B GIO DC V O TO

TRNG I HC CN TH
VIN NGHIN CU V PHT TRIN CNG NGH SINH HC

CHUYN TNG HP
MN PHNG PHP NGHIN CU KHOA HC

TINH SCH ENZYME KERATINASE

CN B HNG DN

SINH VIN THC HIN

TS. TRNG TRNG NGN

NGUYN NGC THANH

MSSV: 3102779
LP: CNSH TT K36

Cn Th, Thng 10/2013

B GIO DC V O TO
TRNG I HC CN TH
VIN NGHIN CU V PHT TRIN CNG NGH SINH HC

CHUYN TNG HP
MN PHNG PHP NGHIN CU KHOA HC

TINH SCH ENZYME KERATINASE

CN B HNG DN

SINH VIN THC HIN

TS. TRNG TRNG NGN

NGUYN NGC THANH

MSSV: 3102779
LP: CNSH TT K36

Cn Th, Thng 10/2013

Chuyn Tng Hp Mn Phng Php Nghin Cu Khoa Hc

Trng HCT

TM LC
Keratinase l protease c kh nng phn hy nhiu loi protein, trong c keratin
vn l protein bn v khng b thy phn bi cc protease khc. Keratinase c xem
nh l gii php thay th cho phng php truyn thng trong x l rc thi lng v,
ng thi em n nhiu hng ng dng khc. Bn cht ca keratinase thng l serine
protease hoc metalloprotease. Keratinase thng hot ng vng pH kim v nhit
tng i cao. Vic tinh sch keratinase l cn thit cho vic nghin cu enzyme v ng
dng chng vo cc lnh vc. Keratinase c th c tinh sch nh vo mt qui trnh kt
hp cc k thut kt ta bng mui trung tnh, sc k v in di SDS-PAGE.
T kha: Hot tnh, keratin, keratinase, tinh sch.

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MC LC
TM LC...................................................................................................................... i
MC LC ....................................................................................................................... ii
DANH SCH BNG......................................................................................................iii
DANH SCH HNH.......................................................................................................iii
CC T VIT TT.......................................................................................................iii
CHNG 1: T VN ............................................................................................1
CHNG 2: KHI QUT V ENZYME PROTEASE V KERATIN.....................2
2.1. S lc v enzyme protease...................................................................................2
2.2. S lc v keratin..................................................................................................3
2.3. S lc v rc thi cha keratin...........................................................................4
CHNG 3: ENZYME KERATINASE........................................................................5
3.1. Ngun gc thu nhn................................................................................................5
3.2. c im ca keratinase........................................................................................5
3.3. C ch tc ng.......................................................................................................6
3.4. Tnh c hiu c cht.............................................................................................7
3.5. S hot ha v bt hot keratinase.......................................................................7
3.5.1 Cc yu t hot ha.................................................................................................7
3.5.2 Cc yu t km hm.................................................................................................7
3.6. ng dng ca keratinase.......................................................................................8
CHNG 4: CC K THUT C BN TRONG QU TRNH
TINH SCH ENZYME...................................................................................................9
4.1. Nguyn tc ca tinh sch enzyme..........................................................................9
4.2. Phng php ta protein.......................................................................................9
4.3. Phng php sc k................................................................................................9
4.3.1. Sc k lc gel..........................................................................................................9
4.3.2. Sc k trao i ion..................................................................................................9
4.3.3. Sc k i lc............................................................................................................9
4.3.4. Sc k tng tc k nc.......................................................................................10
4.3.5. Sc k lng hiu nng cao.....................................................................................10
4.4. K thut in di SDS-PAGE.................................................................................10
CHNG 5: KT LUN..............................................................................................11
TI LIU THAM KHO..............................................................................................12

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DANH SCH BNG

Trang
Bng 1: c tnh sinh ha ca mt s keratinase c la chn6

DANH SCH HNH

Trang
Hnh 1: Phn ng thy phn lin kt peptide..............................2
Hnh 2: Cu hnh -helix ca keratin..3
Hnh 3: Cu hnh -sheet ca keratin. Lin kt hydro gi cc chui polypeptide
li vi nhau.4

CC T VIT TT
DTT:
EDTA:
PMSF:
SDS-PAGE:

Dithiothreitol
Ethylene diamine tetraacetic acid
Phenylmethylsulfonyl fluoride
Sodium dodecylsulfate-Polyacrylamide Gel Electrophoresis

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iii

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Chuyn ngnh Cng ngh Sinh hc Tin Tin

iv

Trng HCT

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CHNG 1: T VN
Trn th gii ni chung v Vit Nam ni ring, cng vi s gia tng dn s, nhu
cu v thc phm cng thc y s pht trin ca mt s ngnh trong c ngnh
chn nui. ng thi vi s tng dn s v pht trin ca cc ngnh kinh t, lng rc
thi ra cng ngy cng tng v dn n nhiu tc ng xu n mi trng. Cc cht thi
rn, c bit l ph phm t ngnh chn nui v git m gia sc, gia cm nh lng, mng,
sng, v.v. tr thnh mt vn ng lo ngi. Loi cht thi ny c thnh phn ch yu l
keratin (90%), mt loi protein kh b phn hy bi cc protease thng thng, nhng
chng hu ht li c thi ra mi trng m khng qua x l. Trong khi , s phn hy
trong t nhin ca loi rc thi ny to ra nhng hp cht cha lu hunh v lm trm
trng thm tnh trng nhim mi trng hin nay (Romano v Russo, 2008). Bn cnh
s nhim mi trng, vic thi trc tip mt lng ln lng gia sc, gia cm ra mi
trng cn gy lng ph ngun protein di do c trong .
Tuy cc phng php vt l v ha hc c th phn no x l c loi cht thi ny,
nhng chng li gy ra s hao tn nng lng v thi gian, lm gim gi tr dinh dng
ca sn phm v khng thn thin vi mi trng (Romano v Russo, 2008; Park v Son,
2009). Nhu cu v vic nghin cu v tm ra mt bin php hiu qu c kh nng gii
quyt cc vn trn ang ngy cng tng. Bin php x l sinh hc ang l hng i mi
y tim nng, gip tit kim c thi gian, chi ph, tn dng ngun protein v c bit
l thn thin hn vi mi trng. Trong , vi sinh vt hoc enzyme li trch t chng
c s dng nh tc nhn ph hy cu trc bn vng ca keratin v phn hy keratin.
c th phn hy keratin, vi sinh vt cn s xc tc ca enzyme keratinase.
Keratinase l enzyme protease c kh nng thy phn keratin v ch c to ra khi c
mt c cht ny (Agrahari, 2013). Keratinase tinh sch t cc vi sinh vt s em n
nhiu hng ng dng, khng ch trong vic tng hiu qu x l rc thi m cn trong cc
ngnh cng nghip v k c y hc. Dng protein tinh khit cng rt cn thit cho vic
nghin cu trnh t chui amino acid, chc nng sinh ha v cc c tnh khc ca chng
(Berg et al., 2012). Tuy nhin, v keratinase t vi sinh vt rt a dng, nn cc nghin cu
vn cha th hiu r ht c ch ca enzyme cng nh cc yu t nh hng n qu trnh
tinh sch enzyme ny. V vy, iu kin ti u cho vic tinh sch keratinase cn c
nghin cu y hn.

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CHNG 2: KHI QUT V ENZYME PROTEASE V KERATIN

2.1.

S lc v enzyme protease
Nhm enzyme protease (cn gi l peptidase hay proteinase) xc tc phn ng thy
phn lin kt peptide (-CO~NH-) trong protein hoc polypeptide to thnh nhng on
nh hn nh peptone, oligopeptide, dipeptide hoc amino acid (Berg et al., 2012; Salleh
et al., 2006). Cc enzyme ny khc nhau r rt v mc c hiu c cht ca chng
(Berg et al., 2012). Ngoi ra, nhiu protease cng c kh nng xc tc phn ng thy phn
lin kt ester (Berg et al., 2012).

Hnh 1: Phn ng thy phn lin kt peptide

(Ngun: Berg et al., 2012)

Nhng nghin cu gn y v cc protease cho thy s lin quan ca chng n

nhiu qu trnh sinh hc tt c cc sinh vt v vai tr thit yu ca chng trong hot
ng ca t bo (Lpez-Otn v Bond, 2008). Bn cnh , s thay i ca h thng phn
gii protein gy ra bnh ung th, ri lon thoi ha thn kinh, cc bnh vim nhim v
bnh tim mch (Lpez-Otn v Bond, 2008). V th nhiu loi protease c ch trng
nghin cu lm mc tiu ca thuc (drug target) hoc du n sinh hc (biomarker) cho
vic chn on v cho triu chng bnh (Lpez-Otn v Bond, 2008). Protease c th c
ngun gc thc vt, ng vt hoc vi sinh vt (Rao et al., 1998).
Da vo cc loi phn ng m enzyme xc tc, y ban Danh php (The
Nomenclature Commission) ca Hip hi Ha sinh v Sinh hc phn t Quc t (The
International Union of Biochemistry and Molecular Biology) xp protease vo lp th 3,
phn lp 4 (EC 3.4). Theo Rao et al. (1998), protease c chia thnh hai nhm,
exopeptidase v endopeptidase, da vo v tr ct ca chng:
- Exopeptidase ct lin kt peptide gn u mch polypeptide. Da vo v tr
tc ng u N t do hay C t do, chng tng ng c phn loi thnh
aminopeptidase v carboxypeptidase.
- Endopeptidase ct lin kt peptide v tr gia mch popypeptide. Cn c vo
nhm chc v tr tm hot ng, endopeptidase li c chia thnh bn nhm nh:
Serine protease (hay serine endopeptidase): Serine protease cha gc serine
tm hot ng v c xem l enzyme quan trng vi sinh vt v s ph bin ca chng
virus, vi khun, v c sinh vt nhn thc. Serine protease thng hot ng vng pH
trung tnh v kim tnh vi pH ti u 7-11, trong serine protease kim tnh l ph bin
nht. Serine protease c tnh c hiu c cht rng v c th ct t c lin kt ester hay
lin kt C-N. Nhm ny gm hai nhm nh l serine protease c tnh kim v subtilisin.

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Aspartic protease: Aspartic protease l protease c tnh acid hot ng pH

thp (pH 3-4), c hot tnh ph thuc vo gc aspartic acid.
Cysteine protease (hay thiol protease): Hot ng ca tt c cysteine protease
ph thuc vo nh nguyn gm cysteine v histidine. Cysteine protease thng hot ng
ti u pH trung tnh.
Metalloprotease: Metalloprotease c c trng bi s cn thit ca ion kim
loi ha tr hai cho hot ng ca chng. Metalloprotease hot ng vng pH trung tnh
hoc pH kim.
Bn cnh cch phn loi ny, protease cn c th c phn loi da trn trnh t
amino acid v mi quan h trong tin ha hoc vng pH ti u cho hot ng ca chng
(Rao et al., 1998).
2.2. S lc v keratin
Theo Garrett v Grisham (2010), keratin thuc nhm protein dng si, v c hai
dng cu trc bc hai l -helix (cu hnh ch yu ca -keratin) (Hnh 2) v -sheet (cu
hnh ch yu ca -keratin) (Hnh 3). -Keratin l thnh phn ch yu trong mng, vut,
guc, sng, biu b, tc, lng mao ca ng vt hu nh trong khi -keratin cng hn keratin v c tm thy nhiu t nhn hoc lng v, m, mng, vy ca loi chim hay
b st (Garrett v Grisham, 2010; Chen et al., 2012).
a

Hnh 2: Cu hnh -helix ca keratin.

(a) M hnh rng. (b) M hnh c. Mu vng: gc cysteine.
(Ngun: Parry v North, 1998)

Theo Chen et al. (2012), mt loi amino acid quan trng trong cu to ca keratin l
cysteine (chim khong 22%), cha nhm ph thiol hnh thnh s lin kt cho disulfide
vi gc cysteine khc. Lin kt hydro, tng tc k nc, s sp xp trong khng gian ca
cc chui peptide cng hai loi cu ni gia chng gm ni i disulfide v lc ht tnh
in ca dicarboxylic acid (gc COO-) vi diamino acid (gc NH3+ hoc NH2+), to nn
cu trc keratin bn vng, cng, khng th ko di, khng tan trong nc, v khng b
phn gii bi cc protease thng thng nh pepsin, trypsin hay papain (Garrett v
Grisham, 2010; Goddard and Michaelis, 1934). Theo Whitford (2005), do kh nng to

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cu ni disulfide bn ca gc cysteine, cng ca keratin ph thuc vo hm lng lu

hunh (s gc cysteine) trong protein . iu ngha l keratin c nhiu gc cysteine
l keratin cng v kh b phn gii hn keratin mm vi t gc cysteine (Whitford, 2005).
Nghin cu ca Goddard v Michaelis nm 1934 cho thy khi hai loi cu ni nu trn b
ph v, keratin c th b phn ct bi cc protease nh nhng loi protein khc.

Hnh 3: Cu hnh -sheet ca keratin. Lin kt hydro gi cc chui

polypeptide li vi nhau (By Irving Geis)
(Ngun: http://www.sciencedirect.com/science/article/pii/S0079642512000242,
ngy 22/10/2013)

2.3.

S lc v rc thi cha keratin

Hng nm, trn th gii c hng triu tn lng v vi thnh phn chnh l keratin
(khong 90%) c thi ra t cc nh my ch bin gia cm (Saibabu et al., 2013). Theo
Romano v Russo (2008), s phn hy chm ca rc thi ny to ra cc hp cht cha lu
hunh gp phn gy nhim mi trng. Mehta et al. (2013) cho rng cht thi cha
keratin c nhiu ng dng ng lu , v mt trong nhng ng dng l s dng lng
v sau khi qua qu trnh x l (ch yu bng nhit v ha cht) sn xut thc n chn
nui. Vic tn dng ngun protein t rc thi cha keratin c th gip gim chi ph cho
cc nguyn liu t tin khc v trnh lng ph ngun protein di do t rc thi. Tuy
nhin, phng php x l ny cng cn nhiu hn ch nh s dng thit b t tin trong
thi gian di, tn nhiu nng lng, ph hng mt s amino acid to ra sn phm kh tiu
ha v ngho gi tr dinh dng, v c bit l cch x l ny khng thn thin vi mi
trng (Romano v Russo, 2008; Park v Son, 2009; Xu et al., 2009). V th, phng
php x l rc thi keratin mt cch thn thin vi mi trng li tit kim nng lng v
tn dng c ngun protein l cn thit i vi tnh hnh rt nhiu rc thi cha keratin
c thi ra nh hin nay.

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CHNG 3: ENZYME KERATINASE

3.1. Ngun gc thu nhn
Nhiu nhm vi sinh vt c kh nng phn gii keratin nh enzyme keratinase do
chng to ra (Mehta et al., 2013). Bn cnh , vic nui cy vi sinh vt tit kim nhiu
khng gian v vi sinh vt kh nhy vi cc k thut di truyn (Rao et al., 1998). V vy, s
dng vi sinh vt phn hy keratin l mt cch hiu qu thay th cho phng php x l
bng ha hc v c hc vn c nhiu hn ch (Park v Son, 2009; Saibabu et al., 2013).
Keratinase (EC 3.4.21-/24/99) l enzyme protease c kh nng thy phn keratin,
c to ra bi mt s loi cn trng v nhiu loi vi sinh vt (Saibabu et al., 2013;
Gradiar et al., 2005). Mt s loi thc vt cng c th tng hp enzyme keratinase (Rao
et al., 1998). Theo Agrahari (2013), c nhiu vi sinh vt sinh keratinase c phn lp
v kho st gm:
- Vi khun: ch yu thuc chi Streptomyces (thuc h x khun) v Bacillus (trc
khun); ngoi ra cn c mt s chng Vibrio, v.v.
- Nm da v nm hoi sinh: Aspergillus sps., Rhizomucor sps., mt s chng thuc
chi Trichophyton v Microsporum, v.v.
Trong cc dng x khun, cc x khun chu nhit c u th l kh nng tch ly
sinh khi v enzyme nhanh so vi cc dng x khun a nhit trung bnh (Selvam v
Vishnupriya, 2012). Hu ht cc chng Bacillus c phn lp t rc thi gia cm v th
hin kh nng phn gii lng 50-60oC (Park v Son, 2009). Mt s chng vi khun khc
nh Bacillus sp. chng kr16, Vibrio sp. chng kr2 li c th phn gii lng v 30-37 oC
(Park v Son, 2009).
3.2. c im ca keratinase
Tnh cht ca keratinase ph thuc vo vi sinh vt tng hp ra chng, v a s cc
keratinase l enzyme ngoi bo (Selvam v Vishnupriya, 2012). Keratinase c hot tnh
mnh trn nhng c cht protein khng tan nh keratin v ch c tng hp khi c s
hin din ca keratin (Agrahari, 2013; Bckle et al., 1995). Theo Selvam v Vishnupriya
(2012), hu ht keratinase thuc h subtilisin ((EC 3.4.21.62) ca nhm serine protease
(EC 3.4.21) v s tng ng v trnh t trong peptide cng ni ln mi quan h . Bn
cnh , theo y ban Danh php v cc nghin cu t trc n nay, mt s keratinase li
thuc nhm metalloprotease (EC 3.4.24) v mt s l endopeptidase m c ch xc tc
vn cha c hiu r (EC 3.4.99).
Keratinase l enzyme mnh hot ng trong vng nhit v pH rng, c kh nng
phn ct cu ni disulfide trong keratin mt cch d dng. Mt s c tnh sinh ha ca
cc keratinase chn c trnh by trong bng 1. Kiu xc tc ca nhiu loi keratinase
c xc nh bi cc c cht c bit v cht c ch (Selvam v Vishnupriya, 2012).
Hu ht cc keratinase ca vi sinh vt l protease kim tnh hoc trung tnh vi ph pH ti
u 7.5-9.0 v nhit ti u 40-85oC (Selvam v Vishnupriya, 2012). c bit, keratinase
ca Nocardiopsis TOA-1 vn bn vng trong khong pH t 1.5 n pH ln hn 12 trong
24 gi 30oC, vi nhit ti u 70-75 oC v pH ti u 11.0-11.5 (Selvam v
Vishnupriya, 2012). Cc keratinase t nm men (nh Candida albicans, Candida
tropicalis, v.v.) l ngoi l v chng hot ng mnh vng pH thp v thuc nhm

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aspartic protease. Cc enzyme thng c khi lng phn t khong t 20 kDa n 60

kDa (Gradiar et al., 2005). Keratinase chu nhit vi phn t khi ln hn cng c
tm thy (Gradiar et al., 2005).
Bng 1: c tnh sinh ha ca mt s keratinase c la chn
Vi sinh vt

Kiu xc tc

pH
ti u

Khi lng phn t

(KDa)

Nhit
ti u (oC)

Bacillus halodurans PPKS-2

Keratinase 1

Disulphide
reductase

30

11.0

60-70

Keratinase -2

Serine protease

66

11.0

70

Bacillus sp

Serine

32

8.0

50

Bacillus sp.JB 99

Serine protease

66

10.0

65

Pseudomonas sp.,MS 21

Serine

30

8.0

37

Pseudomonas aeruginosa

Serine

33

7.0

50

Vibrio sp.Kr 2

Serine

30

8.0

55

Doratomyces microspores

45-70

9.0

Streptomyces sclerotialus

46

9.0

55

Streptomyces chng BA 7

Serine

44

8.5

50

Aspergillus oryzae NRRL-447

39.7

7.0

70

Ahm 1

Metalloprotease

19

6.0-8.0

50

Ahm 2

Metalloprotease

40

6.0-11.0

60-65

Chrysobacterium sp. chng Kr 6

20

50-60

Penicillium sp

(Ngun: Selvam v Vishnupriya, 2012)

Ni chung, enzyme keratinase c mt s tnh cht sau:

- Nhit ti u cho hot tnh xc tc: 40-85 oC.
- pH ti u cho hot tnh xc tc: vng pH kim hoc trung tnh i vi enzyme thuc
nhm serine protease hoc metalloprotease; vng pH acid i vi cc keratinase thuc
nhm aspartic protease.
- im ng in: thng t 7.0 n 9.0.
- Khi lng phn t: t 20 kDa n 60 kDa, mt s keratinase chu nhit c khi
lng phn t ln hn.
3.3. C ch tc ng
C ch tc ng ca keratinase vn cha hon ton c hiu r. Nghin cu cho
thy s hp thu keratin ca keratinase l nh cc tng tc in t gia chng (Selvam v
Vishnupriya, 2012). C ch phn hy ca enzyme kh phc tp, gm s kt hp ca phn

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ng ct lin kt disulfide (sulfitolysis) v phn ng phn hy protein (proteolysis)

(Selvam v Vishnupriya, 2012). Bckle v Mller (1995) cng khng nh s phn ct
cc lin kt disulfide c nh hng ln n s phn hy ca keratin.
Trong cc th nghim, s phn hy keratin v lng keratinase c tng hp khng
t l thun vi nhau. Bn cnh , theo Bckle v Mller (1995), keratinase ngoi bo ca
Streptomyces pactum c th lm r lng v nhng khng th phn hy keratin mt cch
ng k. Hu ht cc keratinase c chng minh l c kh nng thy phn protein nhng
khng c kh nng kh lin kt disulfide. Cc enzyme tinh khit cn cc tc nhn kh
nh DTT ct t ni i disulfide cho qu trnh phn hy keratin xy ra (Bckle v
Mller, 1995). Nghin cu trn qu trnh thy phn keratin ca vi sinh vt cho thy s
kh lin kt disulfide cn c s tham gia ca enzyme disulfide reductase ni bo kt hp
cng h thng oxi ha-kh trn b mt t bo hoc thnh phn kh c t bo tit ra mi
trng (Bckle v Mller, 1995). Tuy nhin, vn c mt s keratinase c hot tnh
disulfide reductase nh keratinase-1 ca Bacillus halodurans PPKS-2.
3.4. Tnh c hiu c cht
Tnh c hiu c cht ca protease ni chung thng l s c hiu i vi cc gc
amino acid c cha lin kt peptide b thy phn. Cc keratinase khc nhau th hin s
khc bit trong tnh c hiu ca chng. Trong th nghim s dng cc peptide tng hp,
cc keratinase phn lp c th hin tnh chuyn bit vi nhng gc amino acid mch
thng, k nc, b ion ha nh Arginine, hoc gc amino acid thm v k nc nh
Tyrosine, Phe, Alanine, Valine, Leucine (Bressollier et al., 1999; Gradiar et al., 2005).
Nhn chung, cc keratinase c khong c hiu c cht rng. Ngoi kh nng thy gii
keratin, chng cn c th phn gii cc loi protein si khc nh fibrin, elastin, collagen
v c c cht khng phi protein si nh casein, gelatin, myoglobin, albumin, v.v.
(Bressollier et al., 1999; Jaouadi et al., 2013; Lin et al., 1992). Mt s keratinase khng
th hoc phn hy collagen rt km (Cai et al., 2008; Jaouadi et al., 2013).
3.5. S hot ha v bt hot keratinase
3.5.1 Cc yu t hot ha
Da trn bn cht ca cc loi keratinase, cc yu t hot ha v km hm c tc
dng khc nhau ln enzyme. DTT c th hot ha keratinase ngoi bo bng cch ct t
lin kt disulfide trong keratin, to iu kin cho keratinase d thy phn keratin hn.
Da trn cc th nghim, Cai et al. (2008) cho rng cc ion nh Ca 2+, Mg2+ v Mn2+ c tc
dng hot ha mt s loi enzyme keratinase. EDTA c th kch thch hot tnh keratinase
ca enzyme mang bn cht serine protease nh keratinase ca chng Bacillus subtilis KDN2, nhng li c ch mt s loi keratinase khc (Cai et al. 2008; Balaji et al., 2008).
3.5.2 Cc yu t km hm
Hu ht cc enzyme keratinase b c ch mt phn bi cht c ch serine protease
nh PMSF, trong khi mt s keratinase t Paecilomyces marquandii, Doratomyces
microspores, Xanthomonas maltophilia, Streptomyces albidoflavus b c ch hon ton
bi tc nhn ny (Cai et al., 2008; De Toni et al., 2002; Gradiar et al., 2005; Bressollier
et al., 1999).
Khc vi keratinase c tm hot ng l serine, keratinase mang bn cht
metalloprotease b km hm bi s thm tch hoc tc nhn to phc kim loi (metal-

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chilating agent) nh 1,10-phenanthroline hay EDTA. Tt c cc metalloprotease u b c

ch bi EDTA, iu ny l c s nhn din loi enzyme ny.
Nhn chung, cc ion kim loi nng nh Cu 2+, Zn2+, Hg2+ c th c ch hot tnh phn
gii keratin ca enzyme (Cai et al., 2008).
3.6. ng dng ca keratinase
Keratinase th v keratinase tinh sch mang n nhiu ng dng trong mt s
lnh vc nh cng nghip, y hc, v mi trng.
- Trong cng nghip:
Ngnh dt, thuc da: Ngn s co rt ca len, lm sch lng v mm da thay
cho cc ha cht.
Cng nghip cht ty ra: Sn xut cht ph gia ty ra phm nhum cha
protein, cht ty ra cht bn trn vi, v.v. Cc keratinase kim tnh c nhiu li th bi
ph pH v nhit hot ng rng cng nh hot tnh mnh ca chng.
Cng nghip thc phm: Sn xut cc amino acid thit yu nh lysine, thy
phn protein nhm nng cao gi tr dinh dng, ch bin thc n chn nui, v.v.
M phm: Sn xut du gi tr gu, thuc tr mn, thuc bi da, v.v.
- Trong y hc:
Lm vc-xin cha bnh nm da.
Tng cng phn phi thuc qua mng trong liu php cha bnh qua mng.
Phn hy cc prion: Prion l ht protein tng t nh virus vn c xem l
nguyn nhn gy bnh Creutzfeldt-Jakob, bnh b in v thoi ha h thn kinh.
Keratinase tinh khit c s dng phn hy cc ht prion ny. y l ng dng mi
v ni bt ca keratinase.
- Bo v mi trng v qui trnh sinh hc:
Phn hy rc thi cha keratin v cc protein khc.
Qui trnh x l sinh hc phim X-quang hoc phim nh thu hi bc.
Chuyn ha cht hu c t rc thi thnh ngun nng lng nh biohydrogen.

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CHNG 4: CC K THUT C BN TRONG

QU TRNH TINH SCH ENZYME
4.1. Nguyn tc ca tinh sch enzyme
Tinh sch enzyme nhm tng hot tnh c hiu ca enzyme ln mc ti a. Hot
tnh c hiu l t s ca hot tnh enzyme trn nng protein trong dung dch (Berg et
al., 2012). Nu cc bc tinh sch c tin hnh hiu qu, th hot tnh c hiu s tng
dn v t n hng s khi mu enzyme tr nn hon ton tinh khit.
4.2. Phng php ta protein
Enzyme sau khi c tch khi t bo v n nh trong dch chit th thng c
kt ta tch khi dung dch ln tp cht. K thut ph bin nht l to kt ta bng
mui (Salting out). Hu ht cc protein u t tan nng mui cao v cc loi protein
khc nhau kt ta cc nng mui khc nhau (Berg et al., 2012). V th enzyme c
th d dng c phn on bng cch ny (Berg et al., 2012). Mui v c trung tnh nh
ammonium sulfate thng c s dng ta enzyme. Phng php kt ta mui trung
tnh ny cng gip c c li nhng dung dch protein long t nhng giai on tinh sch
khc (Berg et al., 2012). Phng php thm tch hoc phng php lc gel thng c
s dng loi b mui khi enzyme (Berg et al., 2012).
Ngoi mui trung tnh, cn c cc tc nhn khc to ta protein nh:
- Dung mi hu c: Acetone, ethanol, isopropanol thng c s dng.
- pH: pH c chnh n im ng in lm protein mt in tch v to ta.
- Polymer khng mang in: Polyethylen glycol thng c s dng.
- Nhit .
4.3. Phng php sc k
4.3.1. Sc k lc gel
Phng php ny gip phn on protein da trn kch thc (Berg et al., 2012).
Cht mang ca ct sc k gm cc ht xp lm t dextran hoc agarose hoc
polyacrylamide c tc dng gi cc phn t nh li trong cc l xp v cc phn t c
kch thc cng ln s chy ra khi ct cng nhanh hn (Berg et al., 2012). Nh vy, cc
phn t ln nht s c tch ra trc, n phn t c kch thc trung bnh ri nh. Cc
phn t ri khi ct c tch ra theo kch thc ca chng.
4.3.2. Sc k trao i ion
Sc k trao i ion tch cc phn t da trn in tch ca chng (Berg et al., 2012).
Protein c in tch cng du vi in tch ht trong ct sc k c xu hng ri khi ct
nhanh hn, v protein c in tch tri du bm vo ct ri c gii phng sau (Berg et
al., 2012). Phng php ny gm c sc k trao i ion dng v sc k trao i ion m
(Berg et al., 2012).
4.3.3. Sc k i lc
Phng php ny l phng tin hiu qu tinh sch protein vi tnh chn lc cao
cn c vo i lc ca protein vi cc gc ha hc c hiu (cn gi l ligand) (Berg et al.,
2012; Garrett v Grisham, 2010). Phng php sc k i lc t hiu qu cao nht khi
enzyme tng tc rt c hiu vi c cht (Berg et al., 2012).

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4.3.4. Sc k tng tc k nc
Phng php ny da trn bn cht k nc ca protein (Garrett v Grisham, 2010).
Cht nn trong ct sc k lin kt vi gc k nc ca enzyme v gi chng li trong ct
(Garrett v Grisham, 2010).
4.3.5. Sc k lng hiu nng cao
y l k thut c ci thin t cc k thut sc k ct ni trn. p sut c s
dng gi cho tc dng chy ph hp (Berg et al., 2012). u im ca k thut ny
l s phn on cc protein hiu qu hn v nhanh hn (Garrett v Grisham, 2010).
Cc bo co cho thy, enzyme keratinase sau khi ta bng ammonium sulfate c th
c tinh sch bi nhiu phng php sc k khc nhau nh sc k lc gel, sc k trao i
ion, sc k lng hiu nng cao (Lin et al., 1992; Bressollier et al., 1999; Bckle, 1995).
4.4. K thut in di SDS-PAGE
Theo Berg et al. (2012), SDS-PAGE thng c dng xc nh khi lng phn
t protein. SDS-PAGE thng c s dng cng vi cc nhn t kh nh mercaptoethanol cc cu ni disulfide trong enzyme b ph v (Berg et al., 2012). Tc
di chuyn ca protein trong in trng t l nghch vi khi lng phn t ca chng
(Berg et al., 2012). Da vo thang chun v khong cch di chuyn trong in trng,
khi lng phn t ca enzyme c th c xc nh.

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CHNG 4: KT LUN
Keratin l thnh phn chnh ca cc ph phm t vic git m gia sc, gia cm nh
lng, mng, sng, v.v., v rt kh b phn hy bi cc protease thng thng nh vo cu
trc bn vng ca chng. Cc rc thi cha keratin l mt trong nhng nguyn nhn gy
nhim mi trng, v bin php hiu qu x l cng nh khai thc ngun protein trong
l iu cn thit. Keratinase c xem l gii php l tng cho vn rc thi ny v
chng l cc protease c hot tnh mnh trn nhiu c cht protein khng tan, trong c
keratin. Cc keratinase thu nhn c thng l serine protease hoc metalloprotease hot
ng trong vng pH v nhit rng. Tuy c ch hot ng ca keratinase vn cha c
hon ton hiu r v c th mt s keratinase cn thnh phn oxi ha-kh trong t bo
thy phn c keratin, nhng bn thn enzyme vn c hot tnh mnh trn nhiu loi
protein. Nh vo hot tnh mnh, enzyme ny mang n nhiu ng dng cn bn v ng
dng mi trong cc lnh vc nh cng nghip, y hc, mi trng. Keratinase cn c
tinh sch nghin cu k hn v c ch hot ng v cc tnh cht khc. S kt hp cc
k thut nh kt ta protein, sc k v in di SDS-PAGE c th c dng tinh sch
enzyme ny, to tin cho vic nghin cu su hn v cc loi keratinase.

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Trang web
http://www.chem.qmul.ac.uk/iubmb/enzyme/ (ngy 15/10/2013)
http://www.sciencedirect.com/science/article/pii/S0079642512000242 (ngy 22/10/2013)

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