Purification and characterization of a dextranase from Sporothrix schenckii.
A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 degrees C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the Km for soluble dextran was 0.067 +/- 0.003% (w/v). Salts of Hg2+, (UO2)2+, Pb2+, Cu2+, and Zn2+ inhibited by affecting both Vmax and Km. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 degrees C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 degrees C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 degrees C. All of these isolates grew on glucose at 35 degrees C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA. warnold@kumc.edu
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Arnold
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Nguyen
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Mann
D003910:Dextranase / Q000737:chemistry*; D003911:Dextrans / Q000378:metabolism; D004790:Enzyme Induction / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009005:Monosaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000032:analysis; D012492:Salts / Q000494:pharmacology; D012988:Soil Microbiology; D013175:Sporothrix / Q000201:enzymology*; D013174:Sporotrichosis / Q000382:microbiology; D013696:Temperature
D003911:Dextrans; D009005:Monosaccharides; D009844:Oligosaccharides; D012492:Salts; D003910:Dextranase