Effective purification of Cerrena unicolor laccase using microfiltration, ultrafiltration and acetone precipitation.
Microfiltration followed by concentration and diafiltration on ultrafiltration membranes (Biomax-100, Biomax-10 and Ultracel-10) was used to recover extracellular laccase (EC 1.10.3.2) from culture broth of wood-rotting fungus Cerrena unicolor. Feed, permeate, retentate and membrane wash-out solutions were analysed for the presence of laccase, proteases, protein and brown impurities. An easy, cheap and short-term procedure was proposed to obtain retentates with low yields of total proteins (less than 14%), proteases activity (less than 15%) and brown impurities (from 2% to 29%) with a simultaneous laccase recovery above 73%. The degree of laccase purification varied from 6.7 to 11.0 and depended on the type of membrane used and content of brown pigments in the feed. Subsequent protein precipitation with cold acetone increased the degree of purification about twice and reduced proteases and brown impurities to some extent. Ultracel-10 membrane was recommended as the best solution to prevent fouling of membranes and to obtain laccase-enriched fraction with a very low content of proteases and brown pigments.
Bryjak
Jolanta
J
Department of Bioorganic Chemistry, Wrocław University of Technology, Norwida 4/6, 50-373, Wrocław, Poland. jolanta.bryjak@pwr.wroc.pl
Rekuć
Adriana
A
D000096:Acetone / Q000737:chemistry*; D000818:Animals; D055453:Coriolaceae / Q000201:enzymology*; D004795:Enzyme Stability; D042845:Laccase / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000302:isolation & purification; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods*; D014934:Wood / Q000382:microbiology*
D000096:Acetone; D042845:Laccase; D010447:Peptide Hydrolases