Investigation of the effect of freezing on protease-catalyzed peptide synthesis using cryoprotectants and frozen organic solvent.
In order to investigate the effect of freezing on aqueous protease-catalyzed peptide synthesis systems, the influence of polyethylene glycols as cryoprotecting substances on alpha-chymotrypsin-catalyzed coupling of a N-protected acyl donor ester and various nucleophilic amino components was studied. Changes in S'-specificity of alpha-chymotrypsin in frozen aqueous systems were suppressed by polyethylene glycols even at concentrations below 1% (w/v). Furthermore, the influence of freeze-concentration in organic solvents on protease-catalyzed peptide synthesis was investigated for the first time. In frozen tert-butanol, alpha-chymotrypsin-catalyzed peptide synthesis took advantage from freeze-concentration, but in contrast to frozen aqueous systems, no changes in S'-specificity of the biocatalyst were observed. The results suggest that freeze-concentration is not the only cause of freezing-induced yield improvement in aqueous peptide synthesis systems, but interactions between enzyme and ice structures strongly contribute to the observed effects.
Haensler
M
M
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
Arnold
K
K
D002384:Catalysis; D002918:Chymotrypsin / Q000378:metabolism*; D005615:Freezing; D009930:Organic Chemicals; D010452:Peptide Biosynthesis; D012997:Solvents
D009930:Organic Chemicals; D012997:Solvents; D002918:Chymotrypsin; C117608:alpha-chymotrypsin