High yield electroextraction of proteins from yeast by a flow process.
High yields of intracellular enzymes from yeast can be obtained by application of a series of electric field pulses with a flow process. Up to 80-90% of the total activity can be liberated without any further or previous treatment of cells. The method is based on electroinduced changes in the cell envelope leading to a leakage of part of the intracellular proteins without formation of debris and permits treatment of large volumes. Field parameters require a limited electrical power. Treatment of at least 20% wet weight suspensions is possible. The optimal field conditions must be adjusted to the suspension concentration. Maximal yield is obtained within 4h at 30 degrees C for enzymes from Saccharomyces cerevisiae such as hexokinase, 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase. The extraction of beta-D-galactosidase from Kluyveromyces lactis lasts 10h but can be accelerated by adding dithiothreitol in the postpulse medium. The specific activities of the electroextracted enzymes are higher than those obtained by mechanical disintegration or enzymatic lysis.
Ganeva
V
V
Faculty of Biology, University of Sofia, Bulgaria.
Galutzov
B
B
Teissié
J
J
D004574:Electromagnetic Fields; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation / Q000379:methods; D004795:Enzyme Stability; D005456:Fluorescent Dyes / Q000737:chemistry; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology* / Q000254:growth & development; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014617:Vacuoles / Q000201:enzymology
D005456:Fluorescent Dyes; D005656:Fungal Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006593:Hexokinase; D010735:Phosphoglycerate Kinase