Can conformational changes be responsible for solvent and excipient effects on the catalytic behavior of subtilisin Carlsberg in organic solvents?

Griebenow, Kai; Klibanov, Alexander M.

doi:10.1002/(sici)1097-0290(19970220)53:4<351::aid-bit1>3.0.co;2-m

Cited by 123 publications

(106 citation statements)

References 42 publications

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“…The resulting spectra from both these measurements were similar; with amide I band maxima~1655 cm À1 in agreement with the literature. [19] This indicates that spectral acquisition employing the DRIFT method has little influence on the measurement outcome. Having confidence in the technique, we proceeded to study DRIFT spectra of the same lyophilised SC preparation simply mixed with finely ground KBr.…”

Section: Diffuse Reflectance Infrared Fourier Transform Spectroscopymentioning

confidence: 99%

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

et al. 2009

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Structure of immobilised enzymes: Circular dichroism, infrared and fluorescence spectroscopic methods are used to characterise in situ structural stability of enzymes immobilised on particles. The enzyme subtilisin Carlsberg exhibits a similar secondary structure in solution and in the immobilised state but on the surface of porous silica gel a rigid tertiary structure is preferred (see picture). We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm−1 to 1632 cm−1, indicating a shift from α‐helical structure to β‐sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to β‐sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

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Section: Diffuse Reflectance Infrared Fourier Transform Spectroscopymentioning

confidence: 99%

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

et al. 2009

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“…It might promote structural changes in the protein. The relatively limited study here suggests that any changes are quite small over the lower water activity range, consistent with the idea that the structure is quite rigid (Griebenow & Klibanov 1997 The dashed line shows the effect on catalytic activity. (Partridge et al (1998).…”

Section: How Does Hydration Promote Catalytic Activity?mentioning

confidence: 99%

What can we learn by studying enzymes in non–aqueous media?

Halling

2004

Phil. Trans. R. Soc. Lond. B

110

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What is the role of water in enzyme structure and function? One approach to answers should come from studies in which the amount of water present is a variable. In the absence of bulk liquid water, effective monitoring of enzyme action requires an alternative fluid medium through which substrates and products may be transported. The past 20 years have seen quite extensive study of enzyme behaviour when reactants are transferred via a bulk phase that is an organic liquid, a supercritical fluid or a gas. Some lipases, at least, remain highly active with only a few, if any, residual water molecules. Many enzymes seem to require larger amounts of water, but still not a liquid water phase. There are hysteresis effects on both the amount of bound water and the observed catalytic activity. Increasing hydration promotes mobility of the enzyme molecule, as revealed by various techniques, and there are correlations with catalytic activity. There are other plausible roles for hydration, such as opening up proton conduction pathways.

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“…Although the use of organic solvents in the formulation process deactivates biological activity of the encapsulated protein to a great extent, they are unavoidably required to prepare the protein encapsulated PLGA microspheres [20]. Klibanov and co-workers have shown that the conformation of solid proteins in organic solvents is kinetically trapped [21]. Thus, protein unfolding is extremely slow under these preparation conditions.…”

Section: Resultsmentioning

confidence: 99%

Preparation of “Open/closed” pores of PLGA-microsphere for controlled release of protein drug

et al. 2018

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Poly(D,L-lactic-co-glycolic acid) has been extensively used as a controlled release carrier for drug delivery due to its good biocompatibility, biodegradability, and mechanical strength. In this study, porous PLGA microspheres were fabricated by an emulsion-solvent evaporation technique using poly ethylene glycol (PEG) as an extractable porogen and loaded with protein (lysozyme) by suspending them in protein solution. For controlled release of protein, porous microspheres containing lysozyme were treated with water-miscible solvents in aqueous phase for production of pore-closed microspheres. The surface morphology of microspheres were investigated using scanning electron microscopy (SEM) for confirmation of its porous microstructure structure. Protein property after release was observed by enzymatic activity assay. The pore-closing process resulted in nonporous microspheres which exhibited sustained release patterns over an extended period.

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Can conformational changes be responsible for solvent and excipient effects on the catalytic behavior of subtilisin Carlsberg in organic solvents?

Cited by 123 publications

References 42 publications

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

What can we learn by studying enzymes in non–aqueous media?

Preparation of “Open/closed” pores of PLGA-microsphere for controlled release of protein drug

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