Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems.
Thermostable alpha-amylase with temperature optimum at 80 degrees C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000-20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000-20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of alpha-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60-75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4.
Ivanova
V
V
Institute of Microbiology, Bulgarian Academy of Sciences, Sofia. viva@microbio.bas.bg
Yankov
D
D
Kabaivanova
L
L
Pashkoulov
D
D
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D003911:Dextrans / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D001426:Bacterial Proteins; D003911:Dextrans; D011092:Polyethylene Glycols; D000516:alpha-Amylases; D012697:Serine Endopeptidases