Reaction medium engineering in enzymatic peptide fragment condensation: synthesis of eledoisin and LH-RH.
The influence of different reaction systems on alpha-chymotrypsin-catalyzed synthesis of eledoisin and LH-RH peptides from (7 + 4) and (5 + 5) fragments was investigated. The peptide yield was determined in the following systems: buffered aqueous media, frozen solutions, organic media, and cosolvent mixtures. The experimental set up was tailored to allow the screening of an array of conditions with minimum consumption of peptide fragments (2.1 and 2.5 mM). The best yields (22% yield for eledoisin and 68% yield for LH-RH) were obtained in buffered aqueous solutions. It was found that the choice of buffer had a strong influence on the peptide yield; boric-borate and ammonium acetate buffers at pH 9, gave the best results. In buffered aqueous systems, both syntheses were scaled up by using a 10-fold increase in fragment concentration (21 and 25 mM). Under these conditions the yields rose to 57% and 80% of eledoisin and LH-RH, respectively. Moreover, during the synthesis of eledoisin and in the presence of boric-borate buffer pH 9, the peptide precipitated from the reaction medium preventing a secondary hydrolysis and facilitating the in situ product purification.
Björup
P
P
Department of Peptide and Protein Chemistry, Centro de Investigación y Desarrollo-C.S.I.C., Barcelona, Spain.
Torres
J L
JL
Adlercreutz
P
P
Clapés
P
P
D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000737:chemistry*; D004600:Eledoisin / Q000138:chemical synthesis*; D004800:Enzymes, Immobilized / Q000737:chemistry; D005615:Freezing; D007987:Gonadotropin-Releasing Hormone / Q000138:chemical synthesis*; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry*; D012997:Solvents; D014867:Water
D002021:Buffers; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D012997:Solvents; D014867:Water; D007987:Gonadotropin-Releasing Hormone; D004600:Eledoisin; D002918:Chymotrypsin