Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA

Abstract

The advent of thermostable enzymes has led to great advances in molecular biology, such as the development of PCR and ligase chain reaction. However, isolation of naturally thermostable enzymes has been restricted to those existing in thermophylic bacteria. Here, we show that the disaccharide trehalose enables enzymes to maintain their normal activity (thermostabilization) or even to increase activity at high temperatures (thermoactivation) at which they are normally inactive. We also demonstrate how enzyme thermoactivation can improve the reverse transcriptase reaction. In fact, thermoactivated reverse transcriptase, which displays full activity even at 60°C, was powerful enough to synthesize full length cDNA without the early termination usually induced by stable secondary structures of mRNA.The usefulness of thermostable enzymes is indisputable; they allowed the development of outstanding techniques such as PCR and ligase chain reaction (1, 2). However, the isolation of thermostable enzymes has been restricted to those existing in thermophylic organisms. To expand the availability of thermostable enzymes, we explored a completely new way-the thermal stabilization of those enzymes that are normally thermolabile by the addition of structure stabilizing molecules. In particular, we explored the properties of molecules that are normally involved in heat shock response, and we anticipated being able to confer thermal stability to enzymes. Among them, there is the disaccharide trehalose. Trehalose synthesis is induced by heat shock in the Saccharomyces cerevisiae yeast (3), suggesting its possible role in this response and in desiccation tolerance (4). In fact, yeast mutants defective in trehalose synthesis show a significant reduction in thermotolerance (5). It has been reported that enzymes could be protected from irreversible heat aggregation-heat denaturation in vitro by trehalose, suggesting its chaperonin-like function (6). Trehalose also has been used to confer stability to dried enzymes (7).In the present study, we discovered that trehalose can be used as a reaction additive to stabilize or stimulate enzymatic activity at unusually high temperatures, enabling the use of thermosensitive enzymes as though they would be thermostable. This property should be useful for converting a wide range of thermosensitive enzymes to thermostable and thermoactive ones for wide applications in biological, medical, and industrial fields.To show the power of trehalose-mediated thermal activation, we subsequently applied this new method to the synthesis of full length cDNA. The major obstacle to preparing high quality cDNA libraries has been the low efficiency of reverse transcriptase (RT) to synthesize full length cDNA, which is due to the strong secondary structure of mRNA, which cause the RT to stop the synthesis and subsequently to be released from the hybrid mRNA͞incomplete cDNA. To overcome problems associated with the secondary structure of mRNA, both denaturing of sample before the reaction and increased temper...

Why Is Trehalose an Exceptional Protein Stabilizer?

“…Although the surface tension of water increases to a much larger extent for certain simple electrolytes and carboxylic acid salts (26) compared with trehalose at identical concentrations, it seems that nature has preferred trehalose over these salts as their charged nature could have inhibitory effect on the activity of enzymes. 2 These data suggest why trehalose has been selected by nature as an exceptional stabilizing agent under various stress conditions. Table I shows clearly that trehalose does not affect the stability of various proteins to the same extent.…”

“…Unlike trehalose, other mono-and disaccharides and several of the polyols have not been observed to provide thermostabilization and thermoprotection of proteins to such an extent as trehalose (1,2,10,11). This is essentially due to the differences in their cosolvent molecular structure and their solution physico-chemical properties as described earlier.…”

“…It is a compatible osmolyte that gets accumulated in organisms under stress conditions (12, 13). Because of this unique property, tremendous interest has been generated in understanding the molecular basis of stress management through induction of trehalose biosynthesis (13, 14).Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15, 16) and exposure to high temperatures in solution (2,8,9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15, 17).…”

“…Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15, 16) and exposure to high temperatures in solution (2,8,9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15, 17).…”

“…Different proteins are expected to interact with cosolvent molecules in varied ways depending on their physicochemical properties. In general, trehalose has been observed to provide protection to different proteins to various extents and the efficacy of protection depends on the nature of the protein (2,4). Despite the availability of such data, the exact role of proteins and their physico-chemical properties in trehalose-mediated stability is still not clear.…”