Enzymes, thermophilic

The thermophilic enzyme DszD from Paenibacillus All-2 has been cloned into E. coli and characterized [172], The sequence of this enzyme showed 30% similarity to the major flavin reductase of Vibrio fischeri. The optimum activity was reported to be at 45°C in resting cell cultures and 55°C in cell-free extracts. 

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. 

The books, however, cannot yet be closed. Although the flexibilities of the yeast enzyme at 25 °C and thermophilic enzyme at 65 °C are similar, and although both show unmistakeable evidence of tunneling, the nature of the tunneling process appears to be different. This is another instance in which the temperature dependences of the isotope effects generate a complex and ill-understood picture. 

Increase thermostability and environmental compatibility Increase specificity and activity Develop thermophilic enzymes screen for new microbes clone and overexpress in industrial hosts site-direct mutagenesis... 

Thermophilic enzymes are active and stable at high temperature (> 60 C) but they are generally inactive and extremely stable at low temperature (< 25 C). The molecular basis has not been elaborated to explain such thermophilicity. In general, thermophilic enzymes do not denature at high temperature and their activity is higher due to the Qio rule where a 10°C increase results in a doubling of chemical activity. 

Porcelli, M., Cacciapuoti, G., Fusco, S., et al. Non-thermal effects of microwaves on proteins thermophilic enzymes as model system. FEBS Letters 1997 402, 102-6. 

Employing a multichannel PDMS microreactor [350 gm (wide) x 250 gm (deep) x 6.4 mm (long)], in which the thermophilic enzyme (3-glycosi-dase was immobilized, Thomsen et al. (2007) evaluated the hydrolysis of 2-nitrophenyl-p-D-galactopyranoside. Heating the reactor to 80 °C, the authors were able to continuously hydrolyze 2-nitrophenyl-p-D-galactopyranoside and monitored the reaction efficiency via generation of 2-nitrophenol 97. 

Recently, the primary structure of Thermophilic bacillus AspAT was deduced from its cDNA sequence.24 However, there was no significant sequence homology found between thermophilic enzyme and other AspAT s. After careful comparison among primary structures of AspAT s, we found that catalytically essential amino acid residues located at the active site in AspAT were also assigned in the thermophilic enzyme (Fig. 1.2). Although thermophilic AspAT is presumed to have an entirely different tertiary structure from other AspAT s, catalytically essential amino acid residues may be conserved in its structure. 

Comparisons of psychrophilic, mesophilic, and thermophilic enzymes suggest that a continuum of adjustments accompany adaptation to different temperatures (Davail et al., 1994 Feller and Gerday, 1997). Relative to mesophiles, the same kinds of weakly stabilizing interactions that are found in greater proportion in thermophilic enzymes appear in fewer numbers in their psychrophilic counterparts. 

Fig. 3. Homologous enzymes adapted to different temperatures show a trade-off between catalytic activity at low temperatures (high for enzymes from psychrophilic organisms, but generally low for enzymes from thermophiles) and thermostability (high for thermophilic enzymes, but low for enzymes from psychrophiles). These natural enzymes lie in the darker shaded area, which is bounded on one side by the minimal stability and activity required for biological function. Enzymes that are both highly thermostable and highly active at low temperature (lighter shaded area) are generally not found in nature.

The high thermostability of natural thermophilic enzymes is usually accompanied by increased resistance to other forms of denaturation, such as cleavage by proteases and chemical denaturation by guanidine hydrochloride or urea. A similar trend is seen in the evolved thermostable esterases. Thermostable 8G8 is more resistant than wild type to cleavage by trypsin and to denaturation by guanidine hydrochloride (Gershenson et al, 2000). 

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