Isolation and partial characterization of a glucose-binding protein from Bacillus megaterium

Attempts were made to use the osmotic shock procedure established for Gram-negative micro-organisms on a Gram-positive micro-organisms, Bacillus megaterium ATCC 19213. The procedure did not appear to shock the cells osmotically. The procedure, in fact, appeared to enhance binding of both glucose and fructose by the organism. The binding of glucose was greater by cells, whether grown on fructose or glucose, than it was for fructose. A procedure was developed for removing and isolating a protein capable of binding glucose. This involved solubilizing the cytoplasmic membrane of the organism with the nonionic detergent Triton X-100, and then isolating it by gel filtration on a Bio-Gel A 1.5 column with 0.05 M pyrophosphate containing 1% (v/v) Triton X-100 as the eluting buffer. This is the first isolation of such a protein in a Gram-positive organism. Isolated protein was demonstrated to have a specificity only for glucose. The protein did not bind to either the monosaccharides fructose and galactose, or the disaccharide sucrose. This was in comparison to whole cells which demonstrated ability to take up fructose. The protein was estimated to have a molecular weight between 24,000 and 30,000 daltons by gel filtration techniques.