TP CH PHT TRIN KH&CN, TP 9, S 5 -2006

Trang 59
KHO ST TINH SCH ENZYME CHYMOPAPAIN
TRONG M TRI U VIT NAM
L Th Ph, Nguyn Th Thu Sang
Khoa Khoa Hc ng Dng, i hc BC Tn c Thng, Tp HCM
(Bi nhn ngy 26 thng 12 nm 2005)
TM TT: Kho st hm lng cc enzyme thnh phn trong m tri u ti qua
cc giai on sinh trng ca tri bng sc k trao i ion CM-Cellulose cho thy
chymopapain lun chim t l cao nht v hm lng v hot tnh tng cng. Kt qu tng t
vi m ng kh. Tinh sch chymopapain t m ng kh qua giai on phn on mui tch
sunphat amon cho thy chymopapain cn ln peptidase, tip tc tinh sch qua giai on sc k
trao i ion CM-Cellulose v kim tra qua in di SDS-PAGE cho thy s b phn tch
ring c enzyme ny.
1. M U
Trong m u c ba enzyme chnh bao gm papain, chymopapain v peptidase. Trong
chymopapain chim t l nhiu nht v gn y enzyme ny ang c quan tm nhiu v vic
s dng vo cha tr hu hiu cc bnh nhn b lch m ct sng m khng cn phi gii
phu, bnh v xng hng. c bit, ng dng rt c gi tr trong lnh vc y hc l s dng
chymopapain tinh khit tim vo vng m ct sng b lch, sau mt thi gian ngn bnh hon
ton khi hn. Vn ny c nhiu cng trnh cp ti v ang tip tc c nghin cu.
[1][4][5] [9]
Ti M, dc phm Chimodiatin cha chng au thn kinh ta, c s dng tim trc tip
vo ch a m b thot v tiu ho phn thot v ny, lm gim cn au v nhng vn
khc do a nn p ln dy thn kinh. Qua phn tch Chimodiatin cha 70% chymopapain, 20%
caricain, 4% glycyl endopeptidase v 0,1% papain. Nghin cu ngh nn tch caricain,
glycyl endopeptidase v papain ra khi dc phm ny gim nhng phn ng d ng trong
qu trnh iu tr.[5]
Cc loi u pht trin Vit Nam cha c nghin cu nhiu. c bit cha c cng
trnh no i su nghin cu vic tinh sch tch ring cc enzyme thnh phn ng dng trong
cc ngnh cng nghip cng nh trong Y-Dc ti iu kin Vit Nam, trong khi ngun u
Vit Nam rt nhiu, phong ph v s lng, chng loi ng thi cht lng (hot tnh
enzyme) trong m cao [1][8]
Trn c s , nghin cu ny bc u tin hnh kho st thnh phn ha hc v thnh
phn cc enzyme trong m ti v m ng kh ca tri u trng ti Vit Nam bng cc
phng php ho l, ho sinh, tp trung vo kho st tinh sch enzyme chymopapain.
2. NGUYN LIU & PHNG PHP NGHIN CU
2.1.Nguyn liu
M tri u thu nhn ti Lm ng v Long An 3 giai on sinh trng ca tri:
Tri non (N) di 40 ngy tui
Tri bnh t (BT) 50 -100 ngy tui
Tri gi (G) 100 -120 ngy tui
Latex ng kh (LK) (sy t cc m ti trn)
2. 2. Phng php nghin cu
Kim tra nguyn liu: m, tro, protein th, protein tan, hot lc proteolitic.
Science & Technology Development, Vol 9, No.5 - 2006
Trang 60
Xc nh thnh phn cc enzyme: kho st cc m nguyn liu ti ( cc giai on
sinh trng khc nhau ca tri) v m ng kh qua sc k trao i ion vi CM-Cellulose vi
dung mi ra gii l dung dch m pH5 acetate nng t 0,4M -1,5M.
Kho st chit xut v tinh sch enzyme chymopapain t m ng kh: phn on
(NH
4
)
2
SO
4
bo ha 45% loi tr papain, thu nhn chymopapain ti 65% sau tinh sch
tip theo bng sc k trao i ion CM-Celluolse v kim tra qua in di SDS-PAGE.
Cc phng php phn tch: protein th (Kjendahl); protein tan (quang ph,
Braford); hot lc proteolitic (Kunitz); in di SDS-PAGE.
2. KT QU V BN LUN
2.1. Kho st cc mu m (latex)
2.1.1. Kt qu phn tch s b thnh phn
Bng 1. Kt qu phn tch s b cc mu m ti
Protein th
%
Protein tan
%
Tro
(%)
Hot tnh
Loi
m
Cht
kh
% CP CK CP CK CP CK
UI/mg
protein
UI/mg
CP
N 17,43 9,35 53,64 5,88 33,73 1,44 8,26 4,14 0,24
BT 18,00 9,85 54,72 6,17 34,28 1,25 6,94 5,26 0,38
G 16,97 9,67 56,98 6,61 38,95 1,17 6,89 4,26 0,29
LK 93,13 38,18 41,0 35,91 38,56 6,49 6,97 4,57 1,64
Mu ti c hm lng cht kh 16,97-18,0%, tng i cao, cao nht tri bnh t.
Protein tan gim dn t tri gi n tri non. Hot lc proteolitic cao nht giai on bnh t.
Latex ng kh c hm lng protein tan (35,91%) gp 5-6 ln so vi mu ti ban u
(khong 5,8-6,6%), hot lc ring gn bng hot lc ca ch phm Merck.
2.1.2. Kt qu chy sc k trao i ion
Qua th 1 ta thy cc peak protein cc v tr 1,2,3,4 c ra gii ln lt 0,4M; 0,42-
0,46M; 0,62-0,91M; 1,1- 1,20M. Peak 1 khng c hot tnh, peak 2 khng c tri non, peak 3
lun chim hm lng cao nht trong qu trnh pht trin ca tri cng nh trong latex kh. Da
vo trnh t v lc ra gii, cng nh thnh phn bng 2, tham kho [1],[6],[8] chng ti
nhn nh peak 2,3,4 ln lt l papain, chymopapain, peptidase.

















Mu banh te
0.0
0.4
0.8
1.2
1.6
2.0
0 5 10 15 20 25 ong
A280
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
M
Protein Hoat lc Nong o ra giai
Mu trai gia
0.0
0.4
0.8
1.2
1.6
2.0
0 5 10 15 20 25 ong
A280
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
M
Protein Hoat lc Nong o ra giai
LATEX KHO
0.0
0.4
0.8
1.2
1.6
2.0
0 5 10 15 20 25 ong
A280
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
M
Protein Hoat lc Nong o ra giai
Mu trai non
0.0
0.4
0.8
1.2
1.6
2.0
0 5 10 15 20 25 ong
A280
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
M
Protein Hoat lc Nong o ra giai
Hnh 1. Sc k cc loi m tri u

TP CH PHT TRIN KH&CN, TP 9, S 5 -2006
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Bng 2. T l protein v hot lc cc peak trong cc mu latex ti
T l Protein % T l Hot lc % Th t
peak N BT G LK N BT G LK
Peak 1 20,0 12,3 10,2 12,9 0 0 0 0,0
Peak 2 0,0 7,6 13,4 3,7 0 7,4 14,3 2,3
Peak 3 62,7 60,6 60,2 72,6 57,5 50,8 51,3 59,6
Peak 4 15,3 14,1 13,8 6,8 27,0 16,5 15,1 5,2
2.1.3. Kim chng qua in di SDS-PAGE








Hnh 2. in di SDS-PAGE
latex v phn on
1_ Latex ng kh
2_ Ch phm Merck
3,5_ peak 2 (papain)
4_ peak 3 (chymopapain)
6_ peak 4 (peptidase)


So snh kt qu vi cc nghin cu [3][7], latex ng
kh (ct 1) xut hin vch u tin - vch papain thnh
phn, 3 vch k l chymopapain, vch cui cng peptidase,
thy kt qu hon ton ph hp. Ch phm Merck cng c
3 vng tng ng tuy nhin chymopapain khng 3 vch,
c th qu trnh sn xut ch phm lm thay i thnh phn
chymopapain.
ct 3,4,5,6 l cc vch tng ng vi 3 phn on
sau sc k CM-cellulose. Cc vch xut hin trn in di
cho thy gn nh qua ct sc k trao i ion cc
enzyme thnh phn c tch ra ring bit. Nh vy li
mt ln na cc kt qu khng nh trong cc loi m u
kho st cha 3 loi enzyme c hot tnh proteolitic l
papain, chymopapain v peptidase.
Cc kt qu kho st trn cho thy latex ti cng nh
kh u c hm lng chymopapain rt cao. Tip theo l
qu trnh kho st thu nhn enzyme chymopapain t ch
phm latex ng kh.
2.2. Tinh sch enzyme chymopapain t latex ng kh
2.2.1. Phn on bng (NH
4
)
2
SO
4
(xem kt qu bng 3)
Sn phm sau khi phn on sunphat amon cho qua sc k trao i ion CM-Cellulose
2.2.2.Kt qu chy sc k phn on 65% (NH
4
)
2
SO
4

Kt qu ra gii th hin trn th 2. C 3 peak protein ra gii ln lt ti 0,4M; 0,62-
0,99M; 1,13-1,21M nhng ch 2 trong 3 peak c hot lc proteolitic. So vi kt qu kho st
phn trc cho php chng ti kt lun peak 2 l enzyme chymopapain v peak 3 l peptidase.
Phn on mui loi b papain, tuy nhin vn cn ln peptidase.












1 2 3 4 5 6
PHA N OAN 65%
0.0
0.4
0.8
1.2
1.6
2.0
0 5 10 15 20 25
ong
A280
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
M
Protein Hoat lc Nong o ra giai
Hnh 3. Sc k phn on 65% (NH
4
)
2
SO
4

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Trang 62
Bng 3. T l protein v hot lc phn on
65% (NH
4
)
2
SO
4

Th t
peak
T l Protein
%
T l Hot
lc %
Peak 1 13,6 0,0
Peak 2 75,7 74,9
Peak 3 8,5 5,9


Qua bng 3 ta thy peak chymopapain
chim 75,7% protein tng v 74,9% hot
lc tng ban u. Kt qu nh gi hiu
qu thu hi th hin bng 4.

Bng 4. nh gi hiu qu thu hi chymopapain
Phn on
Protein
(mg)
Hot lc
(UI)
HLR
(UI/mg protein)
T l
hot tnh
HS thu
hi (%)
tinh
sch
Dch lc th 1795,5 8205,4 4,57 59,6 100 1
Dch 65% 433,8 2281,5 5,26 74,9 27,8 1,45
Peak 2 328,5 1710,0 5,21 100 20,8 1,91
2.2.3. Kim chng qua in di SDS-PAGE
Phn on 65% chymopapain cn ln peptidase, kt qu in di ph hp vi kt qu sc k
trao i ion phn on 65% th 2. Peak 2 v 3 c 1 vch tng ng khng nh qua sc k
trao i ion trn ct CM-Cellulose tch ring c peptidase ra khi chymopapain.













3. KT LUN
Trong m tri u non khng c enzyme papain, ch tri giai on trng thnh th
mi xut hin enzyme papain v hm lng ny tng dn ln khi tri giai on ln hn.
Enzyme chymopapain chim t l kh cao trong tng lng enzyme ca tri. Cc enzyme
thnh phn papain, chymopapain, peptidase c ra gii qua ct CM-Cellulose vi h m
acetate pH 5 ln lt gradient 0,4 0,47M; 0,62 - 0,99M; 1,13-1,28M.
Khi tinh sch chymopapain qua phn on hai ln 45% v 65% (NH
4
)
2
SO
4
bo ha loi
tr c hon ton papain, tinh sch tip qua ct trao i ion CM-Cellulose loi tr
peptidase. Hiu sut thu hi 20,1% l kh cao nhng tinh sch ca chymopapain t 1,91 l
cha cao. T kt qu nghin cu ny lm c s cho cc hng tinh sch tip theo, kho st cc
yu t nh hng v th nghim tc dng ca chymopapain tinh khit.

Hnh 3 - in di SDS-PAGE latex
v phn on
1 - Ch phm Merck
2 - Latex ng kh;
3,5 - phn on 65% ;
4,6 - peak 2 (chymopapain);
7 - peak 3 (peptidase)

1 2 3 4 5 6 7
TP CH PHT TRIN KH&CN, TP 9, S 5 -2006
Trang 63
STUDYING THE PURIFICATION OF ENZYME CHYMOPAPAIN IN
VIETNAMESE CARICA PAPAYA L. FRUIT LATEX
Le Thi Phu, Nguyen Thi Thu Sang
Applied Science Faculty, Ton Duc Thang University, Ho Chi Minh City
ASTRACT: Studying the compositions of the individual enzymes in the fresh fruit
papaya latex through the different growing periods of the fruit by CM-Cellulose ion exchange
chromatography revealed that chymopapain is always the most abundant protease for the
portion and the activity in total. The result was the same with the freezed-dried papaya latex.
The purification of chymopapain from the freezed-dried papaya latex in the ammonium sulfate
fraction step showed that chymopapain is still contaminated with peptidase. Continuing to next
ion exchange chromatography step and then examining by SDS-PAGE electrophoresis shows
that this enzyme can preliminarily be isolated.
TI LIU THAM KHO
[1]. Nguyn c Lng (cb), Cng ngh enzyme, NXB HQG Tp HCM, 2004.
[2]. Barrett, Alan J; Buttle, David J.; Rich, Daniel H., Pharmaceutical composition of
purified chymopapain, Patent No. 5468480. 1995.
[3]. , , ..,.., ..,
(Carica papaya.L.),
, , 1994.
[4]. Dando PM et al. Department of Biochemistry, Strangeways Research Laboratory,
Cambridge, UK, unoglobin E antibodies to papaya proteinases and their relevance to
chemonucleolysis, Spine. 1;20(9):981-5, May 1995 .
[5]. Eugene J. Nordby, Manucher J.Javid, Winconsin university, US, Continuing Experience
with Chemonucleolysis, 2000.
[6]. Mitsuo Ebata and Kerry T.Yasunobu, ymopapain, isolation, crystallization and
preliminary characterization, The Journal of Biological Chemistry, vol. 237, No.4
4/1962, p1086-1094.
[7]. Rubens Monti et al. Biological Faculty, Estadual Paulista university, Brasil, Purification
of papain from fresh latex of Carica papaya, Brazilian Archives of Biology and
Technology, v.43, n.5, p. 501-507, 2000.
[8]. Sidney P. Colowick, Nathan O.Kaplan, Methods in Ezymology, vol II, vol XIX,
McCollum-Pratt Institute, the Jonh Hopkins university, Baltimore, Maryland.
[9]. Staley Zucker et al, The proteolytic activities of chymopapain, papain and papaya
proteinase III., Biochim. Biophys. Acta 828,196-204, 1985.