Extraction of Bromelain

IMBAO, MA.
Rio Lauren 3CBC  The beaker was removed from the water
Extraction and Purification of Bromelain bath and pH was adjusted to 6.0 with
Enzyme from Pineapples 0.05N NaOH.
 Ten ml of 37% formaldehyde was
Sample: added with constant stirring. Titration
 Pineapple stem was collected and was done to pH 9.0 with 0.1 N NaOH.
maintained in temperatures of 7-12oC, The titration volume of the test solution
used 14-20 days after storage for 85- was recorded. The blank solution was
95% relative humidity. run concurrently with the test solution.
Extraction of Bromelain:  The bromelain content was calculated
 Parts of the stem was collected and for the crude enzyme (Units/gm)
washed with .1% hydrogen peroxide enzyme
solution Purification:
 The stem was peeled off, cut into small  The crude extracts of stem and fruit
pieces and then weighed. bromelain were centrifuged for 10
 The stem was then blended together with minutes at 2,000 rpm, 4,000 rpm and
sodium acetate buffer 6,000 rpm consecutively at 4 C.
 The juice was collected and filtered  The supernatant was collected and
 500mL of the filtrate was collected enzyme assay was performed.
 1 gram per kilogram stem of Benzoic  Salt Precipitation:
Acid was added as preservative o 6.6 g ammonium sulfate was
 The crude extract was obtained an added in 15mL stem bromelain,
labeled as stem bromelain pinch by pinch after
Sample: centrifugation under ice cold
 Pure ripe and unpeeled pineapple conditions with continuous
Extraction of Bromelain: stirring on a magnetic stirrer for
 The pineapple was washed and cleaned 45 minutes.
then, weighed. (The total weight should o For fruit bromelain, similar
be 600g) activity was carried out for same
 The pineapple was homogenized using a time period.
blender and the juice was extracted o Stem and fruit bromelain sample
 The juice extracted was filtered (the solutions were incubated
juice collected must be approximately overnight at 4 C.
300mL) and .6 grams of sodium o After incubation, the precipitated
benzoate was added enzymes were centrifuged at
Selection of substrate: 10,000 rpm for 10 minutes at 4
C.
 The stem bromelain was used to
o The pellet was collected and
determine rate at which gelatin was
dissolved in 10 ml of 10 mM Tris
degraded
HCl buffer which was later
 5% gelatin. 3% hydrogen peroxide,
subjected to dialysis.
37% formaldehyde, .005N NaOH,
100mM sodium acetate buffer with  Dialysis
o The above obtained solution was
2.6M NaCl was prepared
 1mL of crude stem was taken in beaker placed in a dialysis bag and
and the pH was maintained to 6.0 with checked for the leakage of the
0.05 N NaOH sample in it.
 Twenty-five ml of 5% gelatin was o The dialysis bag was then
added to the test solution and suspended in a beaker containing
equilibrated at 45 C in a water bath. 100 mM phosphate buffer-NaCl
 After 20 minutes of incubation at 45 C, solution. This setup was kept in
0.1 ml of 3% hydrogen peroxide was refrigerator/cool conditions
added and swirled. overnight.
 The solution was incubated for an  Ion Exchange Chromatography
additional 5 minutes. o DEAE cellulose bed, of 1 cm
thickness, was prepared in a
chromatography column and o The absorbance was plotted
equilibrated with 0.5 M sodium against protein concentration to
phosphate buffer solution (pH- get a standard calibration curve.
8.0) The absorbance of unknown
o The dialyzed sample of stem sample was checked and the
and fruit bromelain was poured concentration of the unknown
onto the column, from the sides, sample was determined.
without disturbing the DEAE  Gel Electrophoresis
cellulose bed and allowed to o SDS-PAGE was performed with
settle. different isolated/extracted
o Enzyme was eluted using the enzymes (bromelain).
first eluting buffer o The extracted enzymes were
o Eluate was collected in test tube. concentrated and loaded onto a
Elution was done at a flow rate 3.5% stacking gel and subjected
of 1 ml/min. The same process of to electrophoresis on a 12%
elution was carried out using separating gel at 200 V until the
solutions 2, 3, 4, 5 and 6 coomassie dye stained protein
containing 50 mM, 75 mM, 100 band reached the gel bottom.
mM, 125 mM and 150 mM
NaCl, respectively.
o Finally,ion-exchange eluates of
stem and fruit bromelain were
assayed for their activity
Quantitative Estimation of Bromelain
 Lowry Method
o Different dilutions of BSA
solution were prepared by taking
BSA solution (100 µg/ml) and
distilled water as a standard in
the test tube. The final volume in
each of the test tubes was1 ml.
The BSA range was 0.02 to 0.1
mg/ml.
o The 0.1 ml of crude enzymes
of stem and fruit bromelain,
ion exchange eluate 2nd and 4th
of stem and fruit bromelain
enzymes were taken into test
tubes 7, 8, 9, 10, 11 and 12
respectively and the final volume
was made up to 1 ml with
distilled water. These were
unknown samples.
o Five ml of alkaline copper
sulphate reagent was added to
each tube and mixed well.
These solutions were incubated
at room temperature for 10 mins.
o Then 0.5 ml of Folin Ciocalteau
reagent was added to each
tube and incubated in dark for
30 min.
o The spectrophotometer analysis
was performed and the optical
density i.e. absorbance was
measured at 660 nm for all the
samples.

Extraction of Bromelain

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Extraction of Bromelain

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