Effect of ultrasound on enzymatic digestion

ULTRASOUND-ASSISTED ENZYMATIC DIGESTION 3.5.1. Effect of ultrasound on enzymatic digestion 

Ever since its inception in forensic toxicology [68], enzymatic digestion has been extensively used to isolate drugs from forensic samples. Carpenter [69] was the first to develop an analytical procedure in which subtilisin, a proteolytic enzyme, was used to determine Cd, Cu, Pb and Tl from human liver and kidney tissues. 

The enzymes used for this type of digestion in Analytical Chemistry are mainly hydrolytic enzymes, the catalytic effect of which is based on the insertion of water at a specific bond of the substrate. The hydrolytic enzymes used in analytical applications include lipases (which hydrolyse fats into long-chain fatty acids and glycerol) amylases (which hydrolyse starch and glycogen to maltose and to residual polysaccharides) and proteases (which attack the peptide bonds of proteins and peptides themselves). 

Enzymatic digestion has some advantages over conventional sample pretreatment procedures based on acid or alkaline digestion thus, the former uses mild conditions as regards temperature and pH, which avoids potential analyte losses by volatilization or ohemical transformation to other species, and reduces the risk of contamination. In addition, the selectivity of enzyme catalysis is a powerful tool for distinguishing between fractions of elements associated with different components of the matrix as enzymes act on oertain ohemical bonds only. 

For most enzymes, catalytic activity is temperature-dependent to a maximum level, above whioh they lose their activity. Also, by analogy with other proteins, enzymes are stable only within a limited range of pH. Outside this range, enzymes are denatured by ohanges in the charges of ionizable amino acid residues that alter the tertiary structure of proteins. Enzyme activity reaches a peak or a plateau at a specific pH, so enzymatic digestion is usually performed in a buffered medium. The process is also affected by the enzyme concentration, which must therefore be optimized as well. 

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