aPhysical Chemistry Department, School of Industrial Engineering, University of Castilla-La Mancha, Albacete, Spain
bDepartment of Science and Materials Engineering and Chemical Engineering, University Carlos III of Madrid, Leganés (Madrid), Spain
Edelmira Valeroa, María-Isabel González-Sáncheza and María-Teresa Pérez-Priorb
ENZYME KINETICS
EXPERIMENTAL
ENZYME TECHNOLOGY
THEORETICAL
RESEARCH LINES
ANILINE NH2
Polyurethane foam
Agricultural chemicals
Synthetic paints
Antioxidants
Rubber industry
Herbicides
Varnishes
Explosives
It is a precursor to many industrial chemicals:
Conducting polymer polyaniline
Drugs, e.g. paracetamol
Dyes and pigments
TOXICITY OF ANILINE
Aniline is classified as a toxic substance, dangerous for the environment.
It is harmful by inhalation, ingestion or skin contact.
It induces the formation of methemoglobin in blood (MetHb Fe(III)), which is unable to carry oxygen.
Exposure to high concentrations of aniline can cause cyanosis and, in extreme cases, the worker's death from anoxia. Cyanosis manifests as a bluish more or less extensive on lips, nose, ears and nails.
Chronic exposure may cause central nervous system disorders, anemia and general weakness; these clinical symptoms appear when [metHb] > 10%.
Carcinogenic potential: the EU classifies aniline in category 3, i.e. a substance whose potential carcinogenic effects in humans are disturbing but sufficient information is not available for making a satisfactory assessment.
It is very toxic to aquatic organisms.
TREATMENT IN WASTE WATER
CURRENT METHODS:
Chemical oxidation
Bacterial degradation
Adsorption with activated charcoal
ENZYMATIC METHODS: OXIDOREDUCTASE ENZYMES
Peroxidases
Polyphenol oxidases
Laccases
HEMOGLOBIN
OxyHb Fe(II): Transports O2 from the respiratory organs to the rest of the body.
MetHb Fe(III): Unable to transport oxygen
BOVINE METHEMOGLOBIN
HEMOGLOBIN IS NOT AN ENZYME
MetHb
Peroxidase Catalase
BOVINE METHEMOGLOBIN
Peroxidase
Catalase 2 H2O2 H2O + O2
MetHb
CoICoII
H2O2
SS·
S
S·
Hbi
M.I. González-Sánchez, M.C. Manjabacas, F. García-Carmona and E. Valero. Mechanism of acetaminophen oxidation by the peroxidase-like activity of methemoglobin. Chem. Res. Toxicol., 2009, 22, 1841-1850.
M.I. González-Sánchez, F. García-Carmona, H. Macià and E. Valero. Catalase-like activity of human methemoglobin: A kinetic and mechanistic study. Arch. Biochem. Biophys., 2011, 516, 10-20.
BOVINE METHEMOGLOBIN
OTHER SUBSTRATES THAT CAN BE OXIDIZED:
Aromatic amines
Phenolic compounds
Chlorophenols
Drugs, e.g. paracetamol, chlorpromazine, etc.
Hormones (estrogens)
Polycyclic aromatic hydrocarbon (PAH), e.g. anthracene
Chlorpyrifos
Other
M.T. Pérez-Prior, R. Gómez-Bombarelli, M.I. González-Sánchez and E. Valero. Biocatalytic oxidation of phenolic compounds by bovine methemoglobin in the presence of H2O2. Quantitative structure-activity relationships. J. Hazard. Mater., 2012, 241-242, 207-215.
ANILINE OXIDATION BY THE PEROXIDASE-LIKE ACTIVITY OF BOVINE MetHb
t (min)
0 60 120 180
(%)
AN
IL
0
20
40
60
80
100
metHb
H2O2
t (min)
0 30 60 90 120
(%)
AN
IL
0
20
40
60
80
100A B
[ANIL]0 = 0.5 mM [metHb]0 = 5.0 µM [H2O2]0 = 2.5 mM pH 6.0
pH
4 6 8 10(%
) A
NIL
0
20
40
60
80
100
[ANIL]0 = 1.2 mM [metHb]0 = 5.5 µM [H2O2]0 = 2.5 mM
ANILINE OXIDATION BY THE PEROXIDASE-LIKE ACTIVITY OF BOVINE MetHb
t (min)
0 30 60 90 120
(%)
AN
IL
0
30
60
90
120
[H2O2]0 (mM)
0 1 2 3 4 5 6
(%)
AN
IL
0
20
40
60
80
100A B
t (min)
0 30 60 90 120
(%)
AN
IL
0
30
60
90
120
[metHb]0 ( M)
0 1 2 3 4 5 6 7 8
(%)
AN
IL
0
20
40
60
80
100A B
[ANIL]0 = 0.5 mM [metHb]0 = 5.0 µM pH 6.0
[ANIL]0 = 0.5 mM [H2O2]0 = 2.5 mM pH 6.0
CATALASE-LIKE ACTIVITY OF BOVINE MetHb IN THE PRESENCE OF ANILINE
t (min)
0 2 4 6 8 10 12 14
[O2] t-
[O2]o
(
M)
0
30
60
90
120
A
B
A) [ANIL]0 = 0.7 mM B) [ANIL]0 = 2.0 mM [H2O2]0 = 3 mM [metHb]0 = 1.5 µM pH 6.0
CATALASE-LIKE vs PEROXIDASE-LIKE
V0,p /[metHb]0 (min-1
)
5 20 35 50V
0,c
/[m
etH
b] 0
(m
in-1
)
0
2
4
6
HQ4AP
4TBP
R2 = 0.90
B
MPB
4HBZ
4CP
4HACP
PHE
4MP
4CNP
4HPA4NP
APAP
t (min)
0 10 20 30 40
[O2]-
[O2] 0
(M
)
0
20
40
60
80
100A
metHb + H2O
2
HQ
MPB
4HACP
[SUBSTRATE]0 = 0.9 mM [H2O2]0 = 4 mM [metHb]0 = 3.0 µM pH 6.0
0,0,
0 0
4.22 0.08[ ] [ ]
percat VV
metHb metHb
R2 = 0.90
M.T. Pérez-Prior, R. Gómez-Bombarelli, M.I. González-Sánchez and E. Valero. Biocatalytic oxidation of phenolic compounds by bovine methemoglobin in the presence of H2O2. Quantitative structure-activity relationships. J. Hazard. Mater., 2012, 241-242, 207-215.
IMMOBILIZATION OF THE PROTEIN
Calcium alginate
Eupergit C
IMMOBILIZATION OF THE PROTEIN
t (min)
0 30 60 90 120
(%)
AN
IL
0
30
60
90
120
A
B
time (min)
0 50 100 150
(%)
4-C
P
0
20
40
60
80
100
Calcium alginate Eupergit C
[ANIL]0 = 0.25 mM [H2O2]0 = 0.25 mM [metHb] = 35 pellets pH 7.0
[4-CP]0 = 0.25 mM [H2O2]0 = 0.25 mM [metHb] = 10 mg pH 7.0
CONCLUSIONS AND FUTURE WORK
Finally, from the results here shown it can be concluded that the use of
bovine metHb instead other peroxidase enzymes like HRP might
represent a good alternative at economical level.
To confirm this suitability in a simple way, we performed an experiment
consisting of oxidizing 4CP to the same extent (50 %) by the two proteins
under the same experimental conditions ([H2O2]0 = 2.5 mM and [4CP]0 =
0.5 mM). The required amount of metHb and HRP was 5.0 and 0.02 M,
respectively, which implies a cost of 0.18 and 0.33 €/ml of assay,
respectively (prices updated from Sigma).
Therefore, metHb from animal sources, which can easily be obtained
from blood, a waste of the meat industry, could be used effectively for
aromatic amines and phenols removal.
These prices may seem expensive when extrapolated to industrial scale,
but they may be drastically reduced by immobilizing the protein and
searching for new materials to protect it from H2O2-induced inactivation.
ACKNOWLEDGEMENTS
The authors acknowledge financial support from
The Regional Ministry of Science and Technology
(JCCM, Spain)
POII-10-0235-8597
THANK YOU VERY MUCH FOR YOUR ATTENTION
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