title: Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles.
abstract: Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 microm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.
authors.affiliation: Department of Pure and Applied Chemistry, 295 Cathedral Street, University of Strathclyde, Glasgow G1 1XL, UK.
authors.forename: Ashok
authors.initials: A
authors.lastname: Ganesan
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Price
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Kelly
authors.forename: Inga
authors.initials: I
authors.lastname: Petry
authors.forename: Barry D
authors.initials: BD
authors.lastname: Moore
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Halling
mesh_terms: D001407:Bacillus / Q000201:enzymology; D001703:Biophysics / Q000379:methods*; D002384:Catalysis; D002942:Circular Dichroism; D004800:Enzymes, Immobilized / Q000737:chemistry; D006868:Hydrolysis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012822:Silicon Dioxide / Q000737:chemistry; D013053:Spectrophotometry; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D004800:Enzymes, Immobilized; D012822:Silicon Dioxide; D013381:Subtilisins