Properties and stabilization of an extracellular α-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN 1: enzyme activity at 130°C

Abstract

An extracellular α-glucosidase from the thermophilic archaebacterium

The denaturation and degradation of stable enzymes at high temperatures

“…Even though this work was carried out on small peptides, it is difficult to envisage proteins that are stable enough to withstand this temperature. The most stable proteins found so far have half-lives in excess of 10 min at 130 mC [8], and greater stabilities have been shown for very stable enzymes immobilized and in the presence of stabilizing agents [9,11], so that prospects exist for characterizing denaturation and degradation up to 140 mC or so. Such stable proteins could also be starting points for attempts to engineer higher stability.…”

“…Techniques that restrain free movement of the enzyme at the molecular level, such as immobilization and intramolecular cross-linking, tend to stabilize enzymes [66], including enzymes from extreme thermophiles [9,11]. However, excessive flexibility is the first step towards denaturation, so although an enzyme must be sufficiently rigid to have a reserve of stability, it must also have the flexibility needed for effective catalysis.…”

“…As Figure 1 shows, enzymes are now available which, with or without stabilizing treatments or conditions, have half-lives in excess of 10 min at 130 mC. Furthermore, enzyme activity has been measured at these same high temperatures [8][9][10][11]. This is important because many observations of high-temperature enzyme stability have been performed simply by heating the enzyme, rapidly cooling a sample, and assaying for residual activity at a lower temperature (e.g.…”

Temperature and the catalytic activity of enzymes: A fresh understanding

“…A second implication concerns the stability of enzymes and attempts to manipulate this. Although enzymes are only marginally stable at the growth temperature of the source, we know they can be stable and functional at very high temperatures [21], and that the addition of a single productive stabilising interaction can greatly increase the half-life [22]. With this in mind, and given the considerable importance of stable enzymes in biotechnology and the substantial efforts in this field (published and unpublished), the attempts to increase the useful temperature of enzyme activity by directed mutagenesis have been, with some notable exceptions, disappointing.…”

Purificação e caracterização bioquímica da α-glicosidase termoestável produzida por Thermoascus aurantiacus CBMAI 756

“…Segundo Piller et al (1996), a α-glicosidase purificada da archeae extremófila Thermococcus AN1 apresenta pH e temperatura ótimos de 7,5 e 75°C. A α-glicosidase termoestável parcialmente purificada da bactéria Thermus thermophilus por Zdzieblo e Synowiecki (2002), possui pH ótimo 6,2 e retém 80% da atividade entre os pHs 5,8 e 6,9.…”