|
Enzyme
|
Purification method
|
Fractions
|
Total enzyme activity (units)c
|
Total protein (mg)d
|
Specific activity (U/mg protein)
|
Purification fold
|
Yield (%)
|
|---|
|
cel12B
|
Crude extractb
| |
4.49
|
73.2
|
0.061
|
0
|
100
|
| |
UFe-PESf—MWCg (100 kDa)
|
Permeate
|
3.73
|
38.0
|
0.098
|
1.6
|
83.1
|
|
UF-PES—MWC (50 kDa)
|
Permeate
|
2.76
|
11.0
|
0.251
|
4.1
|
61.4
|
|
UF-PES—MWC (30 kDa)
|
Permeate
|
1.91
|
2.32
|
0.823
|
13.4
|
42.6
|
|
Gel filtrationh
| |
1.32
|
1.24
|
1.06
|
17.4
|
29.3
|
|
cel8C
|
Crude extract
| |
28.7
|
75.3
|
0.381
|
0
|
100
|
|
UF-PES—MWC (100 kDa)
|
Permeate
|
25.8
|
44.5
|
0.579
|
1.5
|
89.7
|
|
UF-PES—MWC (50 kDa)
|
Retentate
|
23.1
|
27.4
|
0.844
|
2.2
|
80.5
|
|
UF-PES—MWC (30 kDa)
|
Permeate
|
18.2
|
7.39
|
2.47
|
6.5
|
63.4
|
|
Gel filtration
| |
14.4
|
6.08
|
2.36
|
6.2
|
50.0
|
|
peh28
|
Crude extract
| |
1.58
|
88.8
|
0.0178
|
0
|
100
|
|
UF-PES—MWC (100 kDa)
|
Permeate
|
1.37
|
52.2
|
0.0262
|
1.5
|
86.5
|
|
UF-PES—MWC (50 kDa)
|
Retentate
|
1.31
|
31.2
|
0.0419
|
2.4
|
82.6
|
|
UF-PES—MWC (30 kDa)
|
Permeate
|
1.00
|
11.3
|
0.0888
|
5.0
|
63.4
|
|
Gel filtration
| |
0.708
|
6.56
|
0.108
|
6.0
|
44.7
|
- All values are given as a mean of triplicates ± SE
-
aCel12B, cel8C, and peh28 are clones of Pcc for genes encoding cellulase B, cellulase C, and polygalacturonase, respectively, that were transformed into E. coli using pTAC-MAT expression vector
-
bCrude extracts are cell-free extracts of E. coli cell-free lysates. The cultures were stimulated for enzyme induction for 5 h, for cel12B and cel8C, and for 7 h, for peh28, at 37 °C using 10 mM IPTG
-
cOne Unit of enzymatic activity is defined as the amount of enzyme releasing 1 µmol of reducing sugars per minute from the substrate under the assay conditions (pH 5.0 at 40 °C, for cel12B and cel8C, and pH 5.0 at 40 °C, for peh28)
-
dAll protein concentrations are in mg per ml of enzyme solution at each fractionation stage
-
eUltrafiltration
-
fPolyethersulfone
-
gMolecular weight cut-off
-
hGel filtration was carried out using Sephadex G-100
