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Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.

Lequerica, JL.; Berjano, E.; Herrero, M.; Melecio, L.; Hornero, F. (2008). Cooled waterirrigated intraesophageal balloon to prevent thermal injury during cardiac ablation: Experimental study based on an agar phantom. Physics in AbstractA great deal of current research is directed to finding a way to minimize thermal injury in the esophagus during radiofrequency (RF) catheter ablation of the atrium. A recent clinical study employing a cooling intraesophageal balloon reported a reduction of the temperature in the esophageal lumen. However, it could not be determined whether the deeper muscular layer of the esophagus was cooled enough to prevent injury. We built a model based on an agar phantom in order to experimentally study the thermal behavior of this balloon by measuring the temperature not only on the balloon, but also at a hypothetical point between the esophageal lumen and myocardium (2 mm distant).Controlled temperature (55ºC) ablations were conducted for 120 s. The results showed that: 1) The cooling balloon provides a reduction in the final temperature reached, both on the balloon surface and at a distance of 2 mm; 2) Coolant temperature has a significant effect on the temperature measured at 2 mm from the esophageal lumen (it has less effect on the temperature measured on the balloon surface); and 3) The precooling period has a significant effect on the temperature measured on the balloon surface (the effect on the temperature measured 2 mm away is small). The results were in good agreement with those obtained in a previous clinical study. The study suggests that that the cooling balloon gives thermal protection to the esophagus when a minimum pre-cooling period of 2 minutes is programmed at a coolant temperature of 5ºC or less.

Although previous studies have established the feasibility of monitoring esophageal temperature during radiofrequency cardiac ablation using an esophageal temperature probe (ETP), some questions remain regarding its efficacy. The aims of this study were to study the effect of the location of the ETP on the temperature reached, and to test the characteristics of ETP as used in clinical practice. We constructed an agar phantom to model the thermal and electrical characteristics of the biological tissues (left atrium, esophagus and connective tissue). The ETP was positioned at 6.5 mm from an ablation electrode and at distances of 0, 5, 10, 15, 20 mm from the catheter axis. A thermocouple was located on the probe to measure the actual temperature of the external esophageal layer during the ablations (55 degrees C, 60 s). The mean temperatures reached at the thermocouple were significantly higher than those measured by the ETP (48.3 +/- 1.9 degrees C versus 39.6 +/- 1.1 degrees C). The temperature values measured with the ETP were significantly lower when the probe was located further from the catheter axis (up to 2.5 degrees C lower when the distance from the probe-catheter axis was 2 cm). The dynamic calibration of the ETP showed a mean value for the time constant of 8 s. In conclusion, the temperature measured by the ETP always underestimates the temperature reached in the thermocouple. This fact can be explained by the distance gap between the thermocouple and probe and by the dynamic response of the ETP. The longer the distance between the ETP and catheter axis, the higher is the temperature difference.