Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.
J Appl Microbiol
; 120(5): 1326-35, 2016 May.
Article
en En
| MEDLINE
| ID: mdl-26808611
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 ANDRESULTS:
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.Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Esporas Bacterianas
/
Vapor
/
Geobacillus stearothermophilus
/
Esterilización
/
Alfa-Glucosidasas
/
Calor
Idioma:
En
Revista:
J Appl Microbiol
Asunto de la revista:
MICROBIOLOGIA
Año:
2016
Tipo del documento:
Article
País de afiliación:
Estados Unidos