Amino acid composition enzymatic methods

For historical reasons many pharmaceutical enzymes are assayed with physiological or biopolymeric substrates (proteins, polysaccharides, bacteria, oil emulsions), which causes a number of theoretical and practical problems. The interpretation of results is difficult when natural substrates are converted into products that are substrates themselves for the next enzymatic attack. Reaction rates often depend on the position of the scissile bonds in the molecule and the chemical nature of the moieties. Hydrolysis can proceed simultaneously on various bonds at various rates. In proteolysis it is assumed that some products are liberated only after denaturation and that during the reaction course new peptide bonds become accessible for hydrolysis. In these cases the enzymatic mechanisms become exceedingly complex, kinetic parameters are apparent values, and experimental results are strongly influenced by the reaction conditions. Reproducibility problems can occur upon assaying proteinases with a limited specificity for particular casein types. Bromelain and pancreatic proteinase, FEP pharmaceutical enzyme standards, are assayed with a casein substrate. The extent of soluble peptide release is a measure of proteolytic activity. However, due to limited specificity, some proteinases release peptides with a nonrandom aromatic amino acid composition. Contamination of casein preparations with protein and of test enzyme substances with other proteinases biases the assay results. Under these conditions, relative assay methods are indicated. 

Catalase has been studied intensively by chemical and physical methods. The protein component is not unusual in amino acid composition. The iron appears to be present only in the ferric form, and cannot be reduced chemically by any procedure that does not denature the enzyme. There is evidence, however, that during an enzymatic reaction with hydrazoic acid and hydrogen peroxide the iron is reduced to the ferrous state. The iron forms compounds with a large number of molecules. Some of these, such as HCN, HNs, and H2S, are inhibitors of the enzyme. The absorption spectrum of catalase shows bands at 403 m/i (Soret), 500 m/i, 540 m/B, and 629 m/t . These shift to give characteristic spectra when HCN, HN3, or HF combines with enzyme. Magnetic... 

Chiral amines have been attracting attention as an important composition, particularly for pharmaceutical products. The organic synthetic methods of optically active amine compounds have been developed through the traditional resolution of racemic amines with the formation of diastereomer salts using an optically active mandelic acid or tartaric acid. Enzymatic synthesis has mainly used lipase and S- or R-stereoselective amine transaminase (AT) [29-31] (Figure 19.7). Turner et al. successfully synthesized chiral (R)- and (S)-amines by kinetic resolution using a combination of stereoselective AT and d- or L-amino acid oxidase (AAOx) [32] (Figure 19.7). However, the theoretical yield of the products has been limited to 50% in the kinetic resolution. 

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