1. RESEARCH POSTER PRESENTATION DESIGN © 2015
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Isolation and purification of alkaline phosphatase
from E. coli determined by specific activity using
procedures as column chromatography, ammonium
sulfate fractionation, heat denaturation, Bradford
assay, SDS page, and others. Methods and
processes required four days to finally have the
enzyme of interest which was not completely
purified but the specific activity is higher in the last
step than at the beginning of the experiment. One
more step would be needed but is not used during
this experiment.
ABSTRACT
INTRODUCTION
A number of methods are available for the
purification of enzymes and therefore the main aim
of enzyme purification is to remove all other
proteins present in the crude. During the first day
lysoszyme was added to the cells to hydrolyze part
of the cell wall and DNase solution to hydrolyze
any DNA liberated from broken spheroplasts,
EDTA was added to the cells to bind divalent
cations to form a soluble complex, and finally
Magnesium sulfate to bind the excess of EDTA.
Centrifugation was key to get Stage 1 of the
experiment from the supernatant which went
through dialysis which allows the exchange of small
molecules while retaining the big molecules. The
second day heat denaturation of proteins was done
to eliminate those unwanted proteins. The solution
from the dialysis sac was transferred to a conical
tube that was placed in a water bath at 80˚C for 15
minutes. After the incubation centrifugation was
needed once again to discard the denatured proteins,
the supernatant was Stage 2. Ammonium sulfate
precipitation was the next step. Ammonium sulfate
is a highly water soluble salt valuable in differential
precipitation of proteins. It is used to concentrate
enzymes. Centrifugation was performed to keep the
pellet and it was resuspended with Tris-HCl buffer
with MgSO4 which was Stage 3 of the experiment.
Dialysis was required after this step. During the
third day EDTA column chromatography was made
with the dialysis solution and buffers that provided
fractions that were used to created a pool as Stage 4.
The fractions selected to created the pool went
through a enzyme activity assay to find the ones
with higher enzyme present. Each day a continuous
assay of alkaline phosphatase was performed to
each Stage to control the process of the purification.
Before the SDS page electrophoresis a Bradford
assay was performed to find out the concentration
of each stage and fill the purification table shown in
the results. SDS page was done the fourth and last
day to see if the enzyme was completely purified.
METHODS
The following graphs and tables show the processes
and results of the experiment:
RESULTS CONCLUSIONS
• The experiments last 4 days and different
methods were used to purify Alkaline
phosphatase from E. Coli K12.
• Dialysis, enzyme assays, Bradford assay were
key processes to analyze the purification of the
enzyme. Lysis of cells, heating, ammonium
sulfate. SDS page chromatography were the
most important steps to purify Alkaline
phosphatase.
• The results show “AP” present in all of the
stages at a size of 51,810 Da. The last stage (4)
shows the enzyme purer than the rest of the
stages but still has other proteins present
meaning that is not 100% pure.
• The purification table shows how protein mass
was lost during the process from Stage 1 which
is after the lysis of cells and the first dialysis
19.07mg to Stage 4 after the SDS page
chromatography 1.30mg with 5.05Units but it
also shows a higher specific activity of
3.92U/mg compared to 0.45U/mg in Stage 1
meaning that it was purified but not completely
(About 85% of the protein present is alkaline
phosphatase).
• To finish the process of purification an ion-
exchange chromatography would be needed but
it was used in this experiment.
REFERENCES
- Ninfa, Alexander J. Fundamental Laboratory
Approaches for Biochemistry and
Biotechnology. First ed. John Wiley & Sons,
1998. Print. Page 176 to 193
- Zulmita Chavez Majluf M. Research Notebook
“BIOT 2933” Book 1, 2016. Page 33 to 41
Alkaline phosphatase is an enzyme that catalyzes
the hydrolysis of phosphate-containing compounds.
This enzyme will be isolated from E. coli. The
strain of E. coli that will be used is K12. The
physiological role of this enzyme in E. Coli is to
cleave phosphoryl groups from a wide variety of
phosphorylated compounds, thereby providing the
cell with a source of inorganic phosphate (Pi). E.
coli mutant is used in which the control of
phosphatase production is defective so that the cells
produce elevated amounts of the enzyme under all
culture conditions. E. coli is heat stable to purify
the enzyme by heat denaturing other proteins in the
solution so the denatured proteins can be removed
by centrifugation. The partially purified enzyme,
after concentration by salting-out with ammonium
sulfate, is further purified by SDS chromatography.
To completely purify this enzyme an ion exchange
chromatography on DEAE would be needed but is
not used in this experiment.
Alkaline Phosphatase Formula:
R-O-PO3H- + H2O  R-OH + H2PO4
-
Oklahoma City Community College, OK
Zulmita Chavez Majluf M.
Purification of Alkaline Phosphatase from E. Coli
[BSA] (mg/ml) Absorbance at 595nm
0 0
0.1 0.131
0.2 0.268
0.4 0.432
0.6 0.604
0.8 0.709
1 0.967
Stage 1 0.905
Stage 2 0.427
Stage 3 0.65
Stage 4 0.254
Sample Volume(ml)
[Protein]
(mg/ml)
Proteinmass
(mg)
Enzymeactivity
(U/ml)
Units(U)
Specificactivity
(U/mg)
mgProtein
yield(%)
Enzymeyield
Stage1 10.2 1.87 19.07 0.84 8.57 0.45 100% 100%
Stage2 14.8 0.44 6.51 1.12 16.58 2.55 34.14% 193.46%
Stage3 2.8 0.67 1.88 2.39 6.69 3.56 9.86% 78.06%
Stage4 5 0.26 1.3 1.02 5.09 3.92 6.82% 59.39%
Sample
Absorbance at
410nm
PNP day 1 0.716
Stage 1 0.605
PNP day 2 0.715
Stage 2 0.804
PNP day 3 0.712
Stage 3 0.862
Stage 4 0.708
75 kD
50kD
37kD
51 kD
Table 1: Absorbance data
for Bradford Assay
Fig 1: Standard curve for
Bradford Assay used to
calculate the concentration of
each Stage.
Table 2: Absorbance data from Alkaline Phosphatase
Assay to calculate Ɛ of PNP and the enzyme activity, units
and specific activity of each stage
Table 3: Purification table of the enzyme Alkaline
Phosphatase shows the protein present in each Stage.
Fig. 2: 15% SDS page gel electrophoresis showing the final
results. The first line shows Precision Plus Protein All Blue
and its sizes on the left side. Lines 2, 3, 4, and 5 shows
Stage 1,2, 3, and 4 and the size of Alkaline Phosphatase on
the right side. A little bit of another proteins still shows up
in the gel in Stage 4.