Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (K m ؍ 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/ 4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.
Eight commercial lipase preparations were examined for the ability to hydrolyze phosphatidylcholine (PC) in hexane solutions. Only the enzymes from Humicola lanuginosa, Rhizopus de/emar and Candida rugosa displayed appreciable activity. Solvent polarity was the largest single factor affecting activity. The H. lanuginosa sample was most active in polar solvents. The R. de/emar preparation was most active in polar (2-hexanone) and nonpolar (decane) solvents and least active in solvents of intermediate polarity (hexane). The solvent dependence of the activity of the C. rugosa enzyme varied with the ratio of substrate to enzyme. Different degrees of activity were retained by the three enzymes after passive immobilization on Celite, controlled-pore glass, polypropylene and Amberlite XAD-7 resins. No single resin yielded the best retained activity for all three preparations. When examined in 2-octanone, hexane and isooctane, the Celite-immobilized R. de/emar and H. lanuginosa enzymes exhibited highest activity in 2-octanone, while immobilized C. rugosa was most active in isooctane. The water content at which maximum activity was observed was relatively independent of solvent polarity and the amount of catalyst but was proportional to the amount of PC in the reaction. The retention of activity by immobilized Rhizomucor miehei lipase (Lipozyme) during multiple hydrolytic cycles required a reduction in the water content of the system below that yielding optimal activity in a single cycle.
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