Elsevier

Journal of Hazardous Materials

Volume 366, 15 March 2019, Pages 520-528
Journal of Hazardous Materials

Acetylacetone extends the working life of laccase in enzymatic transformation of malachite green by interfering with a key intermediate

https://doi.org/10.1016/j.jhazmat.2018.12.028Get rights and content

Highlights

  • Acetylacetone (AA) played double roles in the enzymatic transformation of MG.

  • NTB was identified as a key intermediate in laccase-catalyzed transformation of MG.

  • AA inhibited the transformation of MG by quenching the reactive NTBradical dot+.

  • AA reduced the consumption of laccase by quenching the reactive NTBradical dot+.

  • The inhibition effect of AA was both time and concentration dependent.

Abstract

The potential of acetylacetone (AA) as a mediator of laccase has been tested in the enzymatic transformation of malachite green (MG). AA inhibited the laccase-induced transformation of MG at the beginning of incubation but extended the working life of laccase in long runs. To elucidate the underlying mechanisms, the transformation of MG in the laccase-AA system was systematically investigated. The inhibition of AA on the enzymatic transformation of MG conformed to the partial mixed model. The transformation of N,N,N',N'-tetramethyl-1,1′-biphenyl-4,4′-diamine (NTB) was identified as the rate-controlling step in the laccase system. The generated NTB was oxidized to NTBradical dot+ by laccase, which acted as a redox mediator to accelerate the transformation of MG. The addition of AA to the enzymatic system quenched the NTBradical dot+ by forming an intermediate complex of AA-NTB. This quenching reaction led to two contrary effects: the acceleration caused by NTBradical dot+ in the enzymatic transformation of MG was inhibited whereas the formation of AA-NTB complex enhanced the further transformation at the later stage. As a result, less laccase was consumed, which explained the extended working life of laccase in the long runs. The understanding of these mechanisms are helpful for the better use of laccase as a green biocatalyst.

Introduction

Malachite green (MG) is widely used in aquaculture as a parasiticide and in food, textile and other industries because of its specific structure and property [1]. However, its mutagenicity, carcinogenity and genotoxicity constitute major threats to the aquatic environment and human health [2]. To cope with those threats, various processes have been developed, such as photocatalysis [3], Fenton [4], sorption [5], ozonation [6] and biodegradation [7,8]. Enzyme-based biological degradation has been recognized as one of the most effective processes [9].

Laccases (EC 1.10.3.2) are one type of multi-copper oxidases that can catalyze the oxidation of many phenolic compounds [[10], [11], [12]]. The addition of redox mediators, such as 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT), and 2,2,6,6-tetramethylpiperidinooxy (TEMPO), could expand the workable substrates from phenolic compounds to non-phenolic compounds, including dyes, and other aromatic contaminants [[13], [14], [15]]. Those characteristics endow laccase with great potential for biological and environmental applications [[16], [17], [18], [19]]. However, applications on a large scale is restrained by its high cost and potential toxicity of currently used mediators. Besides these synthetic redox mediators, some natural compounds like syringaldazine (SYR) and acetosyringone, can serve as mediators for laccase and enhance oxidation of recalcitrant dyes [20], bleaching and delignification of kraft pulp [21]. It is worth noting that each redox mediator only works on limited substrates [22]. For example, HBT can efficiently improve the laccase-induced transformation of Reactive Black 5, Bismark Brown R, and Lanaset Grey G, but is not effective for the decoloration of Remazol Brilliant Blue R.

Acetylacetone (AA) is a metabolic intermediate in microbial metabolism of aromatics and terpenes [23]. The combination of AA and laccase has been used as a laccase-mediator system (LMS) to initiate free radical polymerization of vinyl monomers [24,25]. In the enzymatic transformation of malachite green (MG), the laccase-AA system showed a higher turnover number than the laccase-HBT system [26]. After incorporation into polymers through a self-initiated one-pot process, AA still showed redox ability as a laccase mediator [27]. However, in the enzymatic transformation of MG, the presence of AA, no matter in free form or immobilized, led to an inhibition on the transformation kinetics of MG at the beginning stage of incubation. The enhancement effect of AA on the transformation of MG in cycle runs was particularly manifested as a prolonged lifetime of laccase. The mechanism of such a self-contradictory phenomenon is still unclear.

The active sites of laccase are constituted of copper and amino acids moieties/side chains. Chelating agents usually pose a potential threat to the action of enzyme [28]. For example, azide, thioglycolic acid, and diethyl-dithiocarbamate significantly inhibited the activity of laccase while ethylene diamine tetraacetic acid (EDTA) led to a slight inhibition. AA is an effective chelating agent for many transition metals, including copper. Therefore, in this work, our focus was whether the initially inhibitory effect of AA on the transformation kinetics of MG and the ultimately prolonged lifetime of laccase in the laccase-AA system could be attributed to the chelation between AA and the copper centers of laccase. Furthermore, we would like to elucidate the underlying mechanisms of the inhibition/promotion effects of AA in the enzymatic transformation of MG.

Section snippets

Materials

MG of chemical grade was purchased from Shanghai Reagent Station, China. Acetic acid and acetonitrile of chromatography grade and EDTA-2Na, N,N-dimethylformamide (DMF) of analytical grade were supplied by Nanjing Reagent Station, China. N,N,N',N'-tetramethyl-1,1′-biphenyl-4,4′-diamine (NTB) was purchased from Tokyo Chemical Industry Co., Japan. ABTS, SYR, HBT, α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) of analytical grade, Trametes versicolor laccase with a nominal activity ≥0.5 U/mg, and

Effects of AA on laccase-catalyzed transformation of MG

In a previous work [26], cycle runs were conducted with 0.1 mM MG solution in each cycle and 0.07 U/mL laccase (a final laccase concentration) throughout the experiment. The presence of 2.0 mM AA reduced the transformation kinetics of MG in the first two cycles and then surpassed the control from the third cycle until the last. To check whether the concentration of MG or AA would affect the transformation kinetics, the enzymatic reactions were conducted with different concentrations of MG and

Conclusions

The effects of AA on MG transformation in laccase-catalyzed systems were investigated from three aspects: reversibility, inhibition type, and action pathway. The inhibition effect of AA on the laccase-catalyzed transformation of MG was both time and concentration dependent. AA firstly acted as a reversible inhibitor, deriving from the competition for the substrate-binding-pocket of laccase. At a low concentration, the formation of laccase-AA complex dominated, whereas at a high concentration,

Acknowledgments

This research was supported by the National Natural Science Foundation of China (51378254, 21522702, 21677070).

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