| Laccase from Botrytis cinerea 61--34 was purified from the extracellular broth of the culture medium using a facile process. A new method is described herein as a low cost alternative to hydrophobic interaction chromatography. B. cinerea 61--34 exhibited 64-fold greater activity than that of the parent strain 28985. The K m and Vmax for this enzyme were found to be 16.4 muM and 8.99 mukatals/mg protein by monitoring the co-substrate, oxygen. Laccase-catalyzed reactions containing 12--17% dioxane, DMSO, and 2-propanol exhibited 50% residual activity. Laccase was stable at 5°C with a minimal loss in activity over two weeks. The enzyme was tolerant to incubation for 22 hrs at 25°C or for 1hr at 45°C with 25% residual activity. Inhibition studies showed that thioglycollic acid, 1-naphthol, and 4-tert-butyl alcohol inhibited the enzyme. Compounds that modified histidine, cysteine, tryptophan, and methionine were also found to lower catalytic efficiency. However, modifications at aspartate, glutamate, and tyrosine residues did not significantly alter catalytic efficiency. Comparison of laccase substrate oxidation rates using several compounds revealed the effect of the location and size of substitutients on phenolic compounds. N-hydroxy phthalimide was shown to function as a mediator for laccase using a facile method described herein.;This study also describes the gene coding for laccase from B. cinerea 61--34. The sequence contains 1452 nucleotides and one intron containing 66 nucleotides. Translation of the DNA sequence yields a protein containing 461 amino acids. The open reading frame contains 4 cysteine residues that may function in disulfide bridging, 5 putative N-glycosylation sites, and four highly conserved copper binding regions. |
