Purification and molecular characterization of exo-beta-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2.
The extracellular beta-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific beta-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40 degrees C, respectively. The enzyme was significantly stimulated by Li+, Ni2+, and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1,10-phenanthroline. The Km and Vmax values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR which is the conserved motif of the beta-1,3-glucanases from other yeasts.
Peng
Ying
Y
Key Laboratory of Marine Genetics and Gene Resource Exploitation of Ministry of Education, Ocean University of China, Yushan Road, No.5, Qingdao, China.
Chi
Zhen-Ming
ZM
Wang
Xiang-Hong
XH
Li
Jing
J
D000595:Amino Acid Sequence; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D017124:Conserved Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005936:Glucans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014462:Ultrafiltration / Q000379:methods; D055320:Williopsis / Q000201:enzymology* / Q000235:genetics*
D004271:DNA, Fungal; D004187:Disaccharides; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D005936:Glucans; D008670:Metals; D009005:Monosaccharides; D011134:Polysaccharides; C008247:laminaran; D043326:Glucan 1,3-beta-Glucosidase; C117648:beta-1,3-exoglucanase