Influences of the Suspension Method of Thermolysin in Organic Solvents on Enzyme Activity

Thermolysin (EC 3. 4. 24. 4) was suspended in organic solvents containing a small amount of water (aqueous buffer solution) by two different methods, and the catalytic activity as well as the conformational changes in the enzyme were compared. In one method (Method 1), dried powder enzyme was directly suspended in organic solvents containing a small amount of water while in the second method (Method 2), the dried enzyme was first dissolved in the buffer solution and then added to the anhydrous organic solvents containing the same final water content as that used in Method 1. In Method 2, the enzymes showed a catalytic activity 2-10 times as high as that in Method 1. The ratio of the activity obtained in Method 2 to that in Method 1 strongly depended on kinds of organic solvents, and its dependency was reasonably explained by the E_T(30) values, which represent the extent of polarity of an organic solvent. The Fourier transform infrared spectroscopy (FT-IR) analyses indicated that the enzyme conformation in Method 2 was closer to its native one than that in Method 1. The influences of the suspension method on the enzyme activities of the different forms of the enzyme (as donated, dialyzed, and cross-linked) and the courses of the enzyme activities during incubation in Methods 1 and 2 were also investigated.