[24] Assaying activity and assessing thermostability of hyperthermophilic enzymes

Abstract

There is now a wide variety of intra-and extra-cellular enzymes available from organisms growing above 75°C, and having sufficient stability to allow assay well above this temperature. For some of these enzymes, to assay below even 95°C will involve measurement below the optimal growth temperature for the organism. The purpose of this chapter is to cover practical aspects of enzyme assay procedures that are specific to high temperatures. Since by far the commonest routine assessment of enzyme stability is activity loss, and because it is always unwise to measure enzyme activity without being confident of its stability during the assay, we include an outline of procedures for measuring enzyme activity loss/stability at high temperatures.There are a number of useful reviews of the effects of temperature upon enzyme activity [1], and these apply as much to reactions at 100°C as at 37°C. However, enzymes stable at 100°C have a number of advantages as research subjects. For example, they can be used to investigate enzymes that are particularly unstable when isolated from mesophiles, to slow reactions without the use of cryosolvents, and to probe the effect of temperature on enzyme and protein behavior over a very wide temperature range. They can also be used to study enzyme behavior under conditions that would denature most enzymes, since enzymes resistant to heat are also, at room temperature, resistant to organic solvents, 2 chaotropic agents, and proteolysis. Clearly, such stable enzymes also have a variety of applications in biotechnology, where protein stability may be an important practical and economic factor.Enzyme activity increases with temperature, usually by a factor of 1.4 -2.0 per 10°C, depending upon the Arrhenius activation energy. As the temperature is raised, at some point the enzyme will begin to denature during the assay. The extent will depend upon the stability of the enzyme and the temperature, but also on the duration of the assay, and on factors such as the buffer composition and the degree of stabilisation of the enzyme by substrate/cofactor, etc. This combination of activity acceleration and increasing denaturation with temperature has unfortunately led to the production of activity versus temperature graphs that exhibit a so-called "temperature-optimum" peak for the enzyme. The position of this peak can of course be shifted by tens of degrees by varying the assay duration, and these graphs give little biochemical information above the temperature at which denaturation becomes significant during the assay. As a tool to aid in the development of enzyme technology processes, with the assay duration linked to the process time, these graphs may be useful in comparing enzymes. Their use should otherwise be avoided, as should the term "temperatureoptimum" derived from them.However, there is recent evidence [2] that some enzymes may have genuine temperature optima. That is, at some point in the temperature profile, an enzyme may actually become less active as the temperature is raised, but this is...

Structural and dynamical features contributing to thermostability in α-amylases

“…The temperature dependence and the apparent T opt principally depend on the procedures used to assay enzymatic activity (buffers, heat rate and incubation times, thermostability of the substrate etc. ; see for example [53]). However, temperaturedependent activity plots with data taken from the literature allow at least a qualitative comparison (see fig.…”

Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23

“…One use of these measurements is to determine the temperature of maximal enzyme activity, which is an important parameter in characterizing the thermal adaptation process (Fitter et al 2001;D'Amico et al 2003). The temperature dependence and the apparent T opt principally depend on the procedures used to assay enzyme activity (Daniel and Danson 2001). However, temperature-dependent activity plots with data taken from the literature allow at least a qualitative comparison (Fitter 2005).…”

The dependence of enzyme activity on temperature: determination and validation of parameters

“…• C, and since few continuous assay methods are practical at such temperatures, most such assays will have to be discontinuous [4]. Finally, we have demonstrated that the use of initial, zero-time rates enables the ready determination of the Equilibrium Model parameters (except G ‡ inact ) of most non-ideal enzyme reactions.…”

“…Assays at high temperature (and over any wide temperature range) can sometimes pose special problems and may need additional care [4][5][6]. Quartz cuvettes were used in all experiments for their relatively quick temperature equilibration and heat-retaining capacity.…”