Abstract
Laccases that are tolerant to organic solvents are powerful bio-catalysts with broad applications in biotechnology. Most of these uses must be accomplished at high concentration of organic solvents, during which proteins undergo unfolding, thereby losing enzyme activity. Here we show that organic-solvent pre-incubation provides effective and reversible 1.5- to 4.0-fold enhancement of enzyme activity of fungal laccases. Several organic solvents, including acetone, methanol, ethanol, DMSO, and DMF had an enhancement effect among all laccases studied. The enhancement was not substrate-specific and could be observed by using both phenolic and non-phenolic substrates. Laccase preincubated with organic solvents was sensitive to high temperature but remained stable at 25 °C, for an advantage for long-term storage. The acetone-pre-incubated 3-D structure of DLac, a high-efficiency fungal laccase, was determined and confirmed that the DLac protein structure remains intact and stable at a high concentration of organic solvent. Moreover, the turnover rates of fungal laccases were improved after organic-solvent pre-incubation, with DLac showing the highest enhancement among the fungal laccases examined. Our investigation sheds light on improving fungal laccase usage under extreme conditions and extends opportunities for bioremediation, decolorization, and organic synthesis.
Highlights
Laccases are four-copper oxidases that catalyze the oxidation of phenolic units in lignin as well as a wide range of phenolic compounds and aromatic amines[1]
Enzymatic catalysis in the presence of organic solvents is a trend in biotechnology, the polar transition state of the enzyme could be destabilized by the penetration of a water-miscible organic solvent into the active site of enzyme[29,30]
This study aimed to find a technical breakthrough by combining the discovery of organic-solvent tolerance in laccases and the application of optimized reaction conditions for enhancing enzymatic catalysis in the presence of an organic solvent
Summary
Laccases are four-copper oxidases that catalyze the oxidation of phenolic units in lignin as well as a wide range of phenolic compounds and aromatic amines[1]. Fungal laccases are remarkable green catalysts valuable for biotechnology and industry applications such as biodegradation[6,7], detoxification[8], bleaching[9], decolorization[10,11], organic compound synthesis[12,13,14] and enzymatic biosensors[15]. Most of these conversions must be accomplished at high concentrations of organic solvents such as ethanol[16,17,18], acetone[17,19,20], and DMSO17,21–23. We developed a novel approach that could further improve laccase activity for extending its industrial use
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