Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose (original) (raw)
2006, Journal of Biotechnology
Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20 h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD +. In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD +-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.
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Process Biochemistry, 2012
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Methods in molecular biology, 2013
Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme-support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure-activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 • C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.
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Enzyme and Microbial Technology, 2000
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Bioorganic Chemistry, 1986
The holoenzyme crystals of horse liver alcohol dehydrogenase-NADH-DMSO complex were crosslinked with glutaraldehyde. A coupled activity test with ethanol and cinnamaldehyde as substrates was performed on the crosslinked enzyme crystals. The enzymatic activity was preserved and the coenzyme was found to be firmly bound to the enzyme crystals. These crystals can be used as redox catalysts with no addition of coenzyme. The crosslinked crystals were more stable toward dimethoxyethane than the enzyme in solution. Zinc ion salts reinforced this stability. Thirty percent of the initial activity was found in a medium containing 84% (v/v) organic solvents. 0 1986 Academic Press, Inc.
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