Engineering of Enzymes for Peptide Synthesis and Activation

DISCOVERY AND ENGINEERING OF ENZYMES FOR PEPTIDE SYNTHESIS AND ACTIVATION... 

The discovery and engineering of enzymes for peptide synthesis and activation and synthesis of chiral compounds by ene-reductases (ERs) and flavin-dependent enzymes tiiat catalyze the asymmetric reduction of electronically activated carbon-carbon double bonds are described. These enzymes have attracted increased attention from the industry as the enzymatic reduction usually proceeds with enhanced chemoselectivity and elevated stereoselectivity. 

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. 

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