Sulfhydryl peptidases

The application of papain in peptide synthesis is well established [23-25]. Papain can be used for fhe preparation of di- and tripepfides in an aqueous medium wifh cosolvent addition (up to 40%) and at high pH to promote synthetic activity. The enzyme is a sulfhydryl protease with no homology to the trypsin or subtilase families of hydrolases. Since the catalytic nucleophile is a cysteine and because thioesters are relatively more prone to aminolysis than oxo-esters, the enzyme could be very attractive for synfhesis. However, unlike the case with the thiol variants of some serine hydrolases, fhe proteolytic activity is still high, and the broad substrate range of proteolysis makes peptide substrate and product hydrolysis more problematic than trypsin or chymotrypsin. Extensive enzyme engineering studies on papain are lacking, probably due to the laborious procedure for isolation of active papain from inclusion bodies formed in E. coli. 

The enzyme has a rather broad substrate range with a slight preference for Lys and Arg in the SI pocket and for hydrophobic aromatic residues in the S2 pocket. The SI pocket is not selective, which allowed the synthesis of dipeptides with nonpro-teinogenic amino acids [24]. To overcome the SI preference, the substrate-mimetic approach described under Section 15.3.1 was explored with the 4-guanidinophenyl (OGp) leaving group in dipeptide synthesis [25]. 

Fragment condensations with papain have also been reported, for example enkephalin fragment coupling (2 + 3) was achieved in 50% 5tield using equimolar amounts of acyl donor (PhAc-Tyr-Gly-OMe) and nucleophile (H-Gly-Phe-Leu-0 Bu) af pH 9.0 in buffer with 20% methanol [26]. Similar yields were obtained in low water systems such as buffer containing acetonitrile 4% (v/v). 

The synthetic potential of papain in low water systems has been explored with acyl donors bearing the carbamoylmethyl (Cam) leaving group. Dipeptide synthesis proceeded in 80% yield, and yields up to 60% were obtain for the synthesis of bioactive peptides like dermorphin-(l-4) (Boc-Tyr-D-Ala-Phe-Gly-NH2). Papain is also used as a versatile protease in polymer chemistry for the synthesis of amino acid oligomers and cooligomers of a-hydroxy acids and amino acids [22]. 

Homologues of the classical papaya papain that have been tested for peptide S5mthesis are plant-derived enzymes such as bromelain and ficain. Bromelain extracted from pineapple could be applied in Met- and Leu-enkephalin synthesis in a low water system, reaching 97% yield [27]. Ficain isolated from Ficus glabrata latex was tested in di- and tripeptide synthesis in frozen water medium [23] and was used in the synthesis of Lys and Met oligomers starting from the respective ethyl esters [29]. 

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