Crown ether activation of cross-linked subtilisin Carlsberg crystals in organic solvents

Abstract

The activity of cross-linked subtilisin Carlsberg crystals in the catalysis of peptide bond formation can be significantly enhanced by pretreatment of the enzyme crystals with crown ethers. Soaking of the enzyme crystals in a solution of crown ether in acetonitrile followed by evaporation of the solvent results in an up to 13 times enhanced enzymatic activity. The effects of crown ether treatment under various conditions gives support for the hypothesis that removal of bound water molecules from the active site during the drying process is the origin of the observed enzyme activation.

References

1. Enzymatic reactions in organic media, eds. A. M. P. Koskinen and A. M. Klibanov, Blackie academic & professional, Glasgow, 1996.
2. J. S. Dordick, Enzyme Microb. Technol., 1989, 11, 194.
3. (a) D. N. Reinhoudt, A. M. Eendebak, W. F. Nijenhuis, W. Verboom, M. Kloosterman and H. E. Schoemaker, J. Chem. Soc., Chem. Commun., 1989, 399; (b) J. Broos, M.-N. Martin, I. Rouwenhorst, W. Verboom and D. N. Reinhoudt, Recl. Trav. Chim. Pays-Bas, 1991, 110, 222; (c) J. Broos, I. K. Sakodinskaya, J. F. J. Engbersen, W. Verboom and D. N. Reinhoudt, J. Chem. Soc., Chem. Commun., 1995, 255; (d) J. Broos, R. Arends, G. B. van Dijk, W. Verboom, J. F. J. Engbersen and D. N. Reinhoudt, J. Chem. Soc., Perkin Trans. 1, 1996, 1415; (e) J. F. J. Engbersen, J. Broos, W. Verboom and D. N. Reinhoudt, Pure Appl. Chem., 1996, 68, 2171; (f) D. J. van Unen, J. F. J. Engbersen and D. N. Reinhoudt, Biotechnol. Bioeng., 1998, 59, 553.
4. N. L. St. Clair and M. A. Navia, J. Am. Chem. Soc., 1992, 114, 7314.
5. J. Partridge, G. A. Hutcheon, B. D. Moore and P. J. Halling, J. Am. Chem. Soc., 1996, 118, 12873.
6. K. Xu and A. M. Klibanov, J. Am. Chem. Soc., 1996, 118, 9815.
7. (a) R. A. Persichetti, N. L. St. Clair, J. P. Griffith, M. A. Navia and A. L. Margolin, J. Am. Chem. Soc., 1995, 117, 2732; (b) Y.-F. Wang, K. Yakovlevsky, B. Zhang and A. L. Margolin, J. Org. Chem., 1997, 62, 3488.
8. J. L. Schmitke, C. R. Wescott and A. M. Klibanov, J. Am. Chem. Soc., 1996, 118, 3360.
9. N. Khalaf, P. Govardhan, J. J. Lalonde, R. A. Persichetti, Y. F. Wang and A. L. Margolin, J. Am. Chem. Soc., 1996, 118, 5494.
10. G. Gokel, Crown ethers & cryptands, Royal Society of Chemistry, Cambridge, 1991.
11. In cross-linked crystalline thermolysin three out of eleven lysine residues still remain present. Altus Biologics, Personal communication.
12. T. Sakurai, A. L. Margolin, A. J. Russel and A. M. Klibanov, J. Am. Chem. Soc., 1988, 110, 7236.
13. A. Fersht, Enzyme structure and mechanism, 2nd edn., Freeman, New York, 1985.
14. (a) P. J. Halling, Enzyme Microb. Technol., 1994, 16, 178; (b) F. Secundo, S. Riva and G. Carrea, Tetrahedron: Asymmetry, 1992, 3, 267.
15. K. Ryu and J. S. Dordick, J. Am. Chem. Soc., 1989, 111, 8026.
16. P. A. Fitzpatrick, A. C. U. Steinmetz, D. Ringe and A. M. Klibanov, Proc. Natl. Acad. Sci. USA, 1993, 90, 8653.
17. F. de Jong, D. N. Reinhoudt and C. J. Smit, Tetrahedron Lett., 1976, 1371.
18. L. P. Golovkova, A. I. Telyatnik, V. A. Bidzilya, N. E. Akhmetova and V. I. Konovalova, Theor. Exp. Chem. (Engl. Transl.), 1985, 2, 248.
19. Our earlier studies revealed that it is indeed possible for 18-crown-6 to penetrate into the active site of proteases like subtilisin Carlsberg and α-chymotrypsin. It was observed that the hydroxymethyl-18-crown-6 ester of N-acetyl-D,L-phenylalanine could be ransesterified and hydrolyzed enantioselectively using respectively propan-1-ol in cyclohexane and an aqueous buffer. These studies proved that hydroxymethyl-18-crown-6 was located in the active site before it was expelled by the enzyme. J. Broos, Ph.D. Thesis, University of Twente, 1994.