The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties.
Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l(-1)) were produced in response to Cu2+. Highest laccase levels (19,000 U l(-1)) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M (w) of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60 degrees C, and at 5 degrees C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4 degrees C. Kinetic constants (K (m), k (cat)) were determined for 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.
Unit of Environmental Biotechnology, International Graduate School Zittau, Markt 23, 02763 Zittau, Germany. amichniewicz@poczta.onet.pl
Anna
A
Michniewicz
René
R
Ullrich
Stanisław
S
Ledakowicz
Martin
M
Hofrichter
D052160:Benzothiazoles; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D004795:Enzyme Stability; D006835:Hydrazones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D020072:Polyporales / Q000201:enzymology*; D011748:Pyrogallol / Q000031:analogs & derivatives / Q000378:metabolism; D012117:Resins, Synthetic; D013057:Spectrum Analysis; D013451:Sulfonic Acids / Q000378:metabolism; D013696:Temperature; D014462:Ultrafiltration
D052160:Benzothiazoles; D003470:Culture Media; D006835:Hydrazones; D007527:Isoenzymes; D012117:Resins, Synthetic; D013451:Sulfonic Acids; D011748:Pyrogallol; C009370:syringaldazine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C010120:pyrogallol 1,3-dimethyl ether; D003300:Copper; C042855:Mono Q; D042845:Laccase