Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: conformational changes that lead to switch in enzyme stereoselectivity
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
22472304
DOI
10.1016/j.bbapap.2012.03.010
PII: S1570-9639(12)00057-X
Knihovny.cz E-resources
- MeSH
- Aldehyde Reductase MeSH
- Aldo-Keto Reductases MeSH
- Alcohol Dehydrogenase chemistry MeSH
- Alcohol Oxidoreductases chemistry MeSH
- Circular Dichroism MeSH
- Enzymes, Immobilized chemistry MeSH
- Spectrometry, Fluorescence MeSH
- Liver enzymology MeSH
- Kinetics MeSH
- Coenzymes chemistry MeSH
- Horses MeSH
- NAD chemistry MeSH
- Nanoparticles chemistry ultrastructure MeSH
- Silicon Dioxide chemistry MeSH
- Protein Unfolding MeSH
- Protein Structure, Secondary MeSH
- Spectroscopy, Fourier Transform Infrared MeSH
- Stereoisomerism MeSH
- Substrate Specificity MeSH
- Protein Structure, Tertiary MeSH
- Tryptophan chemistry MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Aldehyde Reductase MeSH
- Aldo-Keto Reductases MeSH
- Alcohol Dehydrogenase MeSH
- Alcohol Oxidoreductases MeSH
- Enzymes, Immobilized MeSH
- Coenzymes MeSH
- NAD MeSH
- Silicon Dioxide MeSH
- Tryptophan MeSH
FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60-70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-(p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (V(max) and K(m)) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.
Biochim Biophys Acta. 2013 Aug;1834(8):1681 PubMed
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