Action of shear on enzymes: Studies with alcohol dehydrogenase

Abstract

Yeast alcohol dehydrogenase (ADH) solutions (approximately 1 mg/ml, pH 7) were sheared in a coaxial cylindrical viscometer. This was fitted with a lid sealing the contents from the atmosphere and preventing evaporation. At 30°C after a total of 5 hr intermittent shearing at 683 sec⁻¹ no losses of activity were observed. No losses were found after 5 hr continuous shearing and in a no‐shear control. At 40°C and 683 sec⁻¹ there were only small activity losses in 5 hr. Shearing at 3440 sec⁻¹ no measurable losses of activity were found with a 1.03 mg/ml solution in 5 hr at 30°C, a 1.03 mg/ml solution in 8 hr at 5°C, and with a 3.89 mg/ml solution in 3 hr at 5°C. In all these cases, however, a white precipitate formed that was not observed in zero shear control experiments. The sheared 3.89 mg/ml solution was clarified by centrifugation. It was shown that there were no ADH aggregates in the supernatant and that the precipitate was less than 2% of the original protein. At 30°C under adverse pH conditions (pH 8.8) there was no significant difference in activity losses of an approximately 1 mg/ml solution sheared at 65 and 744 sec⁻¹. An approximately 0.5 mg/ml ADH solution, pH 7, was agitated in a small reactor with no free air–liquid interface. Peak shear rates near the impeller were estimated to be about 9000 sec⁻¹. Only a small decrease in specific activity was observed until over 15 hr total running at 5°C.