RELATIONSHIPS BETWEEN MOLECULAR-STATES (CONFORMATION AND ORIENTATION)AND ACTIVITIES OF ALPHA-AMYLASE ADSORBED ON ULTRAFINE SILICA PARTICLES

Bacillus subtilis alpha-amylase, which contains a relatively large amo unt of alpha-helix, was adsorbed on two types of ultrafine silica part icles (silica-1 and -2, average diameter 15 nm) under various conditio ns. The changes in circular dichroism (CD) spectra of alpha-amylase up on adsorption were measured, and the extent of conformational changes was estimated from the reduction in alpha-helix content. In addition, the activities of adsorbed alpha-amylase were measured at pH 5.2 using corn starch and p-nitrophenylbenzyl alpha-maltopentaoside (BG5P). In the ultrafine silica-2 particles, the extent of both activity reductio ns and conformational changes upon adsorption was much larger than tha t in the ultrafine silica-1 particles and increased with decreasing pH and amount of adsorption. The extent of activity reductions correlate d closely with the conformational changes. On the other hand, the effe ct of reduction in alpha-amylase activity upon adsorption measured by BG5P was smaller than that measured by starch, indicating that the lac k of accessibility of the active site to a large substrate also reduce s the activity of adsorbed alpha-amylase. However, the effects of part icle type and adsorption conditions on the extent of activity reductio ns by the accessibility resistance were small. Therefore, variation of the activity of adsorbed alpha-amylase is mainly attributable to the extent of conformational changes upon adsorption. Based on these resul ts, a procedure to prepare adsorbed alpha-amylase with high activity w as investigated.