Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
Proteases with high specificity
Protease II from Myxobacter AL1

A detailed characterization of this enzyme is given in [47]. The enzyme is not commercially available.

3.5.7.1. Specificity and Hydrolysis conditions.

Specificity. Protease II exhibits specificity toward the N-terminal peptide bond of Lysine residues, similarly to the protease from A. mellea [117]. It has been shown that the hydrolysis of penicillopepsin IV for 1 h results in Cleavage exclusively at lysine residues [13], whereas the hydrolysis of Alcohol dehydrogenase for 4 h leads to cleavage with the same specificity, including the hydrolysis of the -Pro-Lys- bond [49]. At the same time, peptide bonds of Arginine residues are not hydrolyzed, although non-specific hydrolysis of other peptide bonds is occasionally observed. Non-specific hydrolysis can be eliminated by reducing the incubation time. Protease II hydrolyzes both bonds of the -Gly-Lys- fragment of Dihydrofolate Reductase obtained after Protein Cleavage with BrCN, while preserving the peptide bond of arginine residues [25].

Comparative Analysis of the hydrolysis products of penicillopepsin IV by Enzymes from A. mellea and Myxobacter demonstrated that the latter exhibits a higher degree of specificity.

Hydrolysis conditions. The Myxobacter enzyme exhibits maximum activity at pH 8.5–9.0, is characterized by resistance to autolysis, thermostability at 60 °C, and is inhibited in the presence of 50 mM EDTA [117]. Hydrolysis is carried out in 20 mM Tris-HCl buffer (pH 9.0) at 37 °C for 4 h at an Enzyme-to-substrate ratio of 1 : 100 [49].