BrCN-sepharose

C)110 1, BrCN-Sepharose 1011, silica gel11021, or in a hollow fiber ultrafiltration membrane [421. The Eupergit C immobilized pig liver esterase retains 68% of the specific activity of the soluble enzyme. It is easily removed by filtration from the reaction mixture and can be reused several times when stored at 7 °C. In large-scale experiments with pig liver esterase in aqueous solution the enzyme can be stabilized, if necessary, by the addition of inexpensive bovine serum albumin1411. For a determination of the ee value of the monoester, different methods can be used ... 

Wash freshly activated gel (see Protocol 3.6.1) with huge amounts of ice-cold water or in the case of commercially available activated gels, e.g., BrCN-activated Sepharose 6B, with ice-cold 0.001 N HCl. 

Immobilization was carried out using a homogeneous preparation of poly(ADP-ribose) synthetase obtained from calf thymus [6] and commercially available BrCN-activated Sepharose 4B. For successful immobilization of active enzyme, it was important to block a majority (% two-thirds) of the active groups of the gel by pretreatment with dithiothreitol. The treated gel was mixed with the enzyme solution in the presence of 0.35 M KCl, 0.2 mAf dithiothreitol, 20% glycerol, and 0.1 A/K phosphate buffer (pH 8.0). Figure 1 shows the time course of immobilization of the enzyme activity. The decrease in the soluble enzyme activity reflects mainly immobilization of the enzyme, whereas the decrease in the immobilized enzyme activity is indicative of inactivation of the enzyme. In order to avoid the increase of inactive... 

The spacer includes an amine group and so this compound can be attached to a Sepharose gel that has previously been activated by BrCN [5]. Glutaraldehyde can also be used to attach a spacer (hexamethylenediamine) to the activated Sepharose, then an enzyme (alcohol dehydrogenase), and finally NAD by co-immobilization [42]. 

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