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- Title
Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.
- Authors
Setlow, B.; Korza, G.; Setlow, P.
- Abstract
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.
- Subjects
GLUCOSIDASES; ENZYMES; STEAM as a disinfectant; GEOBACILLUS stearothermophilus; FLUORIMETRY; HYDROLYSIS; BACTERIAL spores
- Publication
Journal of Applied Microbiology, 2016, Vol 120, Issue 5, p1326
- ISSN
1364-5072
- Publication type
Article
- DOI
10.1111/jam.13074