Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy.
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 alpha-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.
Natalello
Antonino
A
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Ami
Diletta
D
Brocca
Stefania
S
Lotti
Marina
M
Doglia
Silvia M
SM
D000577:Amides / Q000737:chemistry; D002175:Candida / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry; D006031:Glycosylation; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D000577:Amides; D005656:Fungal Proteins; D012996:Solutions; D002265:Carboxylic Ester Hydrolases; D008049:Lipase