Carboxylic immobilized enzymes

Covalent Attachment The covalent attachment of enzyme molecules via nonessential amino acid residues (that is, amino acids minus water) to water-insoluble, functionalized supports are the most widely used method for immobilizing enzymes. Functional groups of the nonessential amino acid residues that are suitable for the immobilization process are free a-, /3-, or y-carboxyl groups, a- or /3-amino groups, and phenyl, hydroxyl, sulfhydryl, or imidazole groups.2... 

Enzymes catalyze the reversible enantioselective addition of ammonia to a./ -unsaturated carboxylic acids to give L-a-amino acids. Immobilized enzymes and whole-cell bioreactor technology competes well with chemical methods31,32. For practical purposes, immobilized whole cells are preferred over immobilized enzyme because of the added cost of enzyme isolation. 

Mild and selective hydrolysis of diethyl cyanomethylphosphonate leading to carboxylic acid and amide can be achieved under neutral conditions by an immobilized enzyme preparation from Rhodococus sp., and no ester cleavage is detected. ... 

A production process for the synthesis of carboxylic nucleoside precursors, which can be used for the manufacture of anti-HIV-1 agents such as carbovir (128) and analogs, has been developed by Chiroscience (Scheme 39) [ 114]. The process uses jS-lactamhydrolase from Aureobacterium sp, immobilized on a glu-taraldehyde-activated solid support for the optical resolution of lactam rac-126. The biotransformation is conducted as a batch reaction and an aqueous solution of rac-126 is cycled through a fixed bed of immobilized enzyme. This setup guarantees that the enzyme can be used in a steady-state production for more than six months, limited only by the mechanical stability of the carrier. The reaction stops when (-)-126 is completely hydrolyzed and a simple addition of acetone causes only the amino acid (-)-127 to crystallize. The latter can be used... 

A number of other conducting polymer derivatives have been used for immobilizing enzymes and redox mediators for biosensor applications. Among these polymers are poly (tyramine) [167] and poly(l-(5-aminonaphthylethanoic acid) [168], which have accessible amine and carboxyl functionalities, respectively, for covalently linking to complementary groups on available amino acid residues of enzymes. Polypyrrole and polythiophene derivatives have also been widely exploited for covalent immobilization of enzymes, which then function as the sensing layers in efficient biosensors [169,170]. 

Similar behavior of the immobilized enzyme to the free one has been observed in respond to the changes in the substrate s pH (Fig. 21.7). The optimum pH has not shifted, but the immobilized enzyme shows higher activity within acidic region, pH 2.5-3.0. This resistance to pH could be explained due to the presence of negatively charged free carboxylic groups un-binding by calcium ions. This result is in accordance with those obtained by other authors [29]. 

The immobilized enzyme preparation SP409", possessing both nitrilase and amidase activity, has been used for the hydrolysis of glycosyl cyanides. The a-nitrile 20 underwent hydrolysis to the amide 21, whilst the corre nding P-anomer was converted, at a faster rate, into the analogous carboxylic acid. ... 

Cyanohydrins are bifunctional molecules and therefore constitute a particularly useful class of compounds for synthetic purposes. A hydroxyl and a nitrile functional group are available for chemical and enzymatic follow-up reactions (Scheme 25.2 [7, 93-95]), resulting in hydroxy carboxylic acids [96-99], carbamates [KXl], hydroxy-amides [101], primary and secondary hydroxyamines [46,102-104], aziridines [105, 106], aminonitriles [107, 108], diamines [108], azidonitriles [108], a-fluoronitriles [109], hydroxy ketones [110], and many more. The (S)-selective H HNL and the (R)-selective PaHNL, in particular are used on large scale either for the s)mthesis of (S)-3-phenoxybenzaldehyde cyanohydrin, a precursor for p5U ethroids, a class of insecticides (R)- and (S)-mandelonitrile, which can be further converted to man-delic acids (R)-chloromandelonitrile, a precursor for an anticoagulant (R)-2-hydroxy-4-phenylbutyronitrile, which serves as intermediate for the production of angiotensin-converting enzyme inhibitors (ACEi) or (R)-2-amino-l-(2-furyl)ethanol [94]. Several HNLs are commercially available as free or immobilized enzymes. 

The assumption of the association of Hb in the pores of carboxylic cation exchangers has been advanced in Ref. [47] on the basis of electron microscopy at the maximum filling, almost all the pore surface is filled with Hb associates which are ordered star-shaped structures. Interprotein interaction in the adsorption immobilization of enzymes have been reported in Refs. [74, 75]. 

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