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0
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pH 8.3, half-life: 5 min
23
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pH 8.3, half-life: 5 min, essentially full protection against inactivation is provided by either 20% v/v glycerol or by simultaneous presence of Mn2+ and dithiothreitol, Mn2+ and beta-isopropylmalate or Mn2+ and alpha-ketoisocaproate
50
thermal stability curves of wild-type and mutant enzymes at pH 7.6 in presence of 0.5 mM EDTA, overview
60
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5 min, 92% loss of activity in absence of NaCl, 6% loss of activity in presence of 1.5 M NaCl
61
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midpoint of thermal unfolding curve, mutant enzyme A172D
63
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midpoint of irreversible denaturation
65
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midpoint of thermal unfolding curve, mutant enzyme A172V
66
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midpoint of thermal unfolding curve, mutant enzyme A172I and A172E
67
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midpoint of thermal unfolding curve, mutant enzyme A172L and A172F
70
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10 min, 40% loss of activity
79
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midpoint of irreversible denaturation
82
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10 min, 50% inactivation, mutant enzyme A31G/G43A/A709G
83
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10 min, 50% inactivation
85
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10 min, 50% inactivation, mutant enzyme G43A
85 - 95
thermal degeneration curves of wild-type and mutant enzymes at pH 7.6, overview
88
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Tm-value, ancIPMDH-IQ
90
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Tm-value, ancIPMDH-ML
64
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10 min, 50% inactivation
64
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midpoint of thermal unfolding curve, mutant enzyme A172G
87
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denaturation temperature is higher than 87°C
87
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clear correlation was observed between the hyrophobicity and the thermostability of the enzyme
87
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the thermophilic IMPDH was succesfully stabilized by replacing Ala172 with more hydrophobic residues like Glu, Asn and Gln without significant change in the enzymatic activity
87
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the extended hydrogen bonds within the beta-sheet are the major reason for the decreased thermostability of G240A
87
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10 min, 50% inactivation, wild-type enzyme, mutant enzyme C275T and mutant enzyme G376A
97
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melting temperature
97
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pH 7.0, 50% inactivation
additional information
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APS IPMDH denatures significantly at temperatures above 60°C and completely and irreversible loses activity at 70°C within 1 min, Teq: 67°C
additional information
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The thermal denaturation processes of wild-type and mutant enzymes are irreversible under the conditions used. Mutant M256F shows two-phase denaturation curve. The hydrophobic interactions at the subunit interface are critically important for the thermostability of dimeric enzyme.
additional information
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all mutant Bs3-isopropylmalateDHs and the wild-type enzyme are irreversibly inactivated by heat treatments. A linear correlation between thermal stability and hydrophobicity exists for those 3-isopropylmalateDHs containing position 253 residues with hydrophobic side chains
additional information
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two ancestral sequences of 3-isopropylmalate dehydrogenase (ancIPMDH-IQ and ancIPMDH-ML) are designed by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus
additional information
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additional information
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mutant enzyme L204F shows higher thermostability compared to wild-type enzyme, denaturation rates at 68°C and 70°C are slower for the mutant enzyme than for wild-type enzyme
additional information
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high concentrations of KCl considerably protect the enzyme from irreversible thermal denaturation
additional information
time-dependent denaturation-renaturation experiments on IPMDH are presented. Unfolding occurs in a single first-order step with half-times of 1 h. The binding of Mn*IPM (the manganese complex of 3-isopropylmalate) markedly reduces the rates of unfolding. Refolding is a two-step or multistep first-order process involving an inactive intermediate. The restoration of the native structure and reactivation take place with a half-time of a few min
additional information
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thermostabilization by LiCl, NaCl, KCl and to a lesser degree with RbCl, NH4Cl and CsCl