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RESULTADOS:
N
Sol. Std uL
Agua uL
Concentracion
prot g/l
ABS 595nm
1
2
3
4
5
6
7
8
100
80
60
50
40
20
10
0
0
20
40
50
60
80
90
100
0.5
0.4
0.3
0.25
0.2
0.1
0.05
0
0.167
0.15
0.115
0.13
0.061
0.007
0.02
0
1. DETERMINACION DE PROTEINAS
Tenemos la curva de calibrado de Bradford para la determinacin
de protenas.
0.15
absorvancia
0.1
0.05
0
0
0.1
0.2
0.3
0.4
concentracion (g/l)
0.5
0.6
Y=0.3721x-0.0025
Absorbancia =0.3721
concentracin -0.0025
Dilucin de enzima
ABS
(595 nm)
Concentracin
(g/l = mg/ml)
B1
1:2
0.0345
0.0994
B2
1:1
0.0655
0.1827
B3
1:4
0.0340
0.0981
C1
1:1
0.055
0.1411
C2
1:3
0.001
0.0094
C3
1:6
0.005
0.0201
D1
1:2
0.081
0.2244
D2
1:3
0.0285
0.0833
Sol.
Estandar
(mL)
H2O
dest
(mL)
1
2
3
4
5
6
7
0.5
0.4
0.3
0.2
0.1
0.05
0
0
0.1
0.2
0.3
0.4
0.45
0.5
Azuc
ar
red
(g/L)
2
1.6
1.2
0.8
0.4
0.2
0
Abs
540n
m
0.797
0.786
0.487
0.374
0.197
0.11
0
0.6
ABSORVANCIA 0.4
0.2
0
0
0.5
1.5
CONCENTRACION
Absorbancia =0.4179
concentracin -0.00229
2.5
0.3137
CONCENTRA
CION
CORREGIDA*
0.0431
CONCENTRA
CION
AJUSTADA**
0.0000
1.6370
3.2740
0.9311
2.9603
0.839
1.9529
5.8586
1.7408
5.5449
1:4
0.845
1.9672
3.0708
7.5552
1:5
0.91
2.1228
7.8689
10.613
7
3.9923
10.3001
01:01
0.05
01:01
0.025
TIEMPO
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
1.1
0.154
0.3137
1:2
0.707
10
1:3
15
20
Sustrat
o
Enzima
0.1247
GRUPO: C1
0.2276
CONCENTRA
CION
CORREGIDA*
0.0431
CONCENTRA
CION
AJUSTADA**
0
0.5578
1.1156
0.9311
0.888
0.291
0.6415
1.9246
1.7408
1.6977
1:4
0.363
0.8138
3.2553
3.0708
3.0277
1:5
0.372
0.8354
4.1768
3.9923
3.9492
01:01
0.073
01:01
0.027
TIEMPO
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
1.1
0.118
0.2276
1:2
0.256
10
1:3
15
20
Sustrat
o
Enzima
0.18445
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
0.0744
CONCENTRA
CION
CORREGIDA*
-0.0335
CONCENTRA
CION
AJUSTADA**
0.0000
1.1
0.054
0.0744
1:2
0.063
0.0960
0.1919
0.0840
0.0505
10
1:3
0.065
0.1007
0.3022
0.1943
0.1608
15
1:4
0.071
0.1151
0.4604
0.3525
0.3190
20
1:5
0.102
0.1893
0.9464
0.8385
0.8050
Sustrat
o
Enzima
01:01
0.052
01:01
0.016
CONCENTRA
CION
AJUSTADA**
0.0000
0.1079
GRUPO D1:
TIEMPO
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
1.1
0.192
0.4046
0.4046
CONCENTRA
CION
CORREGIDA*
-0.2178
1:2
0.255
0.5554
1.1108
0.4884
0.4884
10
1:3
0.256
0.5578
1.6734
1.0510
1.0510
15
1:4
0.192
0.4046
2.0232
1.4008
1.4008
20
1:5
0.325
0.7229
2.8916
2.2692
2.2692
Sustrat
o
Enzima
01:01
0.155
01:01
0.128
0.6224
TIEMPO
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
0.2108
CONCENTRA
CION
CORREGIDA*
0.0311
CONCENTRA
CION
AJUSTADA**
0
1.1
0.111
0.2108
1:2
0.25
0.5434
1.0869
0.9072
0.8761
10
1:3
0.399
0.9000
2.6999
2.5202
2.4891
15
1:4
0.438
0.9933
3.9732
3.7935
3.7624
20
1:5
0.466
1.0603
5.3015
5.1218
5.0907
Sustrat
o
Enzima
01:01
0.073
01:01
0.025
CONCENTRA
CION
AJUSTADA**
0
0.1797
GRUPO D2:
TIEMPO
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
1.1
0.252
0.5482
0.5482
CONCENTRA
CION
CORREGIDA*
0.3685
1:2
0.506
1.1560
4.6241
4.4444
4.0759
10
1:3
0.664
1.5341
7.6705
7.4908
7.1223
15
1:4
0.425
0.9622
9.6219
9.4422
9.0737
20
1:5
0.515
1.1776
11.5958
11.2273
Sustrat
o
Enzima
01:01
0.088
01:01
0.01
GRUPO B3:
11.775
5
0.1797
TIEMPO
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
0.1606
CONCENTRA
CION
CORREGIDA*
0.0527
CONCENTRA
CION
AJUSTADA**
0
1.1
0.09
0.1606
1:2
0.23
0.4956
0.9911
0.8832
0.8305
10
1:3
0.277
0.6080
1.8241
1.7162
1.6635
15
1:4
0.302
0.6679
2.6715
2.5636
2.5109
20
1:5
0.29
0.6391
3.1957
3.0878
3.0351
Sustrat
o
Enzima
01:01
0.053
01:01
0.015
CONCENTRA
CION
AJUSTADA**
0
0.1079
Diluci
n
ABSORVA
NCIA
Concentra
cin
conc.*f
d
1.1
0.079
0.1342
0.1342
CONCENTRA
CION
CORREGIDA*
-0.0072
1:2
0.203
0.4310
0.8619
0.7205
0.7205
10
1:3
0.214
0.4573
1.3719
1.2304
1.2304
15
1:4
0.231
0.4980
1.9919
1.8504
1.8504
20
1:5
0.224
0.4812
2.4061
2.2647
2.2647
Sustrat
o
Enzima
01:01
0.065
01:01
0.017
0.141
10
10
15
tiempo (minutos)
20
25
y = 0.5039x + 0.2331
R = 0.9964
ACTIVIDAD ENZIMATICA
Actividad volumtrica
P gr
T l min
g
L min
av velocidad RL x
1000ml
2mol
360 g
106 mol
1mol
Consideraciones importantes:
Volumen del reactor: 3ml
Volumen de enzima: 0.1 ml
Peso molecular:
- 1 mol glucosa = 180 g
- 1 mol fructosa = 180 g
Reemplazando
3 g
av 0.5039
x
0
.
1
L min
a v =83.98
1000ml
2mol
x
360
g
10 6 mol
x
1
mol
UI
ml
ACTIVIDAD ESPECIFICA
Ahora, si queremos determinar la actividad especfica,
tendremos que determinar la concentracin de enzima en
mg/ml, a partir de: La curva de calibracin de Bradford.
ABSORVANCIA = A (ENZIMA)+B
y = 0.3721x - 0.0025
R = 0.9125
A: PENDIENTE= 0.3721
B: INTERCEPTO= -0.0025
Despejando las enzimas:
Enzima=
Absorvancia+0.0025
0.3721
Enzima
0.0655 0.0025
gr
0.1827
0.3721
l
Enzima. 0.1827
UI
ml
ae
mg
(0.1827)
mg
ml
83.98
ae
459.66UI
mg
ml
av
(enzima ) mg
ml
Enzima
0.0340 0..0025 gr
0.3721
l
UI
av
ml 856.94 UI
ae
mg ae
mg
mg
mg
Enzima
.
0
.
098
(enzima )
(
0
.
098
)
ml
ml
ml
83.98
Enzima
0.005 0..0025
gr
0.0201
0.3721
l
UI
av
ml
ae
4178.11UI
mg ae
mg
mg
mg
Enzima
.
0
.
0201
(enzima )
(0.0201)
ml
ml
ml
83.98
curva de lineawer-Burk
0.01
0.01
0.01
f(x) = 0.03x + 0
R = 0.99
0.01
0.01
1/ v
0.01
0.00
0.00
0.00
0.00
0.00
0.04
0.06
0.08
0.10
1/s
0.12
0.14
0.16
0.18
y = 0.0267x + 0.0045
Se usa el intercepto en el eje x
1
km
Hallando el intercepto
Y= O
1
=0.1685
km
Km=5.9342
Y para hallar el
1
=0.0045
vm
Vm=222.22
Obteniendo asi
kmax
5.934
2
V max
222.2
2
Usamos la ecuacin
Eucacion de la curva de calibrado (DNS)
Grupo A
N
Tiempo
Dilusio
nes
ABS
Concentraci
on
0
1
2
DNS
0
0.5
1
1
2
0
0.053
0.156
0.313
1.5
0.243
0.331
2.5
0.244
0.348
3.5
10
0.223
10
0.238
0
0
0.60155239
3
1.27856834
8
1.95342820
2
2.71237602
4
2.94307891
3
4.28848641
7
4.45235015
1
4.77576541
6
Grupo B
ANEXOS
p vs t
0.7
0.6 f(x) = 0.04x + 0.03
R = 0.98
0.5
0.4
producto 0.3
0.2
0.1
0
0
10
12
14
16
tiempo
y =0.4179x - 0.00229
PUNTO 1: 2(g/l)
TUBO
N
1
2
t (min)
ABS
0
2
0.018
0.046
dilucin
(1:2)
2
2
producto
(g/L)
0
0.1105527
0.093
10
0.124
15
0.155
6
0.3354869
6
0.4838478
1
0.6322086
6
p vs t
2.5
2
1.5
producto
1
0.5
0
0
10
12
14
16
tiempo
Punto 2: 4 (g/l)
TU
BO
N
t
(m
in)
AB
S
10
0.0
1
0.0
91
0.1
46
0.3
dilu
cin
(1:2
)
2
product
o(g/L)
0.32591
529
0.58913
616
1.32615
458
2
2
15
0.4
47
2.02967
217
Punto 3 : 6 (g/l)
TUB
O N
1
2
t
(min
)
0
2
ABS
0.031
0.078
diluci
on
(1:2)
2
2
0.195
10
0.317
15
0.443
producto(
g/L)
0
0.2636994
5
0.8236420
2
1.4075137
6
2.0105288
3
P vs t
Produc
to
pvs t
3
2
producto (g/l)
8 10 12 14 16
tiempo
t
(min
)
ABS
10
15
0
0.09
8
0.21
1
0.33
5
0.47
7
Diluc
ion
(1:2)
2
2
2
2
2
producto
(g/L)
0
0.359416
13
0.900215
36
1.493658
77
2.173247
19
p vs t
3.0
2.5
2.0
producto (g/l) 1.5
1.0
0.5
0.0
0
10
12
14
16
tiempo(min)
t
(min
)
0
2
6
4
5
10
15
ABS
diluci
on
(1:2)
0.051
2
0.113
2
0.242
2
0.389
0.548
2
2
producto
(g/L)
0.00000
0.43120
1.04858
1.75209
2.51304
p vs t
2.5
2 f(x) = 0.15x + 0.11
1.5 R = 0.99
producto (g/l)
1
0.5
0
0
10 12 14 16
tiempo
t
(min)
ABS
1
2
0
2
0.043
0.137
diluci
on
(1:2)
2
2
0.225
10
0.366
15
0.512
producto
(g/L)
0
0.546063
65
0.967217
04
1.642019
62
2.340751
38
p vs t
3
2
producto (g/l)
10 12 14 16
tiempo
N
TUBO
t
(min)
ABS
1
2
0
2
0.05
0.098
Diluci
on
(1:2)
2
2
0.197
10
0.291
15
0.452
product
o (g/L)
0
0.35941
613
0.83321
369
1.28308
208
2.05360
134
S vs t
3
2.5
2
sustrato (g/l) 1.5
1
0.5
0
0
8 10 12 14 16
tiempo
t
(min
)
0
10
ABS
0.06
7
0.14
6
0.23
4
0.38
diluc
ion
(1:2)
2
product
o(g/L)
0.589136
16
1.010289
54
1.718592
2
2
15
2
0.54
6
96
2.503469
73
S vs t
4
3
10 12 14 16
tiempo
TU
BO
N
1
t
(mi
n)
0
AB
S
0.11
2
0.14
6
diluc
ion
(1:2)
2
producto
(g/L)
0.589136
16
0.32
3
10
0.46
2
15
0.66
1.436228
76
2.101459
68
3.049054
8
s vs t
3
2 f(x) = 0.16x + 0.05
R = 1
sustrato (g/l)
1
0
0
10 12 14 16
tiempo
t
(min
)
0
ABS
0.02
1
0.11
6
diluci
on
(1:2)
2
sustrato
(g/L)
0.445561
14
10
15
0.21
8
0.37
7
0.52
3
2
2
2
0.933716
2
1.694663
8
2.393395
55
5. INMOVILIZACION DE ENZIMAS
Utilizando la ecuacion de calibrado:
ABS=0.4638(CONCEN)+0.0165
GRUPO A: ENZIMA (1:1)
Sacaro
sa
45ml
Sol.Tamp
on.
0ml
Concentrac
ion
20
TI
E
M
P
O
TU
BO
DILUC
ION
ABSO
RBAN
CIA
CONC
ENTR
ACION
COR
RECC
ION
01:01
0.053
0.0000
0.
5
1
01:02
0.156
0.3008
01:02
0.313
0.6393
1.
5
2
01:04
0.243
0.4884
01:04
0.331
0.6781
2.
5
3
01:06
0.244
0.4905
01:06
0.348
0.7147
3.
5
4
01:10
0.223
0.4452
01:10
0.238
0.4776
0.00
000
0.60
155
1.27
857
1.95
343
2.71
238
2.94
308
4.28
849
4.45
235
4.77
577
x (P/si)
0
0.03007762
0.06392842
LN(1-X)
0
-0.03053923
-0.06606333
e
V
V=ke
k
(SI/K)*X
0
0.10136189
0.21543942
45
222.22
5.9347
ket/K
0
0.13190112
0.28150275
0.09767141
0.1356188
0.14715395
0.21442432
0.22261751
0.23878827
-0.10277653
-0.14574141
-0.15917622
-0.24133848
-0.25182278
-0.27284373
0.32915366
0.45703675
0.49591031
0.72261217
0.75022329
0.80471893
0.43193019
0.60277816
0.65508653
0.96395065
1.00204607
1.07756266
Sol.Tamp
on.
Concentrac
ion
35ml
10
15.5
TI
E
M
P
O
TU
BO
0.
5
1
1.
5
2
2.
5
3
3.
5
4
DILUC
ION
ABSO
RBAN
CIA
01:01
0.039
01:02
0.387
01:02
0.744
01:04
0.55
01:04
0.72
01:06
0.62
01:06
0.774
01:10
0.526
01:10
0.582
5
6
7
8
9
e
V
V=ke
k
CONC
ENTR
ACION
COR
RECC
ION
0.048
5
0.798
8
1.568
6
1.150
3
1.516
8
1.301
2
1.633
2
1.098
5
1.219
3
0.04
851
1.59
767
3.13
713
4.60
112
6.06
727
7.80
724
9.79
948
10.9
8534
12.1
9276
45
222.22
5.9347
x (P/si)
LN(1-X)
(SI/K)*X
ket/K
0.00312983
0.10307557
0.20239536
0.29684653
0.3914368
0.50369319
0.63222468
0.70873152
-0.00313473
-0.10878367
-0.22614224
-0.3521801
-0.49665451
-0.70056098
-1.00028307
-1.23350981
0.00817435
0.26920845
0.52860769
0.77529128
1.02233818
1.31552471
1.65121784
1.85103518
0.01130908
0.37799213
0.75474993
1.12747138
1.51899268
2.01608569
2.65150091
3.084545
0.78662939
-1.54472466
2.05448557
3.59921023
Sol.Tamp
on.
Concentrac
ion
25ml
20ml
11.1
TI
E
M
P
O
TU
BO
DILUC
ION
ABSO
RBAN
CIA
0
0.
5
01:01
0.033
01:02
0.166
1
1.
5
01:02
0.379
01:04
0.232
01:04
0.293
e
V
V=ke
k
CONC
ENTR
ACION
COR
RECC
ION
0.035
5757
0.322
3372
0.781
5869
0.464
6399
0.596
1621
0.03
558
0.64
467
1.56
317
1.85
856
2.38
465
9
222.22
5.9347
2.5
01:04
0.355
0.7298404
2.91936
01:04
0.431
0.8937042
3.57482
3.5
01:04
0.581
1.2171194
4.86848
01:04
0.598
1.2537732
5.01509
x (P/si)
LN(1-X)
(SI/K)*X
ket/K
0.003205
0.058079
0.140826
0.167438
0.214833
0.263006
0.322056
0.438602
-0.003210
-0.059834
-0.151784
-0.183247
-0.241859
-0.305175
-0.388690
-0.577324
0.005995
0.108628
0.263396
0.313168
0.401815
0.491914
0.602358
0.820341
0.009205
0.168462
0.415180
0.496416
0.643674
0.797089
0.991048
1.397665
0.451810
-0.601134
0.845046
1.446179
Sol.Tamp
on.
Concentrac
ion
15ml
30
6.67
TI
E
M
P
O
TU
BO
DILUC
ION
ABSO
RBAN
CIA
0
0.
5
01:01
0.016
01:02
0.265
1
1.
5
01:02
0.529
01:04
0.406
2
2.
5
01:04
0.54
01:06
0.332
3
3.
5
01:06
0.394
01:10
0.237
01:10
0.289
e
V
V=ke
k
CONC
ENTR
ACION
COR
RECC
ION
0.000
0
0.535
8
1.105
0
0.839
8
1.128
7
0.680
3
0.813
9
0.475
4
0.587
5
0.00
000
1.07
158
2.21
000
3.35
921
4.51
488
4.08
150
4.88
357
4.75
420
5.87
538
45
222.22
5.9347
x (P/si)
LN(1-X)
(SI/K)*X
ket/K
0
0.06913436
0.14258092
0.216723
0.29128239
0.26332262
0.31506906
0.30672286
0
-0.07164033
-0.15382848
-0.24426888
-0.34429813
-0.30560523
-0.37843727
-0.36632545
0
0.18056222
0.37238686
0.56602803
0.76075911
0.68773496
0.82288414
0.80108588
0
0.25220255
0.52621534
0.81029691
1.10505724
0.99334019
1.20132141
1.16741134
0.3790566
-0.47651535
0.9900041
1.46651944
Si B/k
Si C/k
Si D/k
x (P/si)
ket/K
x (P/si)
ket/K
x (P/si)
ket/K
x (P/si)
ket/K
0.0031
0.0113
0.0032
0.0092
0.0301
0.1319
0.1031
0.3780
0.0581
0.1685
0.0691
0.2522
0.0639
0.2815
0.2024
0.7547
0.1408
0.4152
0.1426
0.5262
0.0977
0.4319
0.2968
1.1275
0.1674
0.4964
0.2167
0.8103
0.1356
0.6028
0.3914
1.5190
0.2148
0.6437
0.2913
1.1051
0.1472
0.6551
0.5037
2.0161
0.2630
0.7971
0.2633
0.9933
0.2144
0.9640
0.6322
2.6515
0.3221
0.9910
0.3151
1.2013
0.2226
1.0020
0.7087
3.0845
0.4386
1.3977
0.3067
1.1674
0.2388
1.0776
0.7866
3.5992
0.4518
1.4462
0.3791
1.4665
INMOVILIZACION DE ENZIMAS
0.9
0.8
0.7
0.6
0.5
X
0.4
0.3
0.2
0.1
0
0
0.5
1.5
2
ket/K
2.5
3.5