title: Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  The results of this work provide new insight into the science behind a rapid test for steam sterilization assurance.
authors.affiliation: Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT, USA.
authors.forename: B
authors.initials: B
authors.lastname: Setlow
authors.affiliation: Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT, USA.
authors.forename: G
authors.initials: G
authors.lastname: Korza
authors.affiliation: Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT, USA.
authors.forename: P
authors.initials: P
authors.lastname: Setlow
mesh_terms: D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005947:Glucose; D005960:Glucosides; D006358:Hot Temperature; D006868:Hydrolysis; D013171:Spores, Bacterial / Q000201:enzymology*; D013227:Steam; D013242:Sterilization; D000520:alpha-Glucosidases / Q000032:analysis* / Q000378:metabolism
chemical_list: D005960:Glucosides; D013227:Steam; D000520:alpha-Glucosidases; D005947:Glucose
keywords: bacillus; bacterial spores; enzymes; glucosidase; sterilization