Characterization and optimization of in vitro assay conditions for (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening

J Antibiot (Tokyo). 1998 Jul;51(7):665-75. doi: 10.7164/antibiotics.51.665.


(1,3)Beta-D-glucan synthase (E.C. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-beta-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans. These included lysis of cells in the following for C. albicans, 100 mM HEPES, pH 8.0, 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 microM GTPgammaS. Resulting low-speed supernatants were used as enzyme sources to determine the optimal in vitro assay conditions. We have characterized the resulting enzyme activities and tested the optimized assays with known (1,3)beta-glucan synthase inhibitors including cilofungin, papulacandin, aculeacin A, and echinocandin B. We have used both optimized assays to screen > 1000 extracts of marine macroorganisms and, using bioassay-guided purification, have identified (1,3)beta-glucan synthase inhibitors.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antifungal Agents / pharmacology*
  • Aspergillus fumigatus / drug effects
  • Aspergillus fumigatus / enzymology*
  • Buffers
  • Candida albicans / drug effects
  • Candida albicans / enzymology*
  • Enzyme Inhibitors / pharmacology*
  • Glucosyltransferases / antagonists & inhibitors*
  • Glucosyltransferases / metabolism*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Membrane Proteins*
  • Reproducibility of Results
  • Schizosaccharomyces pombe Proteins*


  • Antifungal Agents
  • Buffers
  • Enzyme Inhibitors
  • Membrane Proteins
  • Schizosaccharomyces pombe Proteins
  • Glucosyltransferases
  • 1,3-beta-glucan synthase