Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

Abstract

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (±)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V 0 , ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38°C and 56°C, respectively). No relationship between this property and V 0 was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V 0 was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of sub-tilisin activation by crown ethers, as recently suggested.

Reflections on Charge State Distributions, Protein Structure, and the Mystical Mechanism of Electrospray Ionization

“…In fact, several applications in industry have been used to generate kilograms of product in this fashion [10]. Instinctively, one might assume that proteins would be denatured in organic solvents; however, experimental characterization, though limited, has suggested that this is not the case [12,13]. Furthermore, the ultimate test for structural fidelity with proteins is generally considered to be functionality, which can obviously be retained and utilized in organic media.…”

On the relationship between the activity and structure of PEG‐α‐chymotrypsin conjugates in organic solvents

“…There have been a number of studies focusing on increasing the activity of enzymes suspended in organic solvents, for example by controlling the pH-value of the solution from which the enzyme is prepared (Carrea and Riva, 2000;Costantino et al, 1997;Gupta, 2000;Klibanov, 1986;Yang et al, 1993;Zaks and Klibanov, 1985); enzyme lyophilization in the presence of lyoprotectants (various sugars) (Belton and Gil, 1994;Carpenter and Crowe, 1989;Dabulis and Klibanov, 1993;Griebenow and Klibanov, 1997), salts (Khmelnitski et al, 1994;Ru et al, 2000;Sasaki and Kise, 1999), crown ethers (Bross et al, 1995;Griebenow et al, 2001;Reinhoudt et al, 1989;Santos et al, 2001;Van Unen et al, 1998, cyclodextrins (Griebenow et al, 1999a;Montañez et al, 2002;Ooe et al, 1999;Santos et al, 1999), ligands (imprinting with substrates and substrates analogs) (Russell and Klibanov, 1998), crown ether modified peptides (Tremblay et al, 2005); employing cross-linking enzymes crystals (CLECs) (Triantafyllou et al, 1997;Wang et al, 1997); immobilization (Kwon et al, 1999;Suzawa et al, 1995), and hydrophobic ion-pairing (Kendrick et al, 1997;Meyer et al, 1996).…”

Immobilization of Candida antarctica Lipase B on fumed silica

“…Much effort has been focused on strategies to overcome this issue, including enzyme immobilization on porous and non-porous solid supports 12,13 , chemical modification of the enzymes' surfaces to improve compatibility with solvents 14 , protein engineering 2 , and enzyme co-lyophilization with different types of excipients, such as cyclodextrins 10,15 , crown ethers [15][16][17] , and inorganic salts [18][19][20] . Specifically, the co-lyophilization of enzymes with inorganic salts from aqueous solution, termed salt activation, has been remarkably successful.…”