A beta-glucosidase from Sclerotinia sclerotiorum: biochemical characterization and use in oligosaccharide synthesis.
The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one beta-glucosidase (beta-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that beta-glu x may be a homodimer. For p-nitrophenyl beta-d-glucopyranoside hydrolysis, apparent Km and Vmax values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55-60 degrees C and pH 5.0, respectively. beta-Glu x was strongly inhibited by Fe2+ and activated about 35% by Ca2+. beta-Glu x possesses strong transglucosylation activity in comparison with commercially available beta-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50 degrees C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 3C nuclear magnetic resonance spectroscopy.
Unité de Génie Biologique, Institut National de Sciences Appliquées et de Technologie (INSAT), BP 676, 1080 Tunis Cedex, Tunisia.
Smaali M
SM
Issam
Gargouri
G
Mohamed
Legoy Marie
LM
Dominique
Maugard
M
Thierry
Limam
L
Farid
Marzouki
M
Nejib
D001203:Ascomycota / Q000201:enzymology* / Q000254:growth & development; D002475:Cellobiose / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D005947:Glucose / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis / Q000138:chemical synthesis*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification
D005656:Fungal Proteins; D009844:Oligosaccharides; D002475:Cellobiose; D001617:beta-Glucosidase; D005947:Glucose