Purification and characterization of an isomaltotriose-producing endo-dextranase from a Fusarium sp.
An isomaltotriose-producing endo-dextranase was simply purified from cell-free culture broth of a Fusarium sp. by ethanol fractionation and consecutive column chromatographies using DEAE-Toyopearl and Bio-Gel P-100. The purified enzyme was judged to be homogeneous on PAGE and SDS-PAGE as well as isoelectric focusing. The molecular mass of the enzyme was estimated to be about 69 kDa by SDS-PAGE. The enzyme is an acidic protein with a pI of 4.6. The optimum pH and temperature were pH 6.5 and 35 degrees C, respectively. The enzyme was completely stable over the range of pH 4.5-11.8 at 4 degrees C for 24 h and at temperatures below 45 degrees C. Inactivation of the enzyme was found to be partial with 5 mM Cu2+, being about 70% inhibition and complete with 5 mM of Fe3+, Hg2+, Ag+ or NBS. The enzyme split dextran in an endo-lytic action to produce a large amount of isomaltotriose and a slight amount of isomaltose and glucose. The anomeric configurations of the reaction products formed by the enzyme were alpha-form, indicating that the alpha-glycoside linkages in the substrate are retained. The final yield of isomaltotriose from dextran T-2000 was about 62%.
Shimizu
E
E
Novo Nordisk Bioindustry Ltd., Chiba, Japan.
Unno
T
T
Ohba
M
M
Okada
G
G
D000596:Amino Acids; D003910:Dextranase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005670:Fusarium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007526:Isoelectric Point; D008670:Metals; D008968:Molecular Conformation; D008970:Molecular Weight; D013696:Temperature; D014312:Trisaccharides / Q000096:biosynthesis*
D000596:Amino Acids; D003911:Dextrans; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D014312:Trisaccharides; C045441:isomaltotriose; D003910:Dextranase