Alcalase-catalysed Syntheses of Hydrophilic Di- and Tri-peptides in Organic Solvents

1 Alcalase-catalysed Syntheses of Hydrophilic Di- and Tri-peptides in Organic Solvents 

Practical Methods for Biocatalysis and Biotransformations Edited by John Whittall and Peter Sutton 2009 John Wiley Sons, Ltd 

Alcalase (0.3 mL) (Novo Industrial, Denmark 2.5 AU mL ) anhydrous ethanol (2 mL) 

HPLC-mass spectrometry (MS). MS conditions ionization mode, atmospheric pressure ionization-electrospray (API-ES) polarity, positive Vcap, 4000 V nebulizer pressure, 35 psig drying gas, 10 L min gas temperature, 350 °C fragmentor, 70 V scan range, 120-600 atm. 

For pretreatment of the enzyme, alcalase (0.3 mL) and anhydrous ethanol (2 mL) were added to a centrifuge tube and the mixture was agitated for 5 min. The resulting mixture was centrifuged at 3000 rpm for 15 min to separate the enzyme from the solvent and the ethanol was removed by decantation. This procedure was repeated three times. 0.2 mL of a 0.1 m Na2C03-NaHC03 buffer solution (pH 10.0) was added to the pretreated enzyme obtained from 0.3 mL of untreated alcalase and incubated for 10 min at 45 °C. 

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