DR. ROBERTO PARRA SALDÍVAR
“Uso y producción de enzimas lacasas para aplicaciones en remediación de efluentes con contaminantes emergentes”
Magdalena Rostro-Alanisa, Leticia Ramírez-Cavazosa, Carlos Hernández Lunab, Roberto Parraa*a Centro de Biotecnología FEMSA, Escuela de Biotecnología y Alimentos, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, Col. Tecnológico, 64849, Monterrey, NL, Méxicob Laboratorio de Enzimología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Av. Universidad s/n,Ciudad Universitaria San Nicolás de los Garza, NL 64450, México *[email protected].
Emerging contaminants Chemicals in very low concentrations (ng / Lg / L) in wastewater, aquatic ecosystems and even drinking water supplies.
These compounds lack an established regulation and its health effects were unknown.
Recent studies have shown their carcinogenic, highly toxic implications in the etiology of some chronic diseases and high persistence in the environment.
Laccases (p-diphenol/ oxigen oxidoreductases, EC 1.10.3.2)
The mononuclear T1 site extracts electrons from the reducing substrate and mediates their
transfer to the trinuclear T2/T3 center where molecular oxygen is reduced (Bertrand, et.al., 2002)
The catalytic site is quite conserved, the rest of the enzyme structure shows high diversity
(Gochev, et. al., 2007).
Image S
ourc
e: S
olo
mon, E
. 2008
APPLICATIONS, CURRENT AND POTENTIALS
Waste Water
Treatment
Food Industry
Textile Industry (Dyes),
Pulp and Paper
(biobleachingand biopulping)
BiosensorsChemical Synthesis
The use of laccases in different fields has been studied in the last decades.
Laccases from Pycnoporus sanguineus strains
thermostability profiles, tolerance towards salts and organic solvents,
enzymes from a non-GMO could have food-grade status in many countries.
CS43 was able to degrade several dyes:
Remazol Brilliant Blue Reactive (anthraquinonic),
Acid Red 44 (azo),
Tripthenylmethane.
(Hernández-Luna, et al, 2008)
Their production levels reported are usually low or not as high as other no
thermostable wild laccases.
Selection of Strain
Hypothesis
P. sanguineus CS43 has the potential to be used in industrial scale laccase
production; and the produced laccases have interesting properties that have
advantages compared with other laccases previously reported in scientific literature.
Goals
1. Enhance the P. sanguineus CS43 laccase production and evaluate its scale up potential.
2. Characterize the laccases generated with the selected specific production conditions.
3. Perform a proof of concept over the degradation of the Endocrine Disrupting Chemicals (EDCs) nonylphenol and triclosan using the studied enzymes.
Screening
Design of Experiments
Duplicate runs in 10 L ST
Methodology Flow diagram
µ
Materials and Methods
Results
Fig. 1
Screening:
• TJM was selected for the highest laccase titers and low cost compared
with simmilar medium results (A).
• The co-inducer selected was SO with the highest activity measuerd (B).
From the results of the CCD, the ANOVA showed that the most significant factors in
laccase production (P≤0.001) were:
• The tomato juice medium concentration
• CuSO4 concentration
• the interaction of both concentrations
• Also, with a (P<0.05) the interaction of CuSO4 with soybean oil is important
Using a regression over the response surface, a prediction equation was generated
(Eq.1).
Y= 8514.1 + 15222.1*(TJM) - 6749.3*(CuSO4) + 5658.6*(TJM)^2 + 4246.4*(CuSO4)^2 -
3469.8*(TJM) (CuSO4) + 2080.2*(SO)(CuSO4)
Fig. 3 Plot of values observed versus predicted values of laccase activity at culture day 14 from the DOE experimental and model. (r2=94%, P<0.001).
A C
B
Fig. 4 Response surface models for laccase activity at culture day 14 under DOE experimental conditions. X and Y axis variables: (A) Tomato juice medium (TJM) and CuSO4, (B) SO and CuSO4
(SO) and (C) TJM and Soybean Oil.
Fig. 5 Confirmation of best treatment conditions for laccase activity. (A) Laccase levels observed from repetition of the best treatment condition of DOE (A00): () 36.82% (v/v) TJM, 1% (v/v) SO and CuSO4 3 mmol/L. () 36.82% (v/v) TJM no induced. (B) Enzyme activity profile at stirred tank bioreactor level. () Best stirred tank bioreactor run at A00 conditions. Highest activity registered about day 14 () typical pH profile read at secondary axis () flasks at A00 conditions.
The native strain CS43 of P. sanguineus is an ideal candidate for over-production of highly thermostable
laccases in an inexpensive medium composed of
Tomato juice
CuSO4
Soybean oil
The laccase production was successfully enhanced and scalable.
Increment in laccase activity up to 13 times compared with an optimized GP medium.
The cultivation time (at highest laccase activity) was reduced from 28 to 13 d, with a total
productivity increment of 26 times.
To the best of our knowledge, the levels of laccase activity achieved by P. sanguineus CS43 in
stirred tank bioreactor runs (143 000 IU/L) are the highest reported in the literature for this strain (Vanhulle et al., 2007; Dantán-González et al., 2008; Vite-Vallejo et al., 2009; Uzan et al., 2010; Lomascolo et al., 2002; Van Der Merwe, 2002;
Litthauer et al., 2007).
Conclusions
The model from the CCD indicated that laccase titers could be
significantly enhanced by increasing the concentration of the tomato
juice in the medium.
Also, a reduction of the levels of CuSO4 and/or Soybean oil needs to be
tested in future experiments as it is indicated by the model to achieve
higher laccase levels.
Conclusions
Products
Characterize the generated laccases.
Proof of concept degradation of EDCs
Mat &
Met: L
accase P
urific
atio
n
Culture supernatant from the 10 L STR enhanced production, collected at day 15
Filtration (200 micrometer mesh) + flow filtration (5-10 microns)
Freezing and Thawing
Flow filtrations (0.5 + 0.2 microns)
Ultrafiltration 10,000 MWCO
Anionic IEX (QFF HiTrap 5 ml GE). pH 5, 2 steps elution. Two peaks collected, one for each MW form of laccase.
HIC (Phenyl HP, 5ml GE) Gradient elution. One peak collected at around 0.5 M Na2SO4
Mat & Met: Photometric methods
Protein determinationbicinchoninic acid assay kit (QuantiPro BCA Assay Kit, Sigma, USA) using BSA as standard
UV-visible absorption spectrum each laccase recorded between 300 and 700 nm
Ma
t &
Me
t: C
ha
racte
riza
tio
nOptimal condition
pH:
from pH 2 to 8 (B&R buffer)
with 0.3 mM ABTS, 0.3 mM DMP and 0.3 mM guaiacol
@25°C
Temp:
from 25 to 75 °C. measured inmediatelyafter adding laccase
@ pH3 with ABTS .
Stability
pH:
Tested from pH 2 to 8.0 (B&R buffer)
@ room temperature.
Temp:
Tested from 40-70 °C.
@ pH7 Phosphate buffer,
Kinetic Parameters
Km and Kcat:
for ABTS (0.5 µM to 100 µM)
guaiacol ( 20 µM to 1000 µM)
DMP ( 20 µM to 1000 µM)
@ their optimal pH
calculated from NLSF regressions using Michaelis Menten model (Statistica, Softstat, USA)
All experiments were performed in three replicate. The initial laccase activity was around 200 U/L
Stability: Inactivation modeling
The pH (acidic region) and thermal inactivation through time was
modeled using the 3-parameter model of Aymard and Belarbi (2000)
(Ladero, Ferrero, Vian, Santos, & Garcia-Ochoa, 2005).
(1)
The ratio (A)t/(A)0 represents the enzyme activity remaining after time t.
α and β are complex expressions of rate constants.
C constant indicates the weight between both exponential parts of the equation.
The values were obtained by curve fitting of the plot of the residual enzyme activity versus
time using GraphPad Prism 6.0 software.
Half-life values were calculated accordingly to the equation (1) at (A)t/(A)0 =0.5.
Mat
and M
et:
Inh
ibitio
n a
nd D
egra
dation s
tudie
s
Inhibition Study
NaF, NaN3, urea, Na2SO4, NaCl, EDTA, acetonitrile (ACN), DMF, acetone, ethanol and Dimethyl sulfoxide (DMSO).
Incubated for 5 min
@ room temperature
Measured with ABTS @ pH3.
Degradation of EDCs: Nonylphenol (NP) and Triclosan (TS)
Samples prepared in distilled water with:
Culture Supernatant with 100 U/L of Laccase activity
10 ppm of each EDC
@ pH 5
HPLC 1200 system with XDE-C18 column.
Thermostated at 40 °C.
275 nm for NP and 229 for TCS
Flow 1 mL/min, injection volume of 20µL
Gradient of CAN and 10mM phosphate buffer pH5.
Inhibition Study experiments were performed in three replicates and the degradation profile was executed twice.
• For degradation comparison, an efficiency value was
calculated as the
concentration of analyte degraded, in parts per billion,
the laccase activity per Liter, the hours of treatment
Results
Several Isoforms presented
•Molecular mass estimated by SDS-Page:
85, 68 and 66 kDa
•The pI of 68 and 66 are around 4.5-4.7 by 2D
Gel.
Other
Laccase
Other
laccase
Lac I and
Lac II
Table 1. Purification of the P. sanguineus CS43
laccase isoformsPurification Step Volume
(ml)Total
protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification (fold)
Culture filtered 1178.6 1998.86 53591 26.8 100 1.0
Ultrafiltration 7.0 758.94 38212 50.3 71 1.9
IEX:
LacI 8.3 31.67 2284 72.1 4.3 2.7
LacII 9.1 250.96 14812 59.0 27.6 2.2
Total 17096 60.8 31.9 2.3
HIC:
LacI 1.6 6.37 1784 279.9 3.3 10.4
LacII 2.1 34.85 10137 290.9 18.9 10.9
Total 11921 289.3 22.2 10.8
Purification with IEX
QFF HiTrap 5ml, pH5.
Laccases were eluted with a two-step protocol at 3.5% and 10% of 1 M Na2SO4 at a flow rate of 5 mL min-1.
IEX was run at pH 5.0 with 0.2 M piperazine buffer.
0
20000
40000
60000
80000
100000
120000
0
500
1000
1500
2000
2500
3000
3500
4000
0 50 100 150 200 250 300 350 400
Ab
sorb
ance
at
28
0n
m
Volumes through the column (ml)
Typical Profile for IEX pH5 two steps
UV
Activity (U/L)
Act
ivit
y (U
/L)
Lac I
Lac II
Purification with Hydrophobic Interaction Column
Each fraction selected from IEX was process separately in a HIC Column. More active
fractions were selected for characterization studies.
Lac ILac II
Deglycosilation study
Fig. 1 Electrophoresis of laccase isoforms
from P. sanguineus CS43. Lane M Protein
standard; lanes 1 and 3 purified LacI and LacII
respectively; lanes 2 and 4 deglycosylated
isoforms LacI and LacII.
M 1 2 3 4
55
66
97
116
158
212
42
KDa
UV/Vis spectrum of purified P. sanguineus
laccases
Fig. 2 LacI (blue) and LacII (red)
isoforms. Measurements were
performed in triplicates.
The laccase exhibited an absorbance
peak at 610 nm , indicates the presence
of a type 1 copper atom in the molecule.
A shoulder at around 330 nm in this
spectrum suggests the presence of T3
copper site (Shleev et al., 2004).
Sequencing
Table 2. Amino acid sequence of five internal peptides from P. sanguineus CS43 LacI and LacII
isoforms. The underlined amino acids correspond to the differences between the two laccase
isoforms
Amino acid sequence
Peptide LacI LacII Identity (%)
1 FPNGADATLINGLGR FPPGSDATLINGLGR 87
2 TPGTTSADLAVIK SPGTTAADLAVIK 85
3 YSFVLDASQPVDNYWIR YSFVLDASQPVDNYWIR 100
4 ANPPFGNVGFAGGINSAILR ANPSFGNTGFAGGINSAILR 90
5 SAGSSEYNYDNPVFR SAGSSEYNYDNPIFR 93
Optimal conditions
Fig. 3 pH dependency on (A) LacI and (B) LacII
activities with ABTS (♦), DMP (▲), and guaiacol
(+) as substrates at 25°C. (C) Effect of
temperature on laccase activity with ABTS as
substrate at pH3; Lac I (♦), Lac II (■). Data
points represents the means of three replicates
± SD.
A B
C
pH
Sta
bility
Results
Fig. 4 Stabilities of LacI (A), LacII (B) and CS (C) at different pH; pH 2 (▼), pH 3 (♦), pH 4 (∆), pH 5(*), pH 6 (●), pH 7 (■), pH 8(▲). Curves between data points (solid lines) were fitted by least squares regression to a biexponential function. Dotted straight lines are connecting the dots without regression. Laccase activity was measured using ABTS as the substrate at pH 3. Data points represent the means of three replicates ± SD.
A B
C
Therm
osta
bility
Results
Fig. 5 The thermal stability at pH 7 of Lac I(A), LacII (B), CS (C); 40°C (▼), 50°C (♦), 60°C (■), 70°C (▲), 80°C(●). Laccase activity was measured using ABTS as the substrate at pH 3. Data points represent the means of three replicates ± SD.
Table 3. Half Life at specific temperature
or specific pH
Parameter: R2 Half-life (h)
CS LacI LacII CS LacI LacII
pH
2 0.9926 0.9931 0.9946 5.17 7.44 1.52
3 0.9971 0.9918 0.999 31.6 18.29 2.68
4 0.9876 0.9912 0.9979 81.75 24.89 2.49
5 0.9913 0.9943 0.9959 75.29 33.3 2.87
6 0.98 0.9724 0.9867 290.45 263.32 105.02
Temp C
40 NA NA 0.8984 >1012 >1012 1012
50a 0.9862 0.9733 0.9956 73.64 277.77 35.84
60 0.9772 0.9689 0.98 3.25 18 2.25
70 0.9996 0.9917 0.9987 0.18 0.64 0.14
80 0.9984 0.9991 1 0.02 0.03 0.01
Commercial available laccases as the ones produced by Trametes versicolor or Pleourotus ostreatus, do not share the thermostability capacity demonstrated by LacI.
Stoilova et al., (2010) reported a laccase from Trametes versicolor with an approximated half-life of 6 h at 50°C and 3 h at 60°C.
In Han et al., (2005) study, a half-life for same species laccase was less than 1 h at 60°C.
Rancaño et al., reported half-lives of 75 h and 5 h for 50°C and 60°C respectively (2003).
For Pleurotus ostreatus, Palmieri et al, reported for laccases POXA1w, POXA2 and POXC, half-lives of 3.3, 0.2 and 0.5 h respectively at 60°C (1997),
while Okamoto et al(2000). , and Karp (2012) reported half-lives of 20 and 8 minutes respectively at the same temperature
The thermostability of LacI at 60 °C (18 h) and 70 °C (0.64 h) compete in the high end range with others laccases from the genus Pycnoporus already reported, and to the best of our knowledge, the half-life of LacI at 50°C is from the highest reported for purified P.sanguineus laccases (277.7 h)
Discussion
Table 4. Michaelis-Menten kinetic constants of studied P. sanguineus laccases at their optimal pH.
Substrate Km (µM) Kcat (s-1)Kcat/ Km (mM−1 s-1
)Optimal pH
Lac I Lac II Lac I Lac II Lac I Lac II Lac I Lac II
ABTS 6.9 12.2 519.2 447.2 74816.4 36746.1 2.5 2
DMP 89.2 191.6 184.6 155.6 2068.4 812.2 3.5 3
Guaiacol 1484.5 1100.8 2472.5 2084.4 1665.5 1893.5 4 4
Table 5. The effect of inhibitors on purified laccase activity at 25°C. Laccase activity was measured using ABTS as the substrate at pH 3.
Inhibitors IC50 (mM or %a)Complete inhibition
(mM or %a)
LacI LacII LacI LacII
NaF 0.08 0.02 16 8
NaN3 6.2E-06 6.9E-07 16 16
Urea 5810 3930 >8000c >8000 c
Na2SO4 >800b >800b >800 c >800 c
NaCl 65 14 >2000 c >2000 c
Acetonitrilea >64b 33 >64 c >56 c
DMFa 37 26 64 64
Acetonea 47 31 >72 c >72 c
Ethanola 55 52 >64 c >64 c
DMSOa 41 33 72 72
EDTA 49 42 100 80
Degradation of EDCs
Fig. 6 Degradation profile in aqueous phase with 100 U/L of laccase activity from culture
supernatant, and 10 ppm of EDC at pH 5. (A) NP. (B) TCS. Data points represent the means
duplicates ± SD.
Co
nce
ntr
ació
n (
áre
a/ár
ea
inic
ial)
Co
nce
ntr
ació
n (
áre
a/ár
ea
inic
ial)
Tiempo (h)Tiempo (h)
A
Degradation of EDCs
The efficiencies obtained in the degradation of NP and TCS at pH5 and 25°C
(23.6 and 15.3 ppb*L/U*h respectively) were among the range for NP and high
for TCS in comparison with similar studies with other fungi laccases. (Cabana et
al., 2007; Kim and Nicell , 2006; Murugesan et al. 2010; Saito et al. 2004)
The use of the supernatant instead of the purified laccase can be the base to
develop sustainable methodologies with similar efficiency towards pollutant
degradation than with the purified enzymes while eliminating expensive
purification steps.
Conclusions
CS and the purified LacI and LacII isoforms obtained from the P. sanguineus CS43,
have unique properties that make them ideal for industrial applications as wastewater
treatment.
It was possible to effectively produce them with a cheap tomato juice base
medium on a 10 L bioreactor as stated in the previous work;
they have shown to be especially ideal for applications were high temperature
and/or organic solvents are present.
The have outstanding Michaelis-Menten kinetic parameters for typical substrates
They have high biodegradation capability proved for a pair of EDCs.
Products
Laccases Inmobilization
Advantages and Disadvantage of Immobilization
Hypothesis
Systems immobilized laccase P. sanguineus
that have better physicochemical properties
free enzyme.
Supports
Eupergit
Immobead
Lentikats
Immobilization testPreliminar test with commercial enzyme
According to the preliminary tests it was determined that in order of efficiency, it is considered bettersupport the Lentikats> Eupergit> Immobead.
In addition to assessing immobilization techniques for subsequent and final tests Covalent Link methodwas used because the results obtained show good results.
Immobilization test
Immobilization yields using 25 unitsof enzyme from Pycnoporussangineous applying the techniqueof covalent bonds for Eupergit andImmobead while for Lentikats wasfollowed with the manufacturer'sprotocol.
Enzimatic Activity at different pH
In this graph we can see that there is a shift in the performance of the enzyme when it is immobilized, achieving improve between the ranges of pH 3-5 for the case of Lentikats and Eupergit, while the immobead achieves its maximum activity at pH 3.
Enzimatic Activity at different temperature
In the graph it can be seen that in all cases greater activity was achieved at 70° C for the case of Eupergit and immobead, while due to the plastic nature of Lentikats presents deformation to 55° C so that it was impossible to test at higher temperatures.
Reusability test
After 5 cycles of use can see that there is wear on the catalytic capacity of the supports, according to the results obtained by the performance is as follows: Lentikats> Immobead> Eupergit
Reusability test
* Each cycle was performed saturating ABTS and 0.5 mM citrate phosphate bufferA pH in the optimum of each bracket 25 ° C
Conclusions
The laccases immobilized systems and Eupergit Pycnoporus sanguineus, and Lentikats Immobead supports possess better than free enzyme physicochemical and mechanical properties.
The best method of immobilization for Eupergit and Immobead supports is that of covalent bonds and the best method of immobilization for Lentikats is entrapment.
The enzyme-support systems tested have a satisfactory operation range pH between 2-6 and temperature from 25 ° C to 70 ° C, retaining a high percentage of enzyme activity.
Reusability tests showed the value of fixed assets, compared to only use they have free enzymes systems. Immobead Eupergit and retained enzyme activity 86% and 89% respectively at 5 cycles of use and Lentikats retained 89% of its enzymatic activity after 10 cycles.
Conclusions
Our research group is already working
in the characterization of the degradation of different water micropollutants,
in the complete sequencing of the isoforms to understand the differences that confers LacI a markedly better performance than LacIIin pH and thermal stability as well as better resistance to inhibitors.
Application Testing
Supports Economic functionalization
Scaling catalytic processes
1 Professor3 Postdocs
3 PhD candidates5 MSc candidates
5 Specialists10 Undergraduate students
Exchange students: Germany, Ecuador, Spain
30 people
Thank you!!!
Contact:
Dr. Roberto Parra SaldívarTecnológico de Monterrey, Campus MonterreyCEDES 405-ITelephone: +52 (81) 83582000 ext. [email protected]
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