Role of organic solvents on Pa-hydroxynitrile lyase interfacial activity and stability

Catalytic activity and adsorption of Pa-hydroxynitrile lyase (Pa-Hnl) was i nvestigated at various organic solvent/water interfaces. We focused on the role of solvent polarity in promoting activity and stability in two-phase s ystems, specifically for the solvents heptane, dibutyl ether (DBE), diisopr opyl ether (DIPE), butylmethyl ether (BME), and methyl tert-butyl ether (MT BE). Enzyme activity towards mandelonitrile cleavage was determined in a re cycle reactor with a well-defined interfacial area as described by Hickel, et al. 1999. Here the recycle reactor was modified to permit exchange of th e aqueous phase. With this modification, irreversibility of enzyme adsorpti on was determined as a function of the adsorption time at the interface. Ir reversibility of enzyme adsorption was also investigated by measuring the s urface pressure of a sessile-drop upon washout. We find that Pa-Hnl exhibit s the highest stability but the lowest initial catalytic activity at the aq ueous/organic solvent interface with the most polar organic solvents. Thus, DIPE and MTBE display no loss in enzyme activity over a period of several hours. However, the more apolar the solvent is the higher the initial Pa-Hn l activity, but the faster the loss of activity. Dynamic tensiometry reveal s that Pa-Hnl adsorbs more strongly at the interface of the more apolar sol vents. Surprisingly, Pa-Hnl develops some irreversible adsorption after 30 min at the DIPE/water interface, but does not lose catalytic activity. (C) 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 18-28, 2001.