An interesting question concerning the conformation of enzymes in the lyophilised form is whether the structure is native or corresponds to a reversible denatured form [1]. Freezing and dehydration in the lyophilisation process might, in fact, alter enzyme conformation, because water is essential for native structure. Fourier-transform infrared (FT-IR) spectroscopy is a tool useful to study secondary structure of proteins in solution, suspension and solid state, and Griebenow and Klibanov [1] and Prestrelski et al. [2,3] have employed FT-IR to quantify conformational changes following lyophilisation of some proteins. Two regions of IR spectrum called amide I (1600 to 1700 cm-1) and amide III (1215 to 1335 cm-1) have been used to study the individual elements of secondary structure. Although the intensity of amide III band is small, it presents some advantages: 1) it may be analysed in H2O, 2) single bands corresponding to a-helix and b-sheets show well differentiated frequencies and 3) problems accompanying NH/ND exchange are avoided. We have employed the IR amide III band region to investigate the conformational aspects of Candida antarctica lipase B and Pseudomonas cepacia lipase both in aqueous solution and as lyophilised protein in KBr pellets. These enzymes are of notable practical interest since are widely used for synthetic purposes, in the lyophilized state, to catalyse esterification and transesterification reactions in organic solvents. FT-IR spectra were measured with a Jasco 610 instrument. The protein solution spectra were recorded at 15 mg/ml, using a 18 mm CaF2 cell while lyophilised proteins were analysed in KBr pellets. The curve fitting of the amide III band was performed by Grams/32 Galactic program. The table reports the percentages of elements of secondary structure for lipases in solution and lyophilised. We can observe a good correspondence of FT-IR data in solution with those by X-ray, while after lyophilisation the lipases undergo a marked change in secondary structure: the a-helix content decreases and b-sheets content increases. Such behaviour may be due to the formation of intermolecular b-sheets structures. It should be noted that PEG prevents protein conformational modifications during the lyophilisation process [3].

[1] Griebenow, K., Klibanov, A.M., Proc. Natl. Acad. Sci. USA 92, 10969-10976,1995.

[2] Prestrelsky, S.J., Tedeschi, N., Arakawa, T., Carpenter, J.F., Biophys. J. 65, 661-671, 1993.

[3] Vecchio, G., Zambianchi, F., Zacchetti, P., Secundo, F., Carrea, G. Biotechnol. Bioeng., 64, 545-551, 1999.