Abstract
Bacillus subtilis glucose dehydrogenase (EC 1.1.1.47) has been purified from sporulating cell extract to apparent homogeneity (as determined by polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing). The enzyme purified as a single molecular species with no evidence for a multiple form of the enzyme. The B. subtilis glucose dehydrogenase has an apparent isoelectric point of 4.7-4.8 and an apparent Mr = 126,000 and is comprised of four subunits of Mr = 31,500 each. The glucose 2-deoxyglucose and glucosamine substrate specificity of the enzyme is similar to the substrate specificity for B. subtilis spore germination, suggesting that the spore glucose dehydrogenase may play some role in spore germination. The B. subtilis glucose dehydrogenase is extremely dependent on the presence of glycerol or other hydrophobic bond-stabilizing agents (or NAD) for retention of enzymatic activity, and the presence of glycerol (20% w/v) in the extraction and purification buffers was absolutely necessary for the successful purification of this enzyme.
Highlights
1.1.1.47) has been purified from sporulating cell extract to apparent homogenei(tyas determined by polyacrylamide gel electrophoresis,sodiumdodecyl sul- Purification of the B. subtilis Glucose Dehydrogenase-Glufate-polyacrylamide gel electrophoresis, and isoelec- cose dehydrogenase can be purified from B. subtilis spores tric focusing)
An apparentisoelectric point of4.7-4.8 and an apparent M, = 126,000 and is comprised of four subunits of M, = 31,500 each.Theglucose 2-deoxyglucose and glucosamine substratespecificity of the enzymies similar to the substrate specificity for B. subtilis spore germination, suggesting that the spore glucose dehydrogenase may play some role in spore germination
The present paper reports the glycerol stabilization of B. subtilis glucose dehydrogenase, permitting purification of the enzyme to apparenthomogeneity, and reports some of the properties of the purified enzyme
Summary
Acrylamide gel electrophoresis,sodiumdodecyl sul- Purification of the B. subtilis Glucose Dehydrogenase-Glufate-polyacrylamide gel electrophoresis, and isoelec- cose dehydrogenase can be purified from B. subtilis spores tric focusing). Gel filtration of the glucose dehydrogenase of the BG 1722 isolate of Yokota et al [19] (in the presenceof 20% (w/v) glycerol in 0.05 imidazole (pH 6.5)) gave only a single molecular specific species of approximately 125,0002(similar to the results observed for the B. subtilis enzyme; Fig. S-11). We have purified rat liver glucose-6-phosphate dehydrogenasefor comparative purposes and found that the ratliver enzyme is quitelabile in the absencoef glycerol, the B. subtilis glucose dehydrogenase is several orders of magnitude more
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