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β-galactosidase Using Gel-Filtration Chromatography
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
E. coli-PAD is a thiamine auxotroph (Dr. Natividad Ruiz, personal communication) and must be grown in LB broth or agar. It will not thrive on general purpose growth media such as trypticase soy broth or nutrient broth. In addition, this strain must be grown in the presence of ampicillin at a concentration of 125 µg/mL. The antibiotic is necessary to provide positive selective pressure for plasmid pRS415. Furthermore, it is a good idea to propagate this strain on LB agar containing 20 µg/mL x-gal. X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) is a chromogenic substrate of β-galactosidase that, when cleaved, produces a blue color.10 X-gal is commonly used for detecting β-galactosidase made by recombinant vectors such as plasmid pRS415. Individual bacterial colonies that overproduce enzyme are various shades of blue and the bluest of these colonies were preferentially selected for subculturing and subsequent experiments.
Overview of Microbial Methods
Published in Joseph A. Cotruvo, Gunther F. Craun, Nancy Hearne, Providing Safe Drinking Water in Small Systems, 2019
Total coliform bacteria do not conform to a given taxonomic category. Traditionally several genera (Citrobacter, Enterobacter, Escherichia, and Klebsiella) of the family Enterobacteriaceae have been included in this group. The operational definition of a coliform is based upon the method used for detection. Lactose fermentation with the production of acid and/or gas has been considered a positive test for coliform bacteria when using carbohydrate-based media incubated at 35 to 37°C. Lauryl-tryptose broth (LT), with or without an acid indicator, and presence-absence broth (PA) are examples of lactose-based media used in these analyses [Eaton et al., 1995]. Chromogenic media have now been devised in which a positive test is based upon detecting the presence of the enzyme β-D-galactosidase. This enzyme is responsible for the cleavage of lactose to form glucose and galactose. The choromogenic media contain a specific chromogenic substrate which produces a color change due to hydrolysis by the enzyme. Three commonly used chromogenic substrates for β-D-galactosidase are ortho-nitrophenyl-β-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3 indolyl-β-D-galactopyranoside (XGAL), and chlorophenol red β-D-galactopyranoside (CPRG). When hydrolyzed by the enzyme β-D-galactosidase ONPG substrate changes from a clear to a yellow color, XGAL changes from a clear to a blue/green color, and CPRG changes from a yellow to a magenta color to indicate the presence of total coliform bacteria.
Effects of the carbon source and the interaction between carbon sources on the physiology of the industrial Saccharomyces cerevisiae CAT-1
Published in Preparative Biochemistry & Biotechnology, 2020
Valkirea Matos Nascimento, Gustavo Graciano Fonseca
With the presence of two distinct phases, some kinetic parameters were calculated for both sugars (Table 2). The μmax, for example, was higher in glucose than in galactose, as expected (Table 1). The temperature had no apparent influence on the growth rate with glucose. A higher biomass yield on galactose (Yx/GAL) was observed when compared to the other assays (Table 2).