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(sarch )rticl Bio chmistr*





Intrnational Jo+rnal o$ Pharma an" Bio Scincs
ISS,
0975--299

IS./)TI.,0 P1(I2I3)TI., ),4 51),TIT)TI67 ),)/8SIS .2 38ST7I,7
P(.T7)S70 B(.97/)I, 2(.9 ANANAS COMOSUS (PI,7)PP/7)

1*
HEMANT KR. SHARMA,
2
RICHA SRIVASTAVA AND
3
SHUBHANGI SHUKLA

1
Faculty

of Applied Science, Department of Biosciences, Integral Uniersity, !uc"no#, U$%$
&
Faculty of Applied Sciences, Department of Bioengineering, Integral Uniersity, !uc"no#, U$%$
'
Amity Institute of Biotec(nology, Amity Uniersity, !uc"no#, U$%$

ABSTRACT

Proteases were extracted and assayed from the fruit of Ananas comosus (Pineapple).
They were precipitated utilizing variable concentrations of acetone
(40!"0!#0!$00)! ammonium sulphate (4%!"%) and &ello
(40!"0!#0!$00). The extracted enzymes exhibited proteolytic activity which was
determined using other assay techni'ues. (ctivity determined for Ananas comosus was
)*+m,. The enzymes were! in their crude state! analyzed using -.-/(01. 2ands were
observed between the molecular weight range of )4/4% 3.a. The enzymes were
purified from the extract by anion exchange chromatography using -ilica 0el 4olumn
(p5/#.0! -odium Phosphate 2uffer). The elution of Ananas comosus extract resulted in
two bound fractions. -ince the second fraction of the Ananas comosus extract exhibited
no protease activity! it was ignored. The purified samples were analyzed using -.-/
(01! the purified extract of Ananas comosus displays a single band (characteristic of
bromelain! a monomeric molecule).

KEY WORDS: Protease, SDS-PAGE, Silica Gel, Ananas comosus.







*Corresponding author










HEMANT KR. SHARMA
2ac+lt*

o$ )''li" Scinc0 4'artmnt o$ Bioscincs0 Int:ral 1ni;rsit*0 /+c<no#0 1%P%

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INTRODUCTION

The ma6or cysteine protease of pineapple
fruit Ananas comosus is 2romelain.
2romelain is a mixture of enzymes found
naturally in the 6uice and stems of pineapples.
7t is in the same family of thiol proteases as
papain. 2romelain is commonly used as a
commercial meat tenderizing agent8 however!
it has been 9nown to show therapeutic
properties. 2romelain has been suggested as
a complimentary treatment for sinusitis.
Preliminary studies suggest that it may help
reduce congestion! improve breathing and
suppress coughing. 7t is approved by the
4ommission 1 as a complimentary treatment
for nasal and sinus swelling and inflammation
after ear! nose! throat! surgery. ( review of
three small but well designed previously
published studies found that bromelain may
help relieve sinusitis symptoms
.[1]
2romelain
is a popular natural digestive aid due to it:s
ability to digest proteins. 2romelain may help
with mild pain associated with osteoarthirits.
2romelain and other proteolytic enzymes
have been explored as a complimentary
treatment for cancer. (lthough there is some
preliminary research! there isn:t enough
evidence at this time soon the safety or
effectiveness of bromelain for cancer. 7t
should never be used in place of
conventional treatment.





MATERIALS AND METHODS

REAGENTS
(cetone
(mmonium -ulphate
&ello (0elatin)

1) LOWRYS REAGENTS
) -odium 4arbonate in 0.$ ; ;a<5
) 4u-<
4
.%5
)
<
) -odium Potassium Tartarate
(=ochelle -alt)
<=
>olin:s Phenol ? ) ; solutions of >olin:s
Phenol commercially available! yellow in
colour and was used for colour generating
reactions.

2) BRADFORDS REAGENTS
4oomassie 2rilliant 2lue 0
Phosphoric (cid! #% (Caution : It can
burn skin)
2ovine -erum (lbumin (2-()! $0mg+ml in
water! from above

3) SDS-PAGE REAGENTS (381 !! "a#$o
o% ac"&'am$() #o N*N-m)#+&')n) ,$s-
ac"&'am$())

N!E" @ix ingredients 01;T,AB 1nsuring
no bubbles form. Pour into glass plate
assembly 4(=1>*,,A. <verlay gel with iso/
propanol to ensure a flat surface and to
exclude air. Cash off iso/propanol with water
after gel has set (D$% min).

#ES$%&NG GE$" Gels concentrations o' 1(.)* in +.() , !ris--Cl, p--...
/%olu0e1%olu0e2

#eagents 3+0$ 1+0$
40 (crylamide @ixture E.4m, F.$m,
.istilled Cater $).Fm, F.#m,
$ @ Tris 54l (p5/#.#) G.%m, ).%m,
$0 -.- 0.Fm, 0.$m,
$ (mmonium Per -ulphate 0.%m, 0.$Fm,
T1@1. )0micro, #micro,






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S!AC4&NG GE$ " Gels concentrations o' 5.)* in +.1() , !ris--Cl, p--6..
/%olu0e1%olu0e2

#eagents 1)0$ 1+0$
40 (crylamide @ixture $.Gm, $.$m,
.istilled Cater $0.#m, G.$m,
$ @ Tris 54l (p5/#.#) $.Em, $.)%m,
$0 -.- 0.$%m, 0.$m,
$ (mmonium Per -ulphate 0.%m, 0.$Fm,
T1@1. )0@icro, #@icro,

-) SA.PLE B/FFER
4.# ml of deionised water D $.)m, of 0.% @
Tris/54l (p5/".#) D )0m, of $0 -.- D $m,
of 0lycerol D 0.% m, of 0.% of 2romophenol
2lue (w+v in water) were mixed and were
stored at room temperature.

0) RED/12NG B/FFER
%0 @icro/litre of )/@ercaptoethanol was
dissolved in E%0 microlitre of sample buffer.

3) POLY.ER24AT2ON 1ATALYST
>reshly prepared $0 (mmonium per
-ulphate -olution in water T1@1. was used
directly from the supplied bottle which should
be stored in the dar9.

5) ELE1TRODE B/FFER
$% g Tris D G) g 0lycine D %g -.- was
dissolved in $ ,itre of de/ ionised water! p5/
#.F.

8) STA2N2NG OF GELS
C,ASS&E 7#&$$&AN! 7$8E S!A&N&NG
@a9e up stainH 0.) 422 in 4%H4%H$0
methanolH waterH glacial acetic acid. .
6) DESTA2N2NG SOL/T2ON
.e/stain with )%H"%H$0 methanolH waterH
glacial acetic acid mix! with agitation and
observed under white light transilluminator.

17) 18RO.ATOGRAP8Y B/FFER
7899E# A : +.1 , Sodiu0 Phosphate
7u''er, p--..)" F.$ g of ;a5
)
P<
4
.5
)
<
and $0.E g of ;a
)
5P<
4
(anhydrous) was
added to distilled water and the final
volume was made to $ litre with the p5
#.%.
7899E# 7 : +.1 , Sodiu0 Phosphate
7u''er ; 1 , NaCl : Elution 7u''er" (dd
F.$ g of ;a5
)
P<
4
.5)< and $0.E g of
;a
)
5P<
4
(anhydrous) was dissolved in
distilled water and total volume was made
to $ litre and the p5 was ad6usted to #.%!
in this 40 g ;a4l was added.
1 , Sodiu0 Phosphate 7u''er /p--<.+2"
$ @ -odium Phosphate 2uffer was
prepared to dissolve precipitated pellets. 7t
was prepared by mixing $ @ ;a5)P<4.
That is for p5 G.0 buffer! FE0 m, of $ @
;a5)P<4! stoc9 solution and "$0 m, of
$@ ;a5P<4 stoc9 solutions were mixed.

METHODOLOGY

A1ETONE PRE12P2TAT2ON
The outer s9in of pineapple fruit (Ananas
comosus) was peeled off and flesh was
crushed in mixie to form 6uice! use little
water if re'uired.
.ifferent concentrations of acetone were
prepared ? 40! "0! #0 I $00 in
$0m,.
$0m, of 6uice was precipitated with $0m,
of above prepared concentrations of
acetone.
Precipitation was performed with ice cold
acetone only! at 4 deg. 4 overnight! in
order to prevent the denaturation of
enzyme.
(fter overnight treatment with acetone!
the crude enzyme or 6uice samples were
sub6ected to centrifugation at 4 deg. 4
using cooling centrifuge at $0!000 =P@
for $0 minutes.
The supernatant was discarded and the
pellet so obtained was dissolve in 0.$ @
-odium Phosphate 2uffer.


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9igure 1
P)'')# o,#a$n)( $n on) o% #+) )99)n(o"%s a%#)" c)n#"$%u:a#$on (Ac)#on) P")c$9$#a#$on)

A..ON2/. S/LP8ATE
PRE12P2TAT2ON
The outer s9in of pineapple fruit was
peeled off and flesh was crushed in mixer
to form 6uice! use little water! if re'uired.
.issolve G% g of (mmonium -ulfate salt in
$00m, of water to form $00 saturated
solution of ammonium sulfate.
>rom the above formed saturated solution
of ammonium sulfate! final 4% and "%
saturation of ammonium sulfate with fruit
6uice was obtained by adding #.$#$m,
and $#.%Gm, of above $00 saturated
solution to )0m, of 6uice.
The ammonium sulfate precipitation was
performed in cold room at 4 deg. 4 using
magnetic stirrer and magnetic bead
overnight.
(fter overnight treatment with acetone!
the crude enzyme or 6uice samples were
sub6ected to centrifugation at 4 deg. 4
using cooling centrifuge at $0!000 =P@
for $0 minutes.
The supernatant was discarded and the
pellet so obtained was dissolved in 0.$ @
-odium Phosphate 2uffer.



9igure (
R$n:s %o"m)( a%#)" AS P")c$9$#a#$on (O;ERN2G8T TREAT.ENT* - D):< 1)

=ELLO PRE12P2TAT2ON
PREPARAT2ON OF T8E =ELLO
5eat about )%0m, of deionized water to
boiling.
Ceigh about #.%0 g of 6ell/o in a $00m,
bea9er.
(dd )Fm, of boiling water and stir until
the 6ell/o dissolves
4ool the 6ell/o to about %04 before using
it in further experimentation.

P"oc)(u") o% 9")c$9$#a#$on
The outer s9in of pineapple fruit (Ananas
comosus) was peeled off and flesh was
crushed in mixer to form 6uice! use little
water if re'uired.
.ifferent concentrations of gell were
prepared ? 40! "0! #0 I $00 in
$0m,.
Total volume ma9e up ? F.% m,
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(dd different concentrations of 6ell/o in
each of the test tubes with same volume
of sample.
Transfer the obtained sample to )m,
eppendorf and place them for
centrifugation.
4entrifugation should be done at )!000
=P@ for $0 minute at 44 using cooling
centrifuge.
(fter centrifugation! obtained pellet is
should be dissolved in 0.$ @ -odium
Phosphate 2uffer.
,eave it for overnight treatment at 4 deg.
4.



9igure 3
P)'')# o,#a$n)( $n ($%%)")n# conc< o% >)''o (1om9a"$son)

EN4Y.AT21 A1T2;2TY ANALYS2S
Fo'$n-1$oca'#)au R)a:)n# Ana'&s$s
(LOWRYs ASSAY)
PREPARAT2ON OF BSA STO1?
0.$g of 2-( /7o=ine Seru0 Al>u0in2 was
weighed and was dissolved in $00m, of
distilled water. 4oncentration used was $00
mg+m,.

PREPARAT2ON OF WOR?2NG STANDARD
>rom the above prepared solution $0m, was
ta9en and was made upto $00m, of distilled
water. 4oncentration / $00 microgram+m,.
N!E" A$$ !-E #EAGEN!S S-8$D 7E
P#EPA#ED 9#ES- 9# E?PE#&,EN!A$
ANA$@S&S.

LOWRYS ASSAY PROTO1OL FOR
DRAW2NG STANDARD 1/R;E AND
@/ANT2FY2NG /N?NOWN SA.PLES OF
PROTE2NS /S2NG STANDARD 1/R;E
/N?NOWN 1ON1< OF PROTE2N A
O<D<BSLOPE
>or un9nown sample! i.e.! enzyme sample
from pineapple fruit 0.%m, of it was ta9en
and ).%m, of .C was added to it followed
by addition of 4.%m, of al9aline 4u-<
4

solution and followed by addition of 0.%
m, of >olin:s Phenol.
The reading of un9nown samples color
was ta9en using spectrophotometer at
%%0nm I calculated as above mentioned.
/SD&8, DDEC@$ S8$P-A!E -
P$@AC#@$A,&DE GE$
E$EC!#P-#ES&S2
/SDS-PAGE2

ELE1TROP8ORES2S B/FFER
The final tan9 buffer composition is ?
$E" m@ 0lycine
0.$ -.-
%0m@ Tris/54l (p5/#.F)
(@ade by diluting a $0J stoc9 solution. This
goes in both top and bottom tan9s)

PRO1ED/RE
2ase plates and notched plates were
washed and were assembled properly
using spacers fixed with the help of
petroleum 6elly. The assembly was
ensured lea9 proof by pouring water in
between them.
The gel solutions were poured between
plates till the level of gel is )cm below the
notched plate.
)00/)%0 microlitre of water was added to
ma9e surface even.
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(fter gel was set (approximately )0/
F0minutes) it was washed with distilled
water to drain off the water completely.
The stac9ing gel was poured directly into
already polymerized separating gel and
was left for F0 min. for it to be completely
casted.
The comb was inserted into gel solution
without trapping any air bubbles
approximately $ cm above the stac9ing
gel.
)%microlitre of protein sample (pineapple)
and $0microlitre of protein mar9er were
pippeted into individual vial. To each of
these vials $%microlitre of gel loading dye
was added.
The vials were placed in boiling water
bath for % minutes.
(fter the stac9ing gel is set the comb and
bottom spacer was removed and the gel
was washed thoroughly to remove
unpolymerised acrylamide.
The bottom reservoir was filled with $ J
=eservoir 2uffer.
The plates were carefully fixed to P(01
apparatus without trapping any air bubble
between the bottom of the gel and buffer
with notched plate facing top reservoir.
Pineapple fruit samples were loaded in
wells.
The cords were connected to the power
supply according to convention red/
anode! blac9/cathode.
Koltage was set at $00K and power
supply was switched on.
Chen the dye front reached 0.%cm above
the bottom of the gel the power supply!
was switched off.
The plates were removed using spatula to
remove them apart.
The gel was transferred in tray containing
water and was washed for % minutes.
)0 m, of 422 -tain was added to the gel
and was stained for F0/"0 minutes.
Then the gel was de/stained for about )4
hrs to obtain clear bac9ground.

EN4Y.E P/R2F21AT2ON /S2NG AN2ON
EC18ANGE 18RO.ATOGRAP8Y

MATERIALS

$. 2*>>1= (
). 2*>>1= 2
F. .istilled water
4. @icropipette (Pippetemen)
%. Tips (Tarson)
". 2ea9er (2orosil)
G. -odium 5ydrogen Phosphate (@erc9)
#. ;a4l (-odium 4hloride) (@erc9)
E. >ilter Paper
$0. -ilica 0el
$$. 1nzyme -ample

PRO1ED/RE
The precipitated protein samples were
filtered using 0.44nm filter paper.
The resultant sample was loaded onto a
-7,74( 01, 4<,*@; e'uilibrated with
2*>>1= ( for $.) 5rs.
The ma6or protease of the fruit was eluted
from column using 2*>>1= 2. Protein
fraction was collected at its pea9



9igure 5
18RO.ATOGRAP821 1OL/.NS (.ATR2C S2L21A GEL)
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RESULTS



TABLES
!A7$E 1
LOWRYS @/ANT2F21AT2ON D P2NEAPPLE A1ETONE PRE12P2TAT2ON


Concentration o' acetone
ptical densitA
))+ n0
Concentration o' protein
/ug10l2
"0 (() 0.%4
%)%
"0 (2) 0.%$
#0 (() 0.%F
%F%
#0 (2) 0.%4
$00 (() 0.%)
4#0
$00 (2) 0.44

!A7$E (
LOWRYS @/ANT2F21AT2ON D P2NEAPPLE A..ON2/. S/LP8ATE PRE12P2TAT2ON

SA!8#A!&N 9 A,,N&8, S8P$-A!E P!&CA$ DENS&!@
))+ n0
CNCEN!#A!&N 9 P#!E&N
/ug10$2
4% 0.%0 %00
"% 0.4) 4)0

P/R2F21AT2ON /S2NG S2L21A GEL 1OL/.N 18RO.ATOGRAP8Y
>our syringes filled with crude enzyme samples of Ananas comosus were sub6ected to (nion
1xchange 4hromatography8 the resulting fractions were collected and stored at ?)04.










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0Au ---- 8%



9igure <
An$on$c EEc+an:) 1+"oma#o:"am a,oF) ()9$c#$n: #+) 9u"$%$)( %"ac#$ons
o% )nG&m) %"om c"u() )nG&m) )E#"ac#s o% Ananas comosus

GRAP8S
/S!ANDA#D C8#%E-$B#@CS2



9igure <
Lo!"&s G"a9+


SDS-PAGE o% EN4Y.E SA.PLES
The crude samples obtained from precipitation with different acetone and ammonium sulphate
concentration were then sub6ected to -.-/P(01 analysis. 7t was observed that a single band was
observed between )4/4%3.a range which might corresponds to ma6or pineapple protease
L2=<@1,(7;M.
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DISCUSSION

There are some research papers which have
been published detailing the purification of
bromelain from Ananas comosus. 5owever
there have been reports that another
protease present in Ananas comosus similar
in characteristics to actinidain! present in
Actinidia chinensis. 7 am trying to purify
proteases from the fruits and screen for that
particular protease which has not been
wor9ed upon yet. Therefore all the wor9 7
have done and the results discussed are
based on my original wor9. Protein
concentration was 'uantified using ,owry:s
and 2radford method of protein
'uantification. The concentration is
proportional to the absorbance. The different
range of acetone! ammonium sulfate I 6ell/o
were chosen for precipitation and protein
'uantification was performed for precipitated
proteins obtained from this range. >or
acetone from 40 to $00 range was
chosen as from established sources it was
found that 40/$00 acetone range give
maximum concentration of protein. >or
(mmonium -ulphate precipitation! 4% and
"% saturated ammonium sulphate was
chosen as 4%/"% range generally gives
maximum protein concentration. (cetone is
generally used for precipitation as it is
volatile! and easily evaporates after
precipitation. 7t was observed that for
pineapple! #0 acetone and 4% ammonium
sulphate gave maximum concentration of
protein.

CONCLUSION

The protease from the pineapple fruit was
purified using L-ilica 0el (nion 1xchange
4hromatographyM. The fractions obtained!
containing the proteases similar to 2romelain!
the ma6or protease of pineapple as when the
fractions were sub6ected to -.-/P(01
analysis! the multimeric bands were observed
in the range of )4/4% 3.a. 7n case of
pineapple! a single faint band was observed
between )4/4% 3.a also the range in which
molecular weight of 2romelain lies. The
presence of protease in these fractions was
also confirmed using other (ssay:s! where
activity of purified protease was found. The
extra fraction obtained in the chromatogram
of the Ananas comosus! sample fraction
showed no activity when sub6ected to other
assay:s indicating the absence of protease in
the corresponding fraction thus conforming
the absence of any other ma6or protease
similar to that of 2romelain in pineapple.
[(]
7n
future! the purified fractions from both
pineapple and 9iwi is intended to go for
crystallographic or se'uencing analysis to
conform structurally the presence of ma6or
protease actinidain in 9iwi and bromelain in
pineapple
.[3]

REFERENCES

1. Porter P.4! Ph...!
$
Aang T.! 2.-.!
)
,uong
(.! @...! Ph...!
F
.elclos 0.,! @...!
Ph...!
4
(bramson -.,! @...! Ph...!
%!"

3heradmand >.! @...!
$!"
and 4orry ..2.!
@...
$!"
()0$$). Proteinases as Molecular
Adjuvants in Allergic Airwa !isease.
2iochim 2iophys (cta. )0$$
;ovember8 $#$0($$)H $0%E?$0"%.
doiH $0.$0$"+6.bbagen.)0$$.04.0$E.
). ,ee 3.,! (lbee 3.,.! 2ernasconi =.&!
1dmunds T.($EEG). Com"lete amino acid
se#uence o$ ananain and a com"arison
with stem bromelain and other "lant
csteine "roteases. 2iochem &. $EEG
<ctober $8 F)G(Pt $)H $EE?)0). P@4
$)$#G#$.
F. 5usain -.-.! ,owe 0. ($E"#). %vidence
$or histidine in the active sites o$ $icin and
stem&bromelain. 2iochem &. $E"#
;ovember8 $$0($)H %F?%G. P@4 $$#G$0#