Biochem. J. (2005) 385
(511–517) (Printed in Great Britain)
Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy
Antonino NATALELLO*†, Diletta AMI*†, Stefania BROCCA*, Marina LOTTI* and Silvia M. DOGLIA*†1
*Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy, and †Istituto Nazionale per la Fisica della Materia, Unità di Milano-Bicocca, Via Cozzi 53, 20126 Milano, Italy
The secondary structure of lipase 1 from Candida rugosa, a model system for large monomeric enzymes, has been studied by FTIR (Fourier-transform infrared) spectroscopy in water and 2H2O. The secondary structure content, determined by the analysis of the amide I band absorption through second derivative and curve fitting procedures, is in agreement with that estimated by X-ray data and predicts, in addition, the existence of two classes of a-helices. We have also investigated the enzyme stability and aggregation at high temperature by following the protein unfolding. The thermal stability determined by FTIR is in excellent agreement with the temperature dependence of the lipase activity. Furthermore, new insights on the glycosylation of the recombinant protein produced in Pichia pastoris and on its heterogeneity related to different fermentation batches were obtained by the analysis of the IR absorption in the 1200-900 cm-1 carbohydrate region. A drastic reduction of the intensity of this band was found after enzymic deglycosylation of the protein. To confirm that the FTIR absorption in the 1200–900 cm-1 region depends on the carbohydrate content and glycoform distribution, we performed an MS analysis of the protein sugar moieties. Glycosidic structures of the high mannose type were found, with mannoses ranging from 8 to 25 residues.
Key words: Candida rugosa lipase, conformational stability, heterologous protein glycosylation, infrared spectroscopy, MS, secondary structure.
Abbreviations used: CRL1, Candida rugosa lipase 1; FSD, Fourier-self-deconvolution; FTIR, Fourier-transform infrared; MALDI-MS, matrix-assisted laser-desorption ionization MS; NAG, N-acetylglucosamine; PNGase F, peptide N-glycosidase F.
1To whom correspondence should be addressed (email silviamaria.doglia@unimib.it).
Received 30 July 2004/9 September 2004; accepted 14 September 2004
Published as BJ Immediate Publication 14 September 2004, DOI 10.1042/BJ20041296
The Biochemical Society, London ©2005
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