Metalloproteases in Peptide Synthesis

The metalloprotease thermolysin has been widely used for peptide synthesis [66,67]. Thermolysin is produced and secreted by the thermophilic bacterium Bacillus thermo-proteolyticus. Catalysis involves water activation by a zinc ion that is coordinated by side chains of two histidines and a glutamate. Thermolysin specificity is defined by its Sr pocket accepting large hydrophobic, polar, and charged residues. In the SI pocket hydrophobic residues are preferred, and Leu Ala Phe Gly is the preference order for the S2 and S2 pockets. This hydrolytic specificity of thermolysin is reflected in its synthetic specificity. 

Many related so-called thermolysin-like proteinases (TLPs) from various Grampositive strains have been described [47], including neutral proteases from Bacillus subtilis, and some of these variants are applied in peptide synthesis. Several metal-loenzymes acting as carboxy- or aminopeptidase have also been characterized, but these variants have not been extensively used in peptide synthesis. A bovine carboxy-peptidase A [39] and orange carboxypeptidase C [68] have been applied for dipeptide synthesis in water-organic solvent mixtures, both under thermodynamic and xmder kinetic control. 

15 DISCOVERY AND ENGINEERING OF ENZYMES FOR PEPTIDE SYNTHESIS AND ACTIVATION 

Chemoenzymatic peptide synthesis of aspartame using thermolysin under thermodynamic control. The coupling reaction is stereo- and regioselective. The unreacted isomer o-Phe-OMe forms a precipitate with the product, shifting the equilibrium toward the synthesis. After precursor isolation and hydrogenolysis, the o-isomer is chemically racemized and can be reused [69, 70]. 

Another example of thermolysin-catalyzed peptide synthesis is the production of precursors of enkephalins [57], in particular, coupling of nonnatural amino acids, such as halophenylalanines, is also possible [34]. Similar to a-ch5unotrypsin and papain, thermolysin has been used for peptide polymerization [71]. Using the reversibility of thermolysin-catalyzed peptide bond formation, d5mamic combinatorial libraries of peptides could be established and screened for e formation of nanostructures with special properties [72]. 

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