期刊
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
卷 65, 期 1-4, 页码 73-78出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.molcatb.2010.01.028
关键词
Formate dehydrogenase (FDH); Ionic liquid; Chemical modification; Stability
The formate dehydrogenase (FDH, EC: 1.2. 1.2) from Candida boidinii was found to be inactivated and unstable in the presence of high concentration (>50%) of the water soluble dimethylimidazolium dimethyl phosphate ([MMIm][Me2PO4]) ionic liquid. In order to circumvent this problem, the enzyme was chemically modified by cations usually present in ionic liquids: cholinium (1), hydroxyethyl-methylimidazolium (2) and hydroxypropyl-methylimidazolium (3) cations were activated with carbonyldiimidazole before being reacted with the FDH leading to a heterogeneous population of 6-7 biocatalysts. FDH modified by (1) or (3) led to 3-9 modifications while FDH modified by (2) led to 6 proteins presenting 7-12 grafted cations. Specific activity of the modified enzymes was decreased by a 2.5-3-fold factor (0.10-0.15 mu mol min(-1) mg(-1)) compared to the non-modified FDH (0.33 mu mol min(-1) mg(-1)) when assayed in carbonate buffer (pH 9.7, 25 mM). After modification, the FDH still present 0.06 mu mol min(-1) mg(-1) in 70% [MMIm][Me2PO4] (v:v) (30-45% of their activity in aqueous buffer) while the native enzyme is inactive at this ionic liquid concentration, proving the efficiency of this strategy. The half-life of the modified enzyme is also increased by a 5-fold factor after modification by (1) (t(1/2) of 9 days) and by a 3-fold factor after modification by (2) or (3) (t(1/2) of 6 and 5 days respectively) in aqueous solution. When stored in 37.5% [MMIm][Me2PO4] (v:v), both modified and unmodified FDH have an increased half-life (t(1/2) of 6-9 days). This grafting strategy is found to be good methods to mimic and study the stabilizing effect of ionic liquids on enzymes. (C) 2010 Elsevier B.V. All rights reserved.
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