Thermophiles dehydrogenase

Dehydrogenases are usually multimeric enzymes. Thus, under certain conditions even thermophilic dehydrogenases may be easily inactivated by subunit dissociation, being these enzymes an excellent target for the development of stabilization strategies by immobilization and post-immobilization modification techniques as shown in Fig. 6.4.5. 

To test the hypothesis that the conformational flexibility of the thermophilic enzyme is lower at room temperature than at higher temperatures, Kohen and Klinman measured, by FTIR, the time course of H/D exchange of protein N-H sites in deuterium oxide for the thermophilic alcohol dehydrogenase. Their measurements were made at the optimal host-organism temperature of 65 °C and at 25 °C, below the transition temperature. They also included yeast alcohol dehydrogenase at 25 °C, which is the optimal temperature for its own host organism. 

Kohen, A., Cannio, R., Bartolucci, S. and Klinman, J.P. (1999). Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase. Nature 399, 496-499... 

Kohen, A. and Klinman, J.P. (2000). Protein flexibility correlates with degree of hydrogen tunneling in thermophilic and mesophilic alcohol dehydrogenases. J. Am. Chem. Soc. 122, 10738-10739... 

Glucose was determined by the glucose oxidase-peroxidase method. Cellobiose (liberated enzymatically from methylcellotrioside) was determined in a coupled assay using cellobiose dehydrogenase from Sporotrichum thermophile (4). 

V. Rehaver and R. Jaenicke, Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyper-thermophilic eubaderium Thermotoga maritima, J. Biol. Chem. 1992, 267, 10999-11006. 

The results of the temperature dependence of the reaction rates of the enantiomers of secondary alcohols with a secondary alcohol dehydrogenase (SADE1) from the thermophilic bacterium Thermoanaerobacter ethanolicus demonstrated a temperature-dependent reversal of stereospecificity (Pham, 1990) (Figure 5.16). At T < 26°C, (S)-2-butanol was a better substrate than (i )-2-butanol on the basis of kCSLt/KM values however, at T> 26°C, (R)-2-butanol was a better substrate than (S)-2-butanol. (S)-2-Pentanol was the preferred substrate at T < 60°C however, the data predict that (i )-2-pentanol would be preferred at T > 70°C. (S)-2-Elexanol was predicted to be the preferred enantiomer only at T > 240°C. Therefore, the concept of isoinversion temperature is as valid for enzyme reactions as for others only the range of catalytically accessible temperatures is smaller. 

T. Ohshima, C. Wandrey, M. Sugiura, and K. Soda, Screening of thermostable leucine and alanine dehydrogenases in thermophilic Bacillus strains, Biotechnol. Lett. 

D Auria S, Di Cesare N, Gryczynski Z, Gryczynski I, Rossi M, Lakowicz JR. A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor. Biochemical and Biophysical Research Communications 2000, 274, 727-731. 

Schmitz, R. A., Albracht, S. P. J., and Thauer, R. K., 1992, A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei, Eur. J. Biochem. 209 1013nl018. 

Kirino H, Aoki M, Aoshima M, Hayashi Y, Ohba M, Yamagishi A, Wakagi T, Oshima T. Hydrophobic interaction at the subunit 30. interface contributes to the thermostability of 3-isopropyhnalate dehydrogenase from an extreme thermophile, Thermus ther-mophilus. Eur. J. Biochem. 1994 220 275-281. 31. 

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