Purification and characterization of the phospho-alpha(1,1)glucosidase (TreA) of Bacillus subtilis 168.
The intracellular phospho-alpha(1,1)glucosidase TreA from Bacillus subtilis has been overproduced in Escherichia coli and purified by ion-exchange chromatography and gel filtration. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 64 kDa. Isoelectric focusing indicated homogeneity of the protein, and its pI was determined to be 4.3. Characterization of the enzyme showed a protein which is stable up to 44 degrees C after temperature treatment for 15 min. The temperature optimum was found to be 37 degrees C, and the pH optimum was 4.5. TreA activity is stimulated by high salt concentrations with different efficiencies depending on the kind of salt. When increasing amounts of ammonium sulfate are used, the increase of TreA activity is correlated with a conformational change of the protein or dimerization. The substrate specificity of the purified enzyme was characterized, showing additionally that trehalose is also hydrolyzed, but to a much smaller extent than trehalose-6-phosphate. In vitro, the presence of glucose reduces TreA activity, indicating product inhibition of the enzyme.
Gotsche
S
S
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Federal Republic of Germany.
Dahl
M K
MK
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D004186:Disaccharidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism*; D014199:Trehalose / Q000031:analogs & derivatives* / Q000378:metabolism
D011994:Recombinant Proteins; D013403:Sugar Phosphates; D005632:Fructose; C082722:trehalose-6-phosphate; D012965:Sodium Chloride; D011189:Potassium Chloride; D014199:Trehalose; D004186:Disaccharidases; C040631:alpha, alpha-phosphotrehalase; D005947:Glucose