Thermal Stability

A marked improvement in thermal stability as a result of sol-gel encapsulation has been observed for three flavoprotein oxidases: glucose oxidase, lactate oxidase (LOX), and glycolate oxidase (GLyOX) (13). When encapsulated in xerogels, where smaller pore sizes were likely to enhance electrostatic interactions between the silicate and the protein, glucose oxidase was reported to be stabilized by immobilization. The extent of the stabilization was impressive; the half-life at 63°C was increased 200-fold on sol-gel encapsulation compared to enzyme in water. Interestingly, LOX and GLyOX were initially destabilized by sol-gel encapsulation. The three oxidases have different pi values: 3.8 for glucose oxidase, 4.6 for LOX, and 9.6 for GLyOX. It was apparent that electrostatic interactions between the enzyme and charged silicate matrix caused the destabilization since both LOX and GLyOX experienced a dramatic improvement in stability if the enzymes were electrostatically complexed with a base prior to sol-gel immobilization. Once electrostatically complexed, LOX experienced a 150-fold increase and GLyOX a 100-fold increase in enzyme half-life at 63°C compared to enzyme in water (13). These findings show the benefit of increased stability as a result of encapsulation, yet they also under-

Fig. 6. Normalized enzyme activity of butyrylcholinesterase as a function of storage time at 4°C shows that activity was substantially better retained in the sol-gel-immobilized enzyme than in the free enzyme (enzyme in buffer solution).

score the importance of electrostatic interactions between biomolecules with the highly charged surfaces of the sol-gel-derived matrix.

Increased thermal stability has also been observed in the heme protein cyt-c (15). Thermally induced unfolding of proteins in solution, in general, exhibits a sharp transition over a small temperature range, and the transition point at which half of the molecules are denatured is termed Tm. For cyt-c, the unfolding can be monitored using optical absorption of the heme Soret band. As shown in Fig. 5, Tm for cyt-c in liquid buffer was approx 65°C, whereas Tm for cyt-c in the sol-gel matrix was at least 90°C. It is possible that the sol-gel-encapsulated cyt-c was stable beyond 90°C because boiling of the buffer led to gel cracking at approx 95°C.

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