Peptide Production 289
- Z Endoproteinase Lys-C (EC 3.4.99.30)
2. 7.1. General Information
This enzyme is a serine protease isolated from culture filtrates of
Lysobacter enzymogenes (57). - 7.2. Specificity
The enzyme cleaves specifically C-terminal to lysine residues (57).
Some minor nonspecific cleavages, notably at Asn--X bonds, have
also been reported. - 7.3. Molecular Mass
It has a mol mass of about 33,000 Da. - 7.4. pH Optimum
The enzyme has a pH optimum in the range 8.5-8.8. - 7.5. Assay
The assay is based on hydrolysis of Tosyl-Gly-Pro-Lys-4-nitroanilide
(commercially sold as Chromozym ® PL). Chromozym ® PL (9 mg) is
dissolved in 1 mL of redistilled water. This solution (0.05 mL) is
combined with 1.0 mL of buffer (25 mM Tris-HC1, pH 7.7, 1 mM
EDTA) and incubated at 25°C. The reaction is started by addition of
0.05 mL of enzyme (suitably diluted with the above buffer). The increase
in absorbance is monitored at 405 nm, and using an extinction coeffi-
cient of 10.4 mM-locm -1, the enzyme activity of the sample is calcu-
lated. Using this assay, the enzyme is supplied with a specific activity
of 25-35 U/mg, where 1 U is defined as the amount of enzyme that
liberates 1 lxrnol 4-nitroaniline/min at 25°C from Tosyl-Gly-Pro-Lys-
4-nitroanilide. - 7.6. Stability
The enzyme is active in 0.5% SDS, 5M urea, and in 10% acetonitrile.
Aqueous solutions are stable at pH 5-12 for up to 1 d at 4°C, or can be
stored at -20°C for months. The enzyme is stable at 4°C, stored dry. To
avoid autolysis, the incubation temperature should not exceed 37°C (44).
- Inhibitors
The enzyme is inhibited by DFP (1 mM), TLCK (1 rnM), leupeptin
(0.4 mM), and aprotinin (100 gg/mL). The enzyme is not inhibited by
EDTA (2 mM), PMSF (1 mM), or oq-antitrypsin.
- Inhibitors