Effect of Elevated Temperature and Increase in Activity Following Heating

CK in sol-gel monoliths and buffer solutions were stored at 37, 47, and 60°C for varying lengths of time to evaluate enzyme stability. At the elevated temperatures, for both monoliths and solution, there was a faster loss in CK activity as the temperature was increased. There was, however, significantly higher activity for the sol-gel-encapsulated CK compared with CK in solution at all temperatures. For example, at 60°C, no activity was observed in the CK solution after 1 h, whereas the sol-gel-immobilized enzyme still retained 50% activity after 5 h of heating. Similarly, at 47°C, activity in the solution dropped to essentially zero after about 1 d, whereas the sol-gel-immobilized enzyme retained 50% of its activity after 5 d of heating. Furthermore, the activity remained at approx 50% of its maximum value after 12 d. Figure 8 shows a comparison of CK activity in both sol-gel monolith and buffered solution after heating at 47°C.

One unusual and unexpected observation from our results was that CK solgel monoliths that were heated exhibited a sharp initial increase in activity, which was then followed by a gradual decrease. This phenomenon was observed at all elevated temperatures (i.e., 37, 47, and 60°C). The increase in

Fig. 8. (A) Relative activity as a function of time for CK heated at 47°C in solution (•) and in sol-gel silica (■) shows that the activity in solution decreases immediately, but the activity in sol-gel silica increases before it decreases. (B) Activity vs time as a function of heat treatment at 47°C in a CK-doped wet silica gel. (C) Activity vs time as a function of heat treatment at 60°C in a CK-doped wet silica gel. (Reproduced from Ref. 17 with permission.)

Fig. 8. (A) Relative activity as a function of time for CK heated at 47°C in solution (•) and in sol-gel silica (■) shows that the activity in solution decreases immediately, but the activity in sol-gel silica increases before it decreases. (B) Activity vs time as a function of heat treatment at 47°C in a CK-doped wet silica gel. (C) Activity vs time as a function of heat treatment at 60°C in a CK-doped wet silica gel. (Reproduced from Ref. 17 with permission.)

activity as a result of heat treatment is shown in Fig. 8. The apparent activation energy for the activity enhancement was 2.6 ± 0.6 kJ/mol. The activity increased generally about fourfold as a result of heat treatment. Note that heating the enzyme in liquid buffer did not cause an increase in the activity but, rather, caused an immediate decrease.

The average pore sizes of the sol-gel monoliths were determined using nitrogen adsorption and desorption isotherms. The pore size, pore volume, and surface area as a function of temperature are listed in Table 1. There was a relatively narrow pore size distribution in which 80% of the pore volume was within ±10% of the average pore size. The nitrogen adsorption and desorption

Table 1

Pore Size as Function of Heating for Sol-Gel Monoliths

Table 1

Pore Size as Function of Heating for Sol-Gel Monoliths

Heating condition

Average pore size (nm)

(m2/g)

Room temperature

8.3

2.0

900

37°C for 3 h

8.8

1.9

780

47°C for 3 h

9.3

2.2

800

60°C for 3 h

10.9

2.2

730

data indicate that there were heat-induced changes in the silica matrix. The pore size increased as a function of temperature, which offers a materials-based explanation for the increase in enzyme activity in the heat-treated samples. With a larger pore size, the enzyme was able to rearrange to a more desirable conformation. In the synthesis of the sol-gel monoliths, gelation occurred on the order of minutes. Although the material experienced a liquid-to-solid transition at gelation, polycondensation continued to occur. As the matrix forms around the enzyme, the enzyme molecules may not be trapped in their native state. By enlarging the pores, the process of enzyme rearrangement becomes more favorable. Maintaining an elevated temperature for an extended period of time, however, induces enzyme denaturation, resulting in a decrease in activity. There are, therefore, two opposing effects, which may explain why the initial activity increased with short-term heating whereas the activity decreased with long-term heating.

To monitor the structural changes and maintenance of the structural integrity of CK, circular dichroism (CD) spectra were taken. CD utilizes circularly polarized light to probe the secondary structure (a helixes and P sheets) of proteins. CD spectra are sensitive to conformational changes and are a common spectroscopic method for studying protein structure (23). The CD spectra of CK in buffer solution and sol-gel monoliths (with and without heat treatment) were obtained, as shown in Fig. 9 (17). The CD spectrum of the enzyme in liquid buffer, which represents the properties of unconfined enzyme in its native state, exhibited a minimum at 220 nm. The CD spectrum of sol-gel-encapsulated enzyme (no heat treatment) showed a minimum at 225 nm, indicating that a large fraction of the enzyme was in a different, nonnative conformational state. On heating the monoliths for 3 h at 37, 47, or 60°C, however, the CD minimum of 220 nm was restored. We can infer from these results that, with heating, the encapsulated enzyme was able to revert to a more native conformation (i.e., the secondary structure of the enzyme became more like that of the enzyme in solution). Enabling the enzyme structure to match more

210 220 230 240 250 260

Wavelength (nm)

Fig. 9. CD spectra of CK in (1) a freshly made solution, (2) an unheated sol-gel silica monolith, and (3) a monolith heated for 3 h at 47°C. The spectra suggest that the enzyme is confined in a nonnative conformation on initial encapsulation, but after heating the monolith for 3 h, the enzyme conformation becomes more like that of free enzyme (enzyme in solution). (Reproduced from Ref. 17 with permission.)

210 220 230 240 250 260

Wavelength (nm)

Fig. 9. CD spectra of CK in (1) a freshly made solution, (2) an unheated sol-gel silica monolith, and (3) a monolith heated for 3 h at 47°C. The spectra suggest that the enzyme is confined in a nonnative conformation on initial encapsulation, but after heating the monolith for 3 h, the enzyme conformation becomes more like that of free enzyme (enzyme in solution). (Reproduced from Ref. 17 with permission.)

closely its native state may explain why there was an initial increase in its activity.

In a second set of experiments, we monitored the thermal transitions of CK by monitoring changes in the ellipticity at 220 nm (characteristic wavelength) as a function of temperature. Ellipticity is an indication of the a-helical content, and protein unfolding is indicated by an increase in ellipticity. As seen in Fig. 10 (17), on heating the monolith and solution to 90°C, the encapsulated enzyme did not fully denature like the enzyme in solution. The midpoint temperature of the unfolding transition, termed Tm, was 75°C for the CK in solution, whereas Tm could not be determined for the monoliths because the enzyme did not unfold completely. In other words, the sol-gel-immobilized CK did not unfold to the same extent as CK in solution. When the temperature was cooled to 20°C, there was virtually no change in ellipticity, indicating that in both cases protein unfolding was not reversible. Nevertheless, these results clearly show that when immobilized in the pores of the sol-gel matrix, the enzyme was able to better withstand thermal denaturation.

Fig. 10. Thermal unfolding transition of CK in monoliths and in solution monitored by ellipticity at 220 nm: (•) solution during heating; (O) solution during cooling; (■) sol-gel monolith during heating; (□) sol-gel monolith during cooling. The sol-gel-encapsulated enzyme unfolded to a lesser extent compared with free enzyme (enzyme in solution), but in both cases the denaturation was irreversible. The sample was heated at 2°C/min from 20 to 90°C and then cooled from 90 to 20°C. (Reproduced from Ref. 17 with permission.)

Fig. 10. Thermal unfolding transition of CK in monoliths and in solution monitored by ellipticity at 220 nm: (•) solution during heating; (O) solution during cooling; (■) sol-gel monolith during heating; (□) sol-gel monolith during cooling. The sol-gel-encapsulated enzyme unfolded to a lesser extent compared with free enzyme (enzyme in solution), but in both cases the denaturation was irreversible. The sample was heated at 2°C/min from 20 to 90°C and then cooled from 90 to 20°C. (Reproduced from Ref. 17 with permission.)

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