Analysis of α ‐glucosidase enzyme activity used in a rapid test for steam sterilization assurance

Aims This study was to determine the sources, location and identity of α ‐glucosidases in dormant/germinating/outgrowing spores and growing cells of Geobacillus stearothermophilus ATCC 7953, an enzymatic activity in spores used in rapid tests of steam sterilization. Methods and Results α ‐Glucosidase activity in spores and cells was determined measuring methylumbelliferyl‐ α ‐ d‐ glucoside ( α ‐ MUG ) or α ‐ MUG ‐6‐phosphate hydrolysis fluorometrically. While α ‐ MUG ‐6‐phosphate was not hydrolysed by cell or spore extracts, assays with α ‐ MUG showed that: (1) the α ‐glucosidase activity was inside and outside spores, and the activity outside spores was largely removed by buffer washes or heat activation, whereas α ‐glucosidase activity was only inside vegetative cells; (2) most α ‐glucosidase activity in cells and spores was soluble; (3) Western blots and enzyme inhibition using an anti‐ α ‐glucosidase antiserum identified ≥2 α ‐glucosidases in spores and growing cells; (4) α ‐glucosidase‐specific activities were similar in dormant, germinated and outgrowing spore and growing cell extracts; and (5) significant α ‐glucosidase was synthesized during spore germination and outgrowth and cell growth, this synthesis was not repressed by glucose nor induced by α ‐ MUG , but glucose inhibited α ‐ MUG uptake. Conclusions α ‐ MUG hydrolysis by G. stearothermophilus is by α ‐ MUG uptake and hydrolysis by ≥2 α ‐glucosidases associated with dormant spores and synthesized by germinating and outgrowing spores. The enzyme activity observed by sterilization assurance assays appears likely to come from heat‐stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth. Significance and Impact of the Study The results of this work provide new insight into the science behind a rapid test for steam sterilization assurance.