Efficacy of L-Cysteine as an Anti-Oxidant in Papain Catalyzed Synthesis ofOligopeptides in Organic Solvent System

Abstract

Enzymatic peptide synthesis has drawn considerable attention for synthesis of high by-pass oligopeptide feed supplements in animal nutrition. A hardy protease, (papain) with a cysteine moiety at the active site requires the presence of an anti-oxidant in the reaction medium to ensure that the thiol group remains intact. Free cysteine has been the antioxidant of choice for papain-catalyzed synthesis of oligopeptides in aqueous systems. However, due to limited solubility of cysteine in organic solvents, it is generally not a suitable antioxidant for the synthesis of oligopeptides in biphasic solvent systems; instead, mercaptoethanol is often used. Lysine and Methionine is couple of well-known limiting amino acids that find application in cattle feed and poultry. Synthesis of co-oligopeptides of Lysine and Methionine has generally been attempted in bi-phasic solvent systems with Mercaptoethanol as an anti-oxidant. The inherent toxicity of mercaptoethanol when present even in trace amounts make its use undesirable during synthesis of oligopeptides to be used as feed supplements. Use of non-toxic antioxidants such as Cysteine would make the end product more amenable as a nutritional supplement for animal feed. Therefore efficacy of L-cysteine as an anti-oxidant was investigated during papain catalyzed oligomerization of Lys, Arg, Glu and Asp in two organic systems: a three phase micro-aqueous media consisting of n-octane, DFP and water, as well as homogeneous ACN/water mixtures. Reactions were also carried out under an argon atmosphere in the presence and absence of anti-oxidants. The results of the experiments showed that L-cysteine facilitated oligomer synthesis in both the three phase system and the ACN/water mixture. The overall oligomer yields were found to be better than 75% in the presence of L-Cysteine. Oligopeptide yields obtained through reactions carried out under the argon atmosphere were less than 20%.