Determining Optimum Temperature and Ph for Enzymatic Reactions of Alpha Amylase

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Biochem. J. (1995) 305, 17-20 (Printed in Great Britain)



The effect of low temperatures
Nicole MORE, Roy M. DANIEL* and Helen H. PETACH

on enzyme


Thermophile Research Unit, University of Waikato, Private Bag 3105, Hamilton 2001, New Zealand

The stability of two enzymes from extreme thermophiles (glutamate dehydrogenase from Thermococcales strain ANI and f,-


glucosidase from Caldocellum saccharolyticum expressed in Escherichia coli) has been exploited to allow measurement of activity over a 175 °C temperature range, from + 90°C to -85 °C for the glutamate dehydrogenase and from + 90 °C to -70°C for the ,-glucosidase. The Arrhenius plots of these

and those for two mesophilic enzymes (glutamate dehydrogenase from bovine liver and ,)-galactosidase from Escherichia coli), exhibit no downward deflection corresponding to the glass transition, found by biophysical measurements of several non-enzymic mesophilic proteins at about -65 °C and reflecting a sharp decrease in protein flexibility as the overall motion of groups of atoms ceases.

It is now accepted that, in general terms, enzyme activity is dependent on protein dynamics [1-5]: in other words, an enzyme must be flexible to function. A number of biophysical studies [6-12] have shown that the flexibility of mesophilic proteins undergoes a transition at about -65 °C. Below this transition temperature the overall motion of groups of atoms within the protein ceases, and all that is left is the harmonic vibration of individual atoms. These results suggest that protein function will also cease at this temperature. The experiments which come closest to demonstrating this are those of Petsko's group [13], which show that, above the transition temperature, crystals of RNAase A rapidly bind the inhibitor cytidine 2'-monophosphate, but at -61 °C do not. The transition is sharp and is manifested by distinct changes in slope. Furthermore, if the transition, as expected, reflects a sharp decrease in the flexibility of the protein, then less-flexible proteins may undergo the transition at a significantly higher temperature. We know that the degree of flexibility of both mesophilic and thermophilic enzymes is similar at their respective growth temperatures [14-17], but at any given temperature the thermophilic enzyme is less flexible. It may be that, for an enzyme from an organism growing optimally at 75 °C, the transition will occur near -25 °C instead of -65 'C. This would be useful, because it is harder to measure enzyme activities accurately at lower temperatures. Given the exactly parallel behaviour of mesophilic and thermophilic proteins in terms of activity, flexibility and structure, thermophilic proteins are assumed to have a glass transition similar to that described for mesophilic proteins. We report here experiments designed to test whether enzyme activity shows the disproportionate decrease at low temperatures which we might expect from biophysical data, and whether such a decrease is seen at a higher temperature in an enzyme from an extreme thermophile. The effect of temperature on the activity of four enzymes has been determined: a 8-galactosidase from Escherichia coli growing at 37 'C, a 8-glucosidase from Caldocellum saccharolyticum (70 °C), and glutamate dehydrogenases from bovine liver (37 °C) and Thermococcus strain ANI (75 °C). The thermophilic enzymes in this study are hyperthermophilic

proteins because of their stability at temperatures far above the growth temperatures of the organisms.

EXPERIMENTAL Glucosidases The thermophilic fl-glucosidase was purified from an E. coli clone containing the ,8-glucosidase gene from C. saccharolyticum strain Tp8 [21]. The mesophilic ,-galactosidase from E. coli (catalogue no. G6008) was obtained from Sigma. The enzymes were assayed in 50% (v/v) dimethyl sulphoxide (DMSO) by nitrophenol release from an appropriate substrate (50 mM...
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