Sínteses bem sucedidas de dipeptídeos a partir de Ac-L-Tyr-OEt ou Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys ou Thr) e glicina amidada em meio reacional bifásico foram realizadas usando dois tipos de preparações comerciais de lipase pancreática suína (PPL) (bruta (cPPL) e purificada (pPPL)). Nas mesmas condições reacionais, a α-quimotripsina, protease pancreática que também apresenta atividade esterásica, catalisou a síntese de Ac-L-Tyr-Gly-NH 2 com alta produtividade. Na maioria das sínteses também ocorreu a hidrólise do produto formado. Eletroforese em gel de poliacrilamida, ensaios enzimáticos com substratos cromogênicos específicos e cromatografia de exclusão molecular demonstraram que cPPL e pPPL apresentam proteases contaminantes e, portanto, atividades esterásica e amidásica. Em conjunto, esses resultados indicam que tais proteases, e não a PPL, possam ser os catalisadores majoritários da síntese da ligação peptídica quando ésteres de N α -acil-L-aminoácidos e preparações comerciais de PPL são usados. Por outro lado, tais dados não contradizem a possibilidade de usar cPPL como fonte barata de catalisadores para a síntese de dipeptídeos em condições reacionais brandas.Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH 2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when N α -blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.
Keywords: biocatalysis, enzymes, peptide bond synthesis, n-hexane
IntroductionIn the last decade, interest in environment friendly technologies has increased drastically. As a result, the ability of enzymes to catalyze reactions required for the manufacture of fine chemicals has been explored extensively. 1 Proteases that also present esterase activity have been widely used to catalyze reactions related to peptide synthesis, such as ester hydrolysis, esterification, transesterification and peptide bond formation. 2,3 However, it is well known that the main concern in protease-catalyzed peptide bond synthesis is protease-catalyzed product consumption. [4][5][6] Therefore, the following approaches have been used to minimize such an undesirable r...
