Useful methods in enzymatic synthesis of peptides: A comparative study focussing on kinetically controlled synthesis of Ac-Phe-Ala-NH2 catalyzed by alpha-chymotrypsin

The usefulness of different reaction systems for enzymatic peptide synthesi s was evaluated by studying the formation of a dipeptide from equimolar amo unts of acyl-donor and acyl-acceptor. alpha-Chymotrypsin (EC 3.4.21.1.), di ssolved or deposited on celite, was used for the synthesis of Ac-Phe-Ala-NH 2 from Ac-Phe-OEt and H-Ala-NH2. Reactions were carried out at room tempera ture (approximate to 25 degrees C), if not otherwise stated, in aqueous sol utions (less than or equal to 21% yield), frozen solutions at -30 degrees C (less than or equal to 60% yield), suspensions in aqueous media with and w ithout 0.5% (v/v) of Triton X-100 (less than or equal to 82% yield), suspen sions in hydrophobic organic media with Na2CO3. 10H(2)O as water source (le ss than or equal to 90% yield), eutectic mixtures at 37 degrees C (less tha n or equal to 97% yield), and acetonitrile containing 1-5% (v/v) of aqueous buffer (less than or equal to 99% yield). Initial rates of synthesis were in the range of 5.9 x 10(-2)-7.1 x 10(2) mu mol min(-1) (mg of enzyme)(-1). The lowest one was obtained with dissolved reactants in acetonitrile conta ining 1% (v/v) of aqueous buffer, and the highest one was obtained with sus pended acyl-donor in aqueous medium containing 0.5% (v/v) of Triton X-100.