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Table 2 Purification steps of cel12B, cel8C, and peh28 overexpressed in E. coli a

From: Molecular and biochemical characterization of recombinant cel12B, cel8C, and peh28 overexpressed in Escherichia coli and their potential in biofuel production

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
  1. All values are given as a mean of triplicates ± SE
  2. 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
  3. 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
  4. 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)
  5. dAll protein concentrations are in mg per ml of enzyme solution at each fractionation stage
  6. eUltrafiltration
  7. fPolyethersulfone
  8. gMolecular weight cut-off
  9. hGel filtration was carried out using Sephadex G-100