Download An investigation to develop treatment methods for EDCs in

INTRODUCTION
The use of enzymes to treat endocrine disrupting chemicals (EDCs) and other
emerging pollutants in wastewater is extremely attractive but developers must resolve
many technical and economic issues. These include enzyme sensitivity to
environmental conditions, the difficulty of retaining enzymes in practical reactors the
relatively low concentration of EDC in wastewater, and the relatively high cost of
enzymes for use in wastewater treatment systems. Our studies seek to develop
methodologies for treating EDCs in wastewater using laccases in practical reactor
systems
Insolubilization of these biocatalysts as cross-linked enzyme aggregates (CLEAs) has
been demonstrated to be quite promising in enhancing their stability, reusability and
the possibility of continuous utilization in bioreactors (Ahuja et al. 2004). CLEAs of
laccase secreted by the white rot fungus (WRF) Coriolopsis polyzona have been
prepared by Cabana et al. (2007) using glutaraldehyde (GLU) as the cross-linking
agent. These CLEAs have shown high enzyme activity and higher stability than free
laccase against physical, chemical and biological denaturants and good kinetics of
reaction. These biocatalysts have been successfully used for the continuous treatment
of water contaminated by the endocrine disrupting chemicals bisphenol A,
nonylphenol and triclosan (Cabana et al., 2009).
The first objective of this study was to produce CLEAs of laccase and combinations
of different laccases (Combi-CLEAs) by using chitosan as the cross-linking agent.
The second objective was to determine the effects of the conditions of formation (pH,
temperature, reaction time and shaking speed) on the characteristics of the CLEAs
produced by this new approach.
MATERIALS AND METHODS
Trametes versicolor laccase (TvL) was purchased from Sigma-Aldrich (St-Louis,
MO) and a bacterial laccase (MGL) was provided by MetGen Oy (Turku, Finland).
Chitosan from crab shells (65% deacetylation), N-(3-dimethylaminopropyl)N'ethylcarbodiimide hydrochloride (EDC), 2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS), and 2,6 dimethoxyphenol (DMP) were purchased from either
Fluka Biochemika or Sigma Aldrich .
Enzymatic activity of free and insolubilized laccases were determined by
spectrometrically monitoring the oxidation of ABTS at 420 nm, ε=36000 M-1cm-1
[3] for TvL and combi-CLEAs and the oxidation of DMP at 477 nm, ε=36000 M1cm-1 [4] for MGL and combi-CLEAs. One unit (U) of enzyme activity is defined as
the amount of enzyme required to oxidize 1 µM of substrate (ABTS or DMP) per min.
The following procedure was used to prepare the combi-CLEAs A total combined
volume of 1 U mL-1 of TvL and MGL were precipitated on ammonium sulfate (500
g/L) for 30 min followed by an addition of solutions of chitosan (1 g/L) and EDC (50
mM) for aggregation and crosslinking. Acetate buffer at pH5 completed total solution
to the desired volume (100 mL, usually) and stored at 4 °C for 48h. Subsequently,
combi-CLEAs were extracted by centrifugation for 5 min at 10 000g and 4°C. The
aliquots were then washed 3 times with deionized distilled water and used for
experiments
RESULTS AND DISCUSSION
Graph1 shows the impact of pHs on the laccase activity of TvL CLEAs versus the
Combi-CLEAs using ABTS as substrate at 20°C. One notes that both of
these CLEAs have highest relative activity at pHs in the 3- 5 range. The CombiCLEAs have lower relative activity than the TvL CLEAs. Graph 2 shows the
corresponding results using dimethoxyphenol (DMP) as substrate. The pH range has
been extended and relative activities are better for pHs in the range of 8.
Graph 3 and 4 shows the influence of temperature on the laccase activity at pH 4 and
8 using both ABTS and DMP as substrate. We are much more successful at obtaining
high relative activities at ph 8 over a broad range of temperature (20 – 60°C) for DMP
using Combi-CLEAs. Consequently, the Combi-CLEAs produced can be used over a
wide temperature range without decrease in laccase activity. These novel laccasebased biocatalysts increase the potential of laccase utilization for wastewater
treatment.
Free enzymes (Trametes versicol and MetGen laccases) have been successfully
insolubilized as combi-CLEAs. Combi-CLEAs synthesized exhibit optimal activity at
50°C with both ABTS and DMP. Activity retention appears high (50% to higher) with
temperature variation for the biocatalyst except with ABTS at 70°C (>80% activity
loss), which implies a great thermostability Similarly to the free enzymes,
insolubilized fungal enzyme (TvL) is more oxidative in acidic pH than the bacterial
enzyme (MGL) whereas the bacterial enzyme exhibit greater oxidation than the fungal
enzyme in alkaline pH.·
Acknowledgements: The authors are thankful to Antoine Mialon, PhD from MetGen
Oy, Turku, Finland (http://www.metgen.fi) for providing the MetGen Laccase (MGL)
enzymes.