Peptide Production 2911 U is defined as the amount of enzyme that hydrolyzes 1 gmol of
substrate/min at pH 2.0 and 37°C.
2.8.6. Stability
The enzyme is irreversibly inactivated at pH values >6.0 (60). Below
pH 6, it retains activity in 4M urea and in 3M guanidine hydrochloride
(61), and is unaffected by heating to 60°C. The lyophilized enzyme is
stable for months at 4°C.
2.8. Z Inhibitors
Pepsin is inhibited by Pepstatin A (mol mass 685.9 Da), diazoketones,
phenylacyl bromide, and aliphatic alcohols.2.9. Thermolysin (EC 3.4.24.4)
2.9.1. General Information
The enzyme is produced in the culture supernatants of the microor-
ganism Bacillus thermoproteolyticus. It is a metalloenzyme having an
absolute requirement of zinc ions for activity. The enzyme is thermo-
stable (62).
2.9.2. Specificity
In general, thermolysin hydrolyzes peptide bonds N terminal to
hydrophobic amino acids with bulky sidechains (63), e.g., Ileu, Leu,
Val, and Phe. However, the enzyme is not absolutely specific for these
residues, with other workers having identified minor cleavage sites N
terminal to most other amino acids. However, the preferred sites of
cleavage are those N terminal to the bulky hydrophobic residues, and
it is this characteristic that has made thermolysin useful in protein
studies. Also, thermolysin appears to be able to cleave native proteins
to a greater extent than most other proteases. Bonds involving proline
of the type --Pro--Ileu-- are cleaved, but in sequences of the type
X--Phe--Pro--, the X--Phe-- bond is resistant to cleavage. Cleav-
age is also inhibited by an adjacent o~ amino or carboxyl group, so
thermolysin has no exopeptidase activity (63, 64).
2.9.3. Molecular Mass
It is a single polypeptide chain of 316 residues and has a mol mass
of 34,600 Da (65). The complete amino acid sequence has been
determined (65).