Assaying activity and assessing thermostability of hyperthermophilic enzymes.

1. Introduction 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 …