Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: Conformational changes that lead to switch in enzyme stereoselectivity

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• 作者：Galina A. Petkova^a ; Kamil Zá ; ruba^a ; Vladimí ; r Krá ; l^a ; ^b ; ^{vladimir.kral@vscht.cz}
• 关键词：Silica nanoparticle ; Alcohol dehydrogenase ; Immobilization ; Spectroscopy ; Unfolding ; Switch in enzyme stereoselectivity
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2012
• 期刊代码：161_15709639
• 类别：bio
• 出版时间：June, 2012
• 卷：1824
• 期：6
• 页码：792-801
• 文件大小：954 K

摘要

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 222 nm indicating a 伪-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222 nm 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.