purified polymerase was incubated briefly with [ P]NAD (10°C, 30 sec, 5 xM NAD) in the presence of 10 pg/ml Hae IH-digested calf thymus DNA. Incorporation was stopped by the addition of 3-aminobenzamide and unincorporated labeled NAD was removed. Pulse-labeled polymer was originally attached to the enzyme. After alkali treatment it showed a heterogeneous distribution of short chains on a 20% polyacrylamide gel (6). Venom phosphodiesterase digested these to p2p]pR AMP and [ P]AMP, of which AMP represented 4.6% of the counts. After a chase with unlabelled NAD (200 pM NAD, 10 min, 25 °C),... 

---