DSpace at VNU: Tìm hiểu các enzym bảo vệ ôxy hóa của nấm Linh chi Ganoderma Lucidum

TIM HIEU CAC ENZYM BAO VE OXI HOA CUA N A M
LINH CHI GANODERMA LUCIDUM
TA BICH THUAN, PHAM KIEN CUdNG,
DANG QUANG HUNG
Khoa Sinh hoc, Dai hoc KHTN
Mddau
Nain Linh Chi (Ganoderma lucidum) tir lau da duge coi nhu la mot
loai "than dugc" vdi nhiing tac dung gia tri dugc Ueu nhu bd gan, than,
phdi, dieu boa huye't ap, lam giam lugng dudng va colesteron trong mau...
Gan day, nain Linh Chi con dugc diing de dieu tri cac benh ung thu va
dugc xem la nguyen lieu chira nhieu chat chdng oxy boa, lao boa cao.
Nbam ddng gdp them cac dan lieu ve loai nam ed nhieu gia tri
dugc lieu nay, chiing tdi da tien hanh tim hieu cac enzyme bao ve oxi
boa cua nam Linh Chi, trong dd trude he't tap trung vao 3 enzym chinh
dd la eatalase, superoxid dismutase va NADH oxidase. Sau day gidi
thieu mot so ket qua ma chiing tdi da thu dugc.
Nguyen lieu va phi/ctng phap

Nguyen lieu
Nam Linh Chi Ganoderma lucidum - ky hieu GaL dugc la'y tit
phdng Gidng na'm gd'c ciia Trung tam Cdng nghe sinh hoc, Dai bgc
Qudc Gia Ha Ndi.
Phuang phap nghien cicu
- Nudi ca'y nam Ganoderma lucidum dugc trinh bay trong [1,2]
- Protein dugc xac dinh theo phuang phap cua Lowry, diing casein
lam cha't chu£n [7]
- Hoat do SOD dugc xac dinh theo phuang phap McCord va
Fredovicli [3,8]
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- noai ug caiaiase ouge xac oinh theo phuang phap cua Thibodeau
va cdng su [9] va lugng FIjOz con lai duge xac dinh theo phuang phap
cua Mottola va cdng su [10].
- Hoat do cua NOX dugc xac dinh theo phuang phap cua Poole va
Clairborne [11].
- Dien di tren gel polyacrylamide dugc tien hanh theo phuang
phap cua Laemmli [6].
- Tinh sach enzyme bang cot sac ky trao ddi ion qua cdt DE-52,
sac ky Igc gel qua cdt Sephadex GlOO [4,5].
Ket qua thao luan

Sir sinh trudng va phat trien ciia nam GaL
Ket qua nghien ciiu ciia chung tdi cho tha'y he sgi ciia nam GaL
phat trien thich bgp d 26-28°C. Sau 18-24 gid ca'y, he sgi bat dau phat
trien manh tren mdi trudng nudi ca'y long. Chung Ganoderma lucidum
cd td'c do sgi mge tir 150 - 200 ml/gid. Cdn trong dieu kien nudi cay
sinh qua the, ke't qua cho tha'y sau khoang 25-30 ngay nudi ca'y, he sgi
na'm bat dau ben ket. Sau khoang 2-3 thang ke tir khi ca'y gidng, tan
na'm phat trien thanh thue, mdi bich ehi cho 1-2 tan na'm Idn, nang
sua't thu hai tuang ddi thap tir 2,5 - 4%.
Hoat do cac enzym NADH, CAT, SOD ciia nam GaL
Hoat dd enzvm NADH oxidase (NOX) cua nam GaT,

NOX sinh khoi

NOX qua the

Hinh L Hoat do NADH oxidase (NOX) cua nam GaL
785

Ke't qua xac dinh boat do ciia enzym NADH oxidase dugc trinh
bay d hinh 1. Qua day chiing tdi thay rang boat do NOX cua na'm GaL
nhin chung la tuang ddi thap, chi dat 0,049 dan vi/ g nguyen lieu tuoi.
Hoat do NOX cua na'm GaL a dang sinh kbdi cao ban so vdi boat do
NOX d dang qua the dat 0,029 dan vi/g nguyen lieu tuai.
Hoat do enzym eatalase (CAT) cua nam GaL
Catalase (CAT) la enzym chii yeu giii vai trd quan trgng phan
buy cac H2O2 trong hSu bet cac ca the sinh vat. Ke't qua xac dinh boat
do CAT cua mSu GaL (hinh 4) cho thay boat do CAT ciia nam GaL
nhin chung la tuang ddi cao, dat tuang ling la 2,331 dv/mg protein va
2,319 dv/mg protein d dang qua the va dang sinh kbdi.

2.34

s
2.33
'<0
(A

bO
C

•5.

2.32

o
X 2.31

fss»it--S»!;,'if»i*

CAT sinh khoi

CAT qua the

Hinh 2. Hoat do Catalase (CAT) cua nam GaL
Hoat do enzym superoxit dismutase (SOD) ciia nam GAL
Ke't qua phan tich boat do enzym SOD cua nam GaL dugc bieu
Men d hinh 5, nhin vao dd thi cho ta tha'y boat do ridng enzym SOD
cua qua the la 1,517 dv/mgPr, cdn d dang sinh kbdi boat do rieng
tuang ling vdi 10,279 dv/mgPr.

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12

S
D

10

8
c
^^

(«•
o

6
4
2
0
SOD sinh kbdi

SOD qua the

Hinh 3. Hoat do superoxit dismuatse (SOD) cua nam GaL
Tren ca sd xac dinh boat do ciia cac enzym CAT, SOD, NADH
chiing tdi da chgn enzym NADH oxidase de tach, tinh sach va ngbidn
ciiu mot sd tinh chat.
Birdc dau tinh sach va nghien curu mot so tinh chat cua NOX tCr
sinh lTren ca sd xac dinh boat do cua cac enzym CAT, SOD, NOX
chiing tdi da chgn enzym NOX de tach, tinh sach va ngbidn ciiu mot
so tinh cha't.
Tinh sack mot phan NOX tit sinh Ichdi nam GAL
De' tinh sach NOX tii sinh khd'i ciia na'm GaL, chiing tdi tie'n hanh
thu dich chie't, bd sung them 0,1 the tich dem Tris-HCl lOOmM, pH
8,0, de ndng do cudi ciing ciia dem la lOOmM, pH dam bao dat 8,0 va
cho sac ky qua cdt gel DEAE- cellulose DE-52, cdt Sigma cd kich
thude 3x14 cm, dugc can bang vdi ciing dem tren. Phan protein gdn
tren cdt dugc rira chiet bang gradient NaCl tir 50mM- lOOOmM pha
trong dem Tris-HCl lOOmM, pH 8,0.
Ke't qua phan tich protein va boat do NOX cua cac phan doan sac
ky qua cdt cho thay phan dich khdng hap thu khdng ed boat do NOX.

PhSn ha'p thu duge d^y ra dudi dang 3 dinh protein chinh, trong dd chi
cd dinh dSu tien duge d£y ra d ndng do khoang 370mM la cd boat do
NOX. Sir dung SDS-PAGE de danh gia do sach cua che pham NOX

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sau khi qua cdt DE-52 (hinh 4) cho tha'y che' ph^m cd mot bang
protein rd net vdi kich thude khoang 40 kDa va mot sd bang protein
khac nbung it rd net hon (H4. cdt 2).
De tiep tuc tinh sach NOX ciia GaL, chiing tdi da tie'n hanh tap
trung cac phan doan cd boat do NOX qua cdt DE-52, cd dac miu bang
centricon va cho sdc ky qua cdt Igc gel Sephadex G-100. Cdt cd kich
thude lx70em, dugc can bang vdi dem. Ke't qua riia chie't va phan tich
protein va boat do NOX cua cac phan doan qua cdt Sephadex GlOO
cho tha'y cd 2 dinh protein chinh, trong dd dinh thii 1 cd boat do NOX.
Ke't qua kiem tra do sach cua che pham sau khi qua budc Igc gel
cho tha'y ngoai bang protein 40 kDa vSn cdn mot sd bang khac cd mat
trong che' pham, cho thay che' pham chua dugc tinh sach hoan toan va
can tie'p tuc tinh sach.
M
kDa
97
66
45
30

20,1
14,4
Hinh 4: Kit qua dien di phd bang protein cua nam GaL

Cdt M: Thang ebu^n protein.
Cdt 1: Dich mlu thd na'm GaL; Got 2: Mau chay qua cdt DE-52
Cdt 3: Mau chay cdt Igc gel G-100
Nghien curu mot sd tinh chat ciia NOX
Sau khi thu duge che' pham NOX tinh sach mot phan qua budc sac
ky trao ddi ion va sac ky Igc gel chiing tdi da tie'n hanh tim hieu mot
sd tinh cha't ciia enzym.
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• vg ben nniet
Ke't qua phan tich do bin cua enzym NOX bang each ii enzym d
cac nhiet dd khac nhau va sau dd xac dinh boat dd cdn lai (hinh 5) cho
tha'y NOX cua na'm GaL la tuang ddi ben vdi nhiet, d nhiet do 30-60°C
sau 15 pbiit xijf ly enzym vin giii nguyen boat dd ban diu. Tuy vay khi
x\x ly d 80"C, trong 15 phut thi enzym chi cdn 10% boat do ban diu.
1

0.6i

!

0.5^

!

0.4-

:0Hoatd6
(mU/mgPr)

ImU/rngPr 0.3 i
0.2 J
I

0.1 i

I

080

Hinh 5: Do ben vdi nhiet cua enzym NOX
• Anh hudng cua mot sd ion kim loai
Ke't qua phan tich anh hudng cua 5 ion kim loai (Ca^^, Cu^"^, Fe^*,
Mg^"^, Zn^"^) va anion F' len boat do cua NOX (hinh 6) cho tha'y chi cd
Fe"*^ d ndng do 3-4mM cd kha nang lam tang mot phan boat do cua
NOX. Cac ion Ca^"^, Mg^"^ va Zn^^ va anion F' khdng anh hudng den boat
do cua enzym. Cdn ion Cu^* d ndng do 4mM lie che' boat do ciia NOX.
OX)

^

1.5
NaF
1

Fe'

~

10 0 5 ^ ^ "

o
S

0

Cz''

Cu^^

2

4

Nong do (mM)
Hinh 6: Anh hudng cua ion kim loai len hoat do cua NOX
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• Xac dinh NOXcila nam GaL Id thude loai sinh H2O hay sinh H2O2.
Theo cac nghien ciiu khac nhau [10], NADH oxidase cua cac sinh
vat dugc phan thanh hai nbdm: nhdm tbii nha't xiic tac cho phan ling
oxi boa mot phan tit NADH den oxi va hinh thanh nen mot phan tir
H2O2, vi vay dugc ggi la NOX sinh H2O2. Nhdm tbii hai oxi boa 2
phan tir NADH va tao H2O, nen cd ten la NOX sinh H2O. Ke't qua xac
dinh lugng H2O2 sau phan ling giiia NADH va enzym cho thay H2O2
khdng hinh thanh sau phan ling, chiing td NADH oxidase ciia na'm
GAL thude nhdm sinh H2O.
Tdm lai, qua nghien ciiu nay chiing tdi da dieu tra dugc hoat do ba

enzym bao ve oxi boa la SOD, CAT va NOX cua nam GaL d dang qua
the' cQng nhu dang sinh khd'i. Cd su khac nhau ve boat do enzym giiia
hai loai miu nay. Chiing tdi cung da budc dau thu dugc che' pham
NOX d dang tinh sach mot phan bang phuang phap sac ky qua cdt trao
ddi ion DE-52 va cdt Igc gel Sephadex G-100. Cac ket qua xac dinh
tinh chat chiing td NOX cua nam GaL la thude nhdm sinh H2O, kha
ben vdi nhiet, dugc boat boa bdi Fe"** va bi ire che' bdi Cu^"^.
Tai lieu tham khac

1. Nam Idn dViet Nam, Nxb. Khoa hgc Ky thuat - Ha Ndi, 1981.
2. Le Xuan Tham - Nam Linh Chi dugc lieu quy d Viet Nam, Nha
xuat ban Ca Mau, 1996.
3. Beanchamp, C , Fridovich, I., 1971. Superoxide dismutase:
Improved assays and assay applicable to acrylamide gel. Anal.
Biochem., 1: 276-287.
4. Coco, D., Kinaldi, A., savini, I., Cooper, J.M., Bannister, J.V.,
1988. "NADH oxidase from the extreme Thermopbile Thermus
aquatious YT-1. Purification and characteristic". Eur. J.
Biochem., 174:267-271.
5. David Toomey and Stephin, G.M., 1998 "Purification and
characteristic of NADH oxidase from Thermus aquatious YT 1 and evidance that is function tin a peroxidase reduction
system", Eur.J. Biochem, 254: 935 - 945.
6. Leammli, U.K., 1970. Cleavage of structure protein during the
assembly of the head of bacterophage T4. Nature, 227: 680-685.
790


/. Lowry, u.H. Kose orougn, N.J., Farr, A.L., Randall, R.J., 1951.
Protein measurement with the folin phenol reagent. J.
Biochem., 193: 265 - 270.

8. Me Cord, J.M, Fridovich, L., 1969. Superoxide dismutase an
enzymic function forerthoenperia. J. Biochem. 244: 6049 6055.
9. Mottola - Aebi, H.E., 1987. "Catalase" In: Methods of enzymatic
analysis, eds, Bermeyer, J., Grabl, M., VCH Publishers. New
York., 273 - 285.
10. Mottola, H.A., Simpson, B.E., Gorin, G., 1970. Absorptiometric
determination of hydrogen peroxide in submicrogram amout with
leucin crystal violet and peroxodase as catalyst. Anal. Chemistry.,
42:410-411.
11. Poole, L.B., and Claibome, A. 1986. Interactions of pyrimidine
nucleotide with redox forms of the flavin-containing NADH
peroxidase form from Streptococcus faeealis J. Biol. Cbem., 261:
14525 - 14533.
Investigation on some anti-oxidation enzymes of Ganoderma lucidum
For many year, Ganoderma lucidum (GaL) has been considered to
be a valuable medicinal fungus with anticancer and anti-oxidation
activities. In this study, Ganoderma lucidum was selected for
investigation of some anti-oxidation enzymes including catalase
(CAT), superoxide dismutase (SOD), NADH oxidase (NOX).
Our obtained results show that GaL biomass grown in lab
conditions has higher activity of CAT and NOX enzymes than GaL
fruit body. SOD is present at the lowest level in both samples.
The NOX (which was partially purifed by chromatography on
DEAE-celluIose and Sephadex-GlOO columns) appears to work well at
pH 7.5 - 8 as a thermostable enzyme with more than 60% of activity
remained after treatment at 60"C for 15 minutes. However, the enzyme
quyckly losts its activity when being incubated at 80"C. Some ions
such as Mg^", Ca^^ at 1 - 4mM concentrations hardly effect the activity
of enzyme. Fe^"^ at 1 - 4mM concentrations stimulate the activities of
the enzyme whereas Cu^^ at the same concentrations shows to be

strong inhibitor for its activity.

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