Circular dichroism studies of subtilisin carlsberg immobilised on micron sized silica particles

P.J. Halling, A. Ganesan, N.C. Price, S.M. Kelly, I. Petry, B.D. Moore

Research output: Contribution to journalArticle

38 Citations (Scopus)

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.

LanguageEnglish
Pages1119-1125
Number of pages6
JournalBBA - Biochimica et Biophysica Acta
Volume1764
Issue number6
DOIs
Publication statusPublished - 2006

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dichroism
silicon dioxide
silica gel
flattening
holders
enzymes
cells
industries
detectors
electric potential
scattering

Keywords

  • immobilised enzyme
  • circular dichroism
  • subtilisin Carlsberg
  • silica gel
  • differential scattering
  • absorption flattening
  • secondary structure
  • organic media

Cite this

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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.",
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Circular dichroism studies of subtilisin carlsberg immobilised on micron sized silica particles. / Halling, P.J.; Ganesan, A.; Price, N.C.; Kelly, S.M.; Petry, I.; Moore, B.D.

In: BBA - Biochimica et Biophysica Acta , Vol. 1764, No. 6, 2006, p. 1119-1125.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Circular dichroism studies of subtilisin carlsberg immobilised on micron sized silica particles

AU - Halling, P.J.

AU - Ganesan, A.

AU - Price, N.C.

AU - Kelly, S.M.

AU - Petry, I.

AU - Moore, B.D.

PY - 2006

Y1 - 2006

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KW - immobilised enzyme

KW - circular dichroism

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KW - silica gel

KW - differential scattering

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