Diapositiva 1 - Universidad ORT Uruguay

Biomimetic silica nanospheres: a versatile nanotool for protein
immobilization.
Erienne Jackson1, Mariana Ferrari1, Valeria Grazú2, Jesús Martinez de la Fuente3, Lorena Betancor1 .
1 Laboratorio de Biotecnología, Universidad ORT Uruguay, Montevideo, Uruguay;
2 Universidad de Zaragoza, Zaragoza, Spain;
3Institute of Materials Science, Spanish Research Council, Aragón, Spain, Institute of Nano Biomedicine and Engineering.
Biotechnology
Department
Lorena Betancor, PhD
[email protected]
Diatoms inspirate nanotechnology
+ silicic acid
Kroger et al., Science 1999
Biomimetic silica: support for protein immobilization
Silaffin peptide or
poly amine molecule
+ Solution of Silicic acid
+ enzyme
Biomimetic silica: support for protein immobilization
Enzyme
Butyrylcholinesterase
Organophosphate hydrolase
Organophosphate acetic
anhydrolase
Application
Biosensor for nerve agent
detection
Biosensor for nerve agent
detection or decontamination
system
Polyamine
molecule
Immobilization
efficiency (%)
R5
90
R5
25
R5
75
Hydroxylaminobenzene
mutase
Biocatalysis of amino-substituted
aromatics
R5
80
Soybean peroxidase
Biocatalysis
R5
60
Nitrobenzene nitroreductase
Activation of prodrugs
PEI / Lys
48 / 21
Glucose oxidase
Biosensor, Biocatalysis
R5 / PEI / Lys
51/ 71/ 24
b-galactosidase
Biocatalysis
R5 / PEI / Lys
45 / 42 / 16
Catalase
Biosensors and biosynthesis
R5
75
Betancor and Luckarift, (2008) Trends Biotechnol. 26, (10), 566
Protein entrapment in biosilica nanoparticles.
 Rapid. Immobilization occurs within seconds.
 Mild. Formation of the particles occurs at room temperature and pH 8.
 Green. No requirement of organic solvents or high temperatures.
 Nanosized. 300 to 500 nm, Lower diffusion limitations and higher
volumetric activities.
Robust. Physical properties suitable for flow-through applications.
 Stabilizing. Numerous enzymes have been stabilized by entrapment in this
support.
 Polymorphous. Shapes can be tailored by varying the conditions of silica
deposition.
Betancor et al. (2006) Chem. Comm. (34), 3640.
Betancor and Luckarift, (2008) Trends Biotechnol. 26, (10), 566
Betancor et al. (2006) Biomacromolecules., 7(9), 2631.
New perspectives in biomimetic silica nanoparticles
(a)
Foto 15
(b)
Tetramethyl
orthosilicate
Polyethylenimine
(PEI)
+
Betancor and Luckarift, (2008) Trends Biotechnol. 26, (10), 566
Buffer phosphate
0.1M, pH 8
+
Na2HPO4
=
New perspectives in biomimetic silica nanoparticles
Protein templated nanoparticles
Foto 15
(b)
DLS
Z potential
Sample
Size (nm)
Silica-PEI
Foto 15
Silica-PEI-BSA
Tetramethyl
orthosilicate
798.7 ± 100.8 0.639 ± 0.2 38 ± 0.8
374.4
± 36.4
Foto 12
Polyethylenimine
(PEI)
+
(mV)
PDI*
0.409 ± 0.1 32 ± 0.7
Buffer phosphate
0.1M, pH 8
+
Na2HPO4
+
=
protein
Protein entrapment
Foto 12
Jackson et. Protein templated biomimetic silica nanoparticles. (2015) Langmuir. Submitted
Use of external particle surface for protein immobilization:
KDa
200
116
97
66
1
2
3
4
5
6
7
8
9
10
45
29
Protein concentration in the supernatant (mg/mL)
Adsorption of E coli protein extract.
2,5
2,0
1,5
1,0
0,5
0,0
0
50
100
150
200
Time (min)
KDa
116
97
66
45
29
1
2
3
4
5
6
7
Protein concentration in the supernatant (mg/mL)
Desorption of E coli protein extract.
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0
200
400
600
800
NaCl (mM)
1000
1200
Immobilization of Laccase from Trametes versicolor
Betancor, L., et al. (2013). ChemCatChem, 5(1), 46.
Adsorption of Laccase from Trametes versicolor
0,1
16
Adsorption
14
0,08
12
0,07
Activiy (UI)
Activity (UI)
Desorption
0,09
10
8
6
0,06
0,05
0,04
0,03
4
0,02
2
0,01
0
0
0
10
20
30
40
50
60
70
0
200
t (min)
400
600
[NaCl] (mM)
Adsorption
Desorption
13.4 ± 1.5 mg
15.3 ± 0.5 mg
Loading capacity using Laccase Tv.
mg protein/g particles
Silica PEI
42.8 ± 7.4
Silica PEI-BSA
57.3 ± 4.5
800
1000
1200
New perspectives in biomimetic silica nanoparticles
Use of external particle surface for protein immobilization through covalent interaction
Tetramethyl
orthosilicate
Polyethylenimine
(PEI)
+
BSA
+
Silica PEI+BSA
Glutaraldehyde
+
Enzyme
+
NH2
Covalent linkage
Immobilization of lipases on biomimetic silica
nanoparticles.
Lipase sources

Rhizomucor miehei lipase(RML)

Thermomyces lanuginosus lipase (TLL)

Bacillus thermocatenolatus lipase(BTL2
Advantages of nanosized supports in biocatlysis:
 Large functional surface area
 Increased enzyme loading
 Reduction in diffusion limitations
 Enhanced particle mobility
Lipase immobilization in/on biomimetic silica
nanoparticles
Enzyme
Strategy
Immobilization (%)
Yield (%)
Physical entrapment
90,0  0, 8
24,0  0,6
Covalent linkage
87,0  0, 4
84,0  0,7
Physical entrapment
40,0  0,9
30,0  0,7
Covalent linkage
74,0  0,9
100  1
Physical entrapment
91,0  0,1
47,00  0,09
Covalent linkage
95,0  0,1
47,00  0,08
RML
TLL
BTL2
Fig. 1 TEM image of
silica nanoparticles
Thermal stability (60°C) of lipase preparations
Enzyme
Strategy
Half life time (min)
RML
soluble enzyme
Physical entrapment
Covalent linkage
2,5 ± 0,2
70 ± 1
60 ± 1
TLL
soluble enzyme
Physical entrapment
Covalent linkage
140 ± 1
160 ± 1
360 ± 5
BTL2
soluble enzyme
Physical entrapment
Covalent linkage
25 ± 1
125 ± 5
63 ± 1
100
90
80
70
60
50
40
30
20
10
0
RML Covalent linkage
100
Residual activity (%)
Residual activity (%)
RML Physical entrapment
RML Si
RML Sol
80
RML SiGlu
60
RML Sol
40
20
0
0
20
40
60
80
T(min)
100
120
0
50
100
150
200
250
T (min)
300
Stability of RML immobilized against solvents.
Stability in 50% ethanol
120
120
100
100
Residual activity (%)
Residual activity (%)
Stability in 50% ethanol
80
60
40
80
60
40
20
20
0
0
0h
1h
0h
72 h
Stability in 100% tert-butanol
1h
72 h
120
Residual activity (%)
100
80
60
40
20
0
0h
1h
96 h
Stabilities were performed
at 25°C incubating 13 IU/mL
Ascorbic palmitate synthesis
Sin título
0,024
0,022
0,020
Ascorbic acid/Palmitic Acid
0,018
0,016
0,014
AU
0,012
0,010
0,008
H2O
HO
Lipase
0,006
0,004
0,002
0,000
2,00
0,190
4,00
Sample Name: sustrato de RML;
UYT
6,00
Vial: 1;
8,00
Minutes
Injection: 4;
10,00
Channel: 486 ;
12,00
Sin14,00
título
Ascorbic acid
Palmitic acid
Ascorbic palmitate
Date Acquired: 9/24/2014 12:32:18 PM
0,180
0,170
Reported by User: System
Report
0,160Method: Sin título
Report Method ID: 101
101
Project Name: Defaults
Date Printed:
10/14/2014
4:32:58 PM America/Montevideo
0,150
Ascorbic palmitate
0,140
0,130
0,120
0,110
AU
0,100
0,090
0,080
0,070
0,060
0,050
0,040
0,030
0,020
0,010
0,000
-0,010
2,00
0,013
4,00
6,00
Sample Name: PA 5 mM reaccion 6-10-2014;
2:54:41 PM UYT
8,00
Minutes
Vial: 1;
10,00
Injection: 4;
12,00
Channel: 486 ;
0,012
Sin título
14,00
Date Acquired: 10/7/2014
Reaction after 1 hour at 37°C
6 % conversion
Reported by User: System
Report Method: Sin título
0,011Method ID: 101
Report
101
Project Name: Defaults
Date Printed:
10/14/2014
4:44:43 PM America/Montevideo
0,010
0,009
0,008
AU
0,007
0,006
0,005
0,004
0,003
0,002
0,001
0,000
-0,001
2,00
Sample Name: RML 1h ;
Reported by User: System
Report Method: Sin título
Report Method ID: 101
101
4,00
Vial: 1;
6,00
Injection: 1;
8,00
Minutes
Channel: 486 ;
10,00
12,00
14,00
After 24 hours of reaction course, the
immobilized preserved 80% of activity
measured with pNPB
Date Acquired: 9/24/2014 2:34:51 PM UYT
RP-HPLC analysis: Luna C18 column, 250 x 4.56, 1 mL/min, Methanol: H2O: Acetic acid, 95:5:0,5 (v:v:v), UV detection at 266 nm
Project Name: Defaults
Date Printed:
10/14/2014
4:38:12 PM America/Montevideo
GFP immobilization in biomimetic
silica nanoparticles
GFP immobilization
•100% immobilization
•Minimal decrease in fluorescence after immobilization
pH stability of entrapped GFP
Conclusions:
.An inert protein template (BSA) enabled the rapid synthesis of positively charged disperse
200-300 nm particles as opposed to the synthesis without template which produced larger
and less size homogenous nanoparticles.
The particles synthesized in the presence of BSA reversibly and ionically adsorbs a range of
proteins.
. Glutaraldehyde functionalization of silica nanoparticles provided the possibility of covalently
attached proteins on the particle surface.
Silica particles provided stability against temperature, pH, organic solvents . In the case of
lipasas it also provided operational stability.
.
ACKNOWLEDGMENTS
Protein technology group
Collaborators
Mariana Ferrari, PhD candidate
Dr. Jesús Martinez de la Fuente
Erienne Jackson, MSc candidate
Dr. Valeria Grazú
Josefina Louge, Research assistant