Peptide Production 283
forming assays, it should be noted that citrate, borate, Veronal, and
Tris anions partially inhibit esterase activity (18).
2.3. Elastase (EC 3.4.21.36)
2.3.1. General Information
The commercial enzyme is normally prepared from porcine pancreas.
Elastase I (pancreatopeptidase E) is a serine protease and is produced
in the pancreas as the inactive zymogen proelastase. Proelastase is con-
verted to elastase by tryptic cleavage of a single peptide bond (22-24).
The isolation of porcine elastase II has also been described, but elastase
I is the commercially available enzyme (25).
2.3.2. Specificity
The enzyme has rather a broad specificity cleaving C-terminal to
small hydrophobic side chains, e.g., Gly, Ala, Ser, Val, Leu, and Ileu
(26-28).
2.3.3. Molecular Mass
Elastase is a single polypeptide chain of 240 residues crosslinked by
four disulfide bridges, and has a mol mass of 25,900 Da. The complete
amino acid sequence is known (29); the gene has been cloned and mol
mass confirmed (30).
2.3.4. pH Optimum
The enzyme has a pH optimum of pH 8.0-8.5 (31).
2.3.5. Assay
An assay based on the hydrolysis of N-benzoyl-L-alanine methyl ester
(BAME) is used. The reaction conditions consist of 10 mM substrate in
0.1M KC1 and 0.01MTris-HC1, pH 8.0, at 25°C. The enzyme solution is
added and the change in absorbance with time is monitored at 253 nm (24).
Alternatively, an assay based on the liberation of congo red from
elastin-congo red can be used (24). The enzyme is supplied with a
specific activity of 15-20 U/mg using this assay, where 1 U is defined
as the amount of enzyme that solubilizes 1 mg of elastin from elastin-
congo red in 60 min at 25°C in carbonate buffer, pH 8.8.
Also, hydrolysis of succinyl-(L-ala)3-4-nitroanilide by elastase can
be monitored at 410 nm. Using this assay, the enzyme is supplied with
a specific activity of 10-15 U/mg where 1 U corresponds to the amount
of enzyme that liberates 1 grnol of 4-nitroaniline/min at 25°C, pH 7.8.