Doko.vn 341037 Phan Lap Va Tuyen Chon Chung V

S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

I HC THI NGUYN

TRNG I HC S PHM

NGUYN TH MINH

PHN LP V TUYN CHN CHNG VI SINH VT

C KH NNG THY PHN KERATIN T LNG V GIA CM

V THIT K VECTOR TI T HP BIU HIN KERATINASE

TRONG T BO VI KHUN ESCHERICHIA COLI

LUN VN THC S SINH HC

THI NGUYN 2011

S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

I HC THI NGUYN

TRNG I HC S PHM

NGUYN TH MINH

PHN LP V TUYN CHN CHNG VI SINH VT

C KH NNG THY PHN KERATIN T LNG V GIA CM

V THIT K VECTOR TI T HP BIU HIN KERATINASE

TRONG T BO VI KHUN ESCHERICHIA COLI

Chuyn ngnh : Di truyn hc

M s: 60.42.70

LUN VN THC S SINH HC

Ngi hng dn khoa hc: TS. Nguyn Huy Hong

THI NGUYN 2011

Lun vn Thc s khoa hc HVCH: Nguyn Th Minh

Chuyn ngnh Di truyn hc Lp Sinh K17

LI CM N

Em xin by t lng bit n tri n v su sc ti TS. Nguyn Huy Hong

Ph trng Phng Vi sinh vt t, Vin Cng ngh Sinh hc, Vin KH&CN

Vit Nam, l ngi thy_ngi anh tn tnh hng dn, du dt, to mi

iu kin thun li gip v chia s nhng kh khn cng em trong sut qu

trnh nghin cu v hon thnh lun vn.

Trong qu trnh nghin cu va qua, em nhn c s gip v ch

bo tn tnh ca cc cn b Phng Vi sinh vt t, c bit l Th.S. Nguyn

Qunh Mai, CN. Nguyn Thu Hin gip em trong qu trnh tin hnh

th nghim.

Em cng xin chn thnh cm n cc thy, c gio tham gia ging dy ti

Khoa Sinh-KTNN i Hc Thi Nguyn- H S Phm Thi Nguyn- Khoa

o to sau i hc truyn t cho em nhng kin thc b ch trong sut 2

nm qua.

Bn cnh , em cng xin by t lng bit n su sc ti BGH cng ton

th cn b gio vin trng THPT im Thy, Phng ADT ng dng thuc

Phng th nghim trng im CNG to mi iu kin thun li gip em

trong qu trnh hc tp v nghin cu ti Trng v Vin

Cui cng, em xin gi ti b, m v nhng ngi thn trong gia nh

lng bit n su sc, cm n nhng ngi bn chia s kh khn th thch

trong cuc sng v cng vic em c c kt qu ny.

Mt ln na em xin chn thnh cm n!

Thi Nguyn, thng 09 nm 2011

Nguyn Th Minh


Chuyn ngnh Di truyn hc Lp Sinh K17 i

MC LC

Trang

Trang ph ba i

Li cm n ii

Mc lc iii

Danh mc cc k hiu, cc ch vit tt iv

Danh mc cc bng v

Danh mc cc hnh vi

M U .......................................................................................................... 1

PHN I: TNG QUAN TI LIU .............................................................. .3

1.1. Keratin .. ...................................................................................................3

1.2. Keratinase ............................................................................................... .6

1.3. a dng vi sinh vt c kh nng sinh keratinase cao .............................. 8

1.4. Tnh hnh nghin cu ........................................................................... .12

1.4.1 Tnh hnh nghin cu trong nc ..................................................... .12

1.4.2 Tnh hnh nghin cu trn th gii .................................................... 13

1.5. Sn xut v ng dng qu trnh thu phn keratin trong lng v ......... 14

1.5.1. Tng hm lng cht dinh dng trong thc n gia sc ................. 17

1.5.2. To phn bn .................................................................................... 17

1.5.3. To enzyme trong cc ngnh cng nghip ....................................... 18

1.5.4. Ci to t ......................................................................................... 18

1.5.6. To hp cht mu ............................................................................. 19

1.5.7. ng dng trong cng nghip dc v sn xut thc phm chc nng ... 19

PHN II: NGUYN LIU V PHNG PHP ..................................... 20

2.1. Nguyn liu ........................................................................................... 20

2.1.1. Nguyn liu ...................................................................................... 20

2.1.2. Ho cht ............................................................................................ 20

2.1.3. Thit b ............................................................................................. 20


Chuyn ngnh Di truyn hc Lp Sinh K17 ii

2.1.4. Mi trng nui cy ......................................................................... 20

2.1.4.1. Mi trng Broth Peptone.......................................................... 20

2.1.4.2. Mi trng I ............................................................................... 20

2.1.4.3. Mi trng II .............................................................................. 21

2.1.4.4. Mi trng III ............................................................................. 21

2.1.4.5. Mi trng phn lp chng Chryseobacterium ................... .21

2.1.4.6. Mi trng nui lc lng v ...................................................... 21

2.1.4.7. Mi trng MPA ........................................................................ 21

2.2. Phng php nghin cu ....................................................................... 22

2.2.1. Phn lp chng vi khun c kh nng sinh keratinase cao .............. 22

2.2.1.1. Ly mu v o pH t ................................................................. 22

2.2.1.2. Phn lp chng vi khun Chryseobacterium .............................. 22

2.2.1.3. Phn lp chng vi khun Vibrio ................................................. 23

2.2.1.4. Phn lp chng vi khun Bacillus ............................................... 23

2.2.2. Xc nh kh nng thu phn lng v ............................................. 23

2.2.2.1. Nui ging cp 1 vo mi trng MPA ..................................... 23

2.2.2.2. Xc nh mt t bo ............................................................... 25

2.2.3. La chn mi trng thch hp cho cc chng vi khun c kh

nng sinh keratinase cao ............................................................................ 25

2.2.4. nh hng ca nhit v pH ln kh nng sinh trng ca vi

khun thy phn lng v ............................................................................ 26

2.2.5. Xc nh c im hnh thi ca cc chng vi khun ..................... 26

2.2.6. Xc nh gram m v gram dng ca vi khun ............................ 26

2.2.6.1. Phng php Hucker ci tin ..................................................... 26

2.2.6.2. Xc nh bng cch kim tra kt dnh ................................... 27

2.2.7. Phng php phn loi vi khun theo c tnh sinh ho ................. 28

2.2.8. Chn dng gene keratinase vo vector biu hin pET32a .............. 30

2.2.8.1. Tch chit DNA tng s ca chng vi khun ............................ 30

2.2.8.2. Nhn bn on DNA bng PCR ................................................ 32


Chuyn ngnh Di truyn hc Lp Sinh K17 iii

2.2.9. Phn tch hot tnh keratinase ngoi bo ......................................... 38

PHN III: KT QU V THO LUN ................................................... 41

3.1. Kt qu ly mu cc a im ............................................................ 41

3.2. Gi tr pH ca cc mu t .................................................................... 41

3.3. Phn lp v tuyn chn cc chng vi sinh vt c kh thy phn keratin ..... 42

3.4. La chn mi trng thch hp ca cc chng c chn lc............. 44

3.5. Ti u nhit ca cc chng vi khun chn lc ................................ 47

3.6. c im hnh thi nhm vi khun thu phn lng v ......................... 48

3.7. Kt qu xc nh gram ca cc chng vi khun ................................... 49

3.8. Phn loi cc chng phn lp theo phng php sinh ho ................... 50

3.8.1. Phn loi theo kit API 50CHB ca chng vi khun .GT.2.2 ....... 50

3.8.2. Phn loi theo kit API 20NE ca chng vi khun L.SC.1.2 ........... 51

3.9. Phn loi theo kiu gene ....................................................................... 52

3.9.1. Tch DNA tng s ........................................................................... 52

3.9.2. Nhn on gene m ha 16S rARN bng k thut PCR ................. 53

3.10. Chn dng gene keratinase vo vector biu hin pET32a ................. 55

3.10.1. Nhn on gene keratinase bng k thut PCR .......................... 55

3.10.2. Gn sn phm PCR thi gel tinh sch vo vector tch

dng pET32a ........................................................................................... 56

3.10.3. Bin np sn phm lai vo t bo kh bin DH10B ................... 58

3.10.4. Tch DNA plasmid ti t hp ..................................................... 59

3.11. nh gi hot tnh ezyme dch th ca chng chn lc ................... 60

3.11.1. nh lng Protein bng phng php Bradford ....................... 60

3.11.2. Th hot tnh enzyme bng azokeratin ....................................... 62

PHN IV: KT LUN V KIN NGH ................................................... 64

4.1 Kt lun .................................................................................................. 64

4.2 Kin ngh ................................................................................................ 64

TI LIU THAM KHO ............................................................................ 65


Chuyn ngnh Di truyn hc Lp Sinh K17 iv

BNG CH VIT TT

PCR Polymerase Chain Reaction (Phn ng chui trng hp)

EtBr Dung dch Ethidium Bromide

BSA Bovine serum albumin

DNA Deoxyribonucleic axid

rARN Ribosomal ribonucleic acid

LB Luria-Bertani

Ker Keratinase

bp Base pair(cp ba z)

E.coli Escherichia coli.

l Microlit

EtOH Ethanol

Primer-F Mi xui

Primer-R Mi ngc


Chuyn ngnh Di truyn hc Lp Sinh K17 v

DANH MC CC BNG

S hiu bng Tn bng Trang

1.1 S a dng ca vi sinh vt c kh nng sinh

keratinase v cc c tnh ha sinh ca keratinase

9

1.2 ng dng ca enzyme 16

2.1 49 php th ln men API 50 CHB 28

2.2 50 php th sinh ha API 20 CHB 31

3.1 a im ly mu 41

3.2 Gi tr pH ca cc mu t 41

3.3 T l thy phn lng v ca cc chng tuyn chn 43

3.4 Mt t bo v kh nng thy phn 1 gam lng v

cc nhit khc nhau(Sau 4 ngy nui cy lc)

47

3.5 Kt qu xc nh gram m, gram dng 50

3.6 Kt qu php th sinh ha theo kit API 50 CHB ca

chng .G.T.2.2

50

3.7 Kt qu php th sinh ha theo kit API 50 CHB ca

chng L.SC.1.2

52

3.8 Kt qu o gi tr mt quang (OD) ca BSA 61

3.9 Kt qu o gi tr mt quang (OD) v hm lng

protein ca dch ni vi khun

62

3.10 Kt qu xc nh hot tnh enzyme keratinase 63


Chuyn ngnh Di truyn hc Lp Sinh K17 vi

DANH MC CC HNH V

S hiu hnh v Tn hnh v Trang

1.1 Mt s hnh dng lng v 3

1.2 Cu to phn t keratin 5

1.3 nh di knh hin vi cc si bn trong t bo

keratin

6

1.4 Cu to phn t enzyme keratinase 7

3.1 Kh nng thy phn lng v ca 4 chng chn lc

(Sau 4 ngy nui cy lc)

44

3.2 Kh nng sinh trng v thy phn lng v ca

cc chng vi khun trn cc mi trng khc nhau

(Sau 4 ngy nui cy lc)

46

3.3 Kh nng thy phn lng v ca cc chng vi

khun trn cc mi trng khc nhau (Sau 4 ngy

nui cy lc)

46

3.4 Kh nng thy phn lng v ca cc chng vi

khun cc khung nhit khc nhau (Sau 4 ngy

nui cy lc)

48

3.5 Hnh nh t bo vi khun vi phng i 4800

ln

49

3.6 nh in di DNA tng s 53

3.7 Sn phm PCR bng mi 16S 54

3.8 Cy pht sinh chng loi ca 2 chng .GT.2.2

v L.SC.1.2

55

3.9 Sn phm PCR bng mi ker F v ker R 56

3.10 Sn phm thi gel tinh sch gene ker 56


Chuyn ngnh Di truyn hc Lp Sinh K17 vii

3.11 Sn phm tinh sch gene ker v vector

pET32asau khi x l bng 2 enzyme ct

57

3.12 Vector ti t hp pET32a /ker 58

3.13 Sn phm bin np ca 2 chng vi khun vo t

bo kh bin E.coli DH10B

58

3.14 nh in di sn phm tch DNA plasmid ti t

hp ca cc chng sau bin np

59

3.15 Sn phm phn ng ct DNA plasmid ti t hp

bng 2 enzyme ct XhoI v BamHI

60

3.16 ng chun Bradford 61


Chuyn ngnh Di truyn hc 1 Lp Sinh K17 S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

M U

Lng v c lng protein keratin th rt cao (90 - 95%), nhng phn ln

protein trong lng v khng ha tan trong nc. Mc d rt kh b phn hy,

nhng cc cht thi keratin c th b phn hy bi cc chng vi khun, x

khun v nm c kh nng sinh tng hp keratinase. Vi sinh vt sinh tng hp

keratinase c phn lp t nhiu v tr a l khc nhau, t t Nam cc

n sui nc nng, k c iu kin mi trng hiu kh v k kh. n

nay, cc keratinase c nghin cu ch yu c ngun gc t vi khun nh

Bacillus, Chryseobacterium, Pseudomonas,. V vy, vic khai thc a dng

vi sinh vt s cung cp sn phm keratinase ph hp vi tng ng dng c

th.

Keratinase ph v cu ni disunfua ca keratin lm t lin kt ca vng

xon. Sau khi thy phn hp cht keratin trong lng v c phn hu v

chia nh thnh cc axit amin v peptit ngn. Nh vy, gia sc, gia cm c th

s dng lng v sau khi b thy phn lm thc n. Chnh v vy, keratinase

v ang c nghin cu ng dng trong cc qu trnh thy phn lng v

to thnh cc hp cht c th b sung vo thc n gia sc, gia cm hoc lm

phn bn hu c,.

Vit Nam hin nay ngnh cng nghip ti to ngun ph thi lm phn

bn v thc n cho gia sc vn cn non tr v ang trong giai on tm ngun

nguyn liu cng nh nh hng pht trin. Phng php c ng dng

ph bin hin nay x l ph thi lng v l s dng kim hoc acid. Tuy

nhin, phng php ny cn nhiu hn ch (gi thnh cao, hiu sut thp),

do vy vic ng dng enzyme t vi khun x l ph thi c nhiu u im

hn nh to sn phm c gi tr dinh dng cao, gi thnh thp, mt khc cn

rt thn thin vi mi trng. V vy, vic nghin cu enzyme t vi khun

thy phn lng v va c ngha khoa hc, va c ngha thc tin.



Vi nhng u im trn, phc v cho vic nghin cu ng dng vi sinh vt

t v trn lng v c kh nng phn hu lng v, chng ti tin hnh

nghin cu ti: Tn ti Phn lp v tuyn chn chng vi sinh vt c

kh nng thy phn keratin t lng v gia cm v thit k vector ti t

hp biu hin keratinase trong t bo vi khun E.Coli .

Mc tiu ca ti:

- Tuyn chn c b chng vi sinh vt t thuc cc nhm vi sinh vt nh

Bacillus, Chryseobacterium v Vibrio c hot tnh thy phn vi hiu sut 70

80% lng keratin lng v trong mi trng nui cy.

- Thit k c vector ti t hp biu hin keratinase E.Coli.

ngha ca ti:

ng dng x l lng v trong sn xut phn bn.

Thay th hoc tng hm lng protein trong thc n gia sc gia cm.

Ngoi ra, vic nghin cu ca ti cn mang li nhiu ngha thit thc

trong i sng nh: Ci to t trng, gim thiu nhim mi trng v to

ra ngun keratinnase v cng phong ph.

Ni dung:

- Thu thp mu t chn nui gia cm hoc ni git m gia cm.

- Tuyn chn c cc chng Bacillus, Pseudomonas, Chryseobacterium trn

mi trng nui cy.

- Nhn dng v hnh thi v c im ha sinh ca cc chng thu c c kh

nng phn gii mnh keratin.

- Thit k v nhn on gen keratinase t chng c hot tnh mnh.

- Chn dng keratinase trong vector biu hin pET32a biu hin trong t

bo vi khun E. coli.

- Phn tch hot tnh enzyme keratinase (enzyme ngoi bo).



PHN I: TNG QUAN TI LIU

1.1. Keratin

Keratin l mt hp cht sng rt kh phn hu, l thnh phn ch yu ca

lp biu b, tc, lng, mng chn mng tay v men rng. Keratin l mt phc

cht ha hc c cha mt lng ln sulfua, cc acid amin, c bit l cystein.

Ngoi ra cn c tyrosin, histidin, lysin, arginin, cc mui: carbonat canxi,

phosphat canxi.

Hnh 1.1: Mt s hnh dng lng v

Nhn hnh dng bn ngoi ca lng v c th thy c lng v bao gm hai

thnh phn chnh l cc si lng v cung lng (Hnh 1.1). Trong lng v

hm lng protein keratin chim t l rt cao chim t 90 95%. T cc si

keratin c cha hm lng lu hunh ln c p thnh cc b keratin ln

hn. Trn cc si lng, cht keratin rt mm do v d dng b phn ct thnh

nhng si nh v tan ht vo mi trng. Tuy nhin, trn cung lng th

keratin li c hm lng lu hunh cao, to ra nhng hp cht cng, th nn

rt kh c th thu phn c. Khi cy cc vi khun thu phn lng v th

tt c cc cung lng nh u c thu phn hon ton nhng cc cung

lng to th mi ch c phn ct thnh nhng on v nhng si rt nh



trong khong thi gian hn t 7- 10 ngy. Nh vy, hm lng hp cht

keratin trong lng v l nguyn nhn gy nn mi kht rt ln khi t lng v

[13,14,15].

Keratin l thnh phn chnh ca biu b v tc, lng v, sng, mng,

len. Da trn xc nh cu trc c bn bc hai, keratin c phn thnh v

(-helix ca tc v len, -sheets ca lng v) [33]. Cc si keratin c hai

dng -helix v -sheets c xon song song vi nhau m bo bn n

nh ca si [34,35]. Cc keratin c chia thnh keratin cng v keratin

mm ty thuc vo lng sulfur. Keratin cng lng v, tc, mng guc,

mng c nhiu cu ni disulfur v khng th ko di ra. Trong khi , keratin

mm nh da, chai da c t cu ni disulfur v mm do hn. Cc enzyme

c bit n nh trypsin, pepsin v papain c kh nng ct cc cu ni trong

keratin phn hy [36]. Mc d rt kh phn hy, nhng cc cht thi

keratin c th b phn hy bi vi khun, x khun v nm do chng to ra cc

protease keratinase.

Thy phn keratin t vi sinh vt c nghin cu t nm 1959 [37],

tuy nhin c ch thy phn t vi khun vn cha c nghin cu nhiu,

nhng vic gim cu ni disulfur c th c nh hng ng k n s phn

hy keratin [38,39]. Keratinase [EC 3.4.21/24/99.11] l serine hay l cc

metalloprotease c kh nng phn hy cu trc protein keratin kh tan. Cc

keratinase tham gia thy phn cc cht thi keratin t lng v trong cng

nghip chn nui gia cm to ra cc sn phm c gi tr nh thc n chn

nui, phn bn, cc polymer v cc amino acid him nh serine, cysteine v

proline.

Trong mng chn tay v men rng th hm lng keratin tn ti dng

cng nn rt kh phn hu. Ngoi ra, keratin cn tn ti trong sng v mng

ca cc loi ng vt c v.Cn 1 s ng vt chn t, lp gip xc thng

c cc b phn ging nh chic o gip hoc ging b xng ngoi lm



nhim v bo v u c cu to bng keratin, ch mt phn rt nh cu to

t chitin.

Tnh cht

Keratin c cu to hnh si xon. Chng xon xung quanh nhau to thnh cc

si trung gian, iu ny to ln cu ni disulfua, hnh thnh ln cu trc rt

bn vng. Trong phn t ca keratin c cha t l cao phn t lu hunh v

nhiu loi axit amin. c bit trong c mt loi axit amin l cysteine, chnh

iu ny lm ni bt tnh cht ca keratin l rt kh ha tan (Hnh 1.2).

Hnh1.2: Cu to phn t keratin

Theo cu to ca keratin c 2 loi chnh sau:

Anpha- keratin (-keratin): Trong tc (k c lng cu), sng, mng tay, mng

chn, mng v mng guc ca ng vt c v. Cu to l cc si n protein

lin kt vi nhau bng lin kt hydro.

Beta keratin (-keratin): Trong mng tay v mng vut ca loi b st, h v

chelonians (ba ba, ra, ), v trong lng, m, mng vut ca chim v nhm,

c hnh thnh ch yu trong cc tm . Cu trc cng, chc hn cu trc

do cc tm c ni vi nhau bng cc cu ni disulfide.



Keratin c to thnh bi keratinocytes, cc t bo sng to nn mt

phn ln ca da, tc, mng tay mng chn, v keratin khc c cha trong cc

b phn ca c th. Cc t bo t t y ln trn b mt, cui cng cht v to

thnh mt lp bo v ca cc t bo. Hng ngn cc t bo ny c th c

sn sinh ra trong cc iu kin khc nhau, chng hn nh bnh vy nn. Do

gy h hi cho cc lp bn ngoi ca keratin, cho nn c th lm, tc, mng

tay mng chn hay gy v da bong ra tng mng (Hnh 1.3).

Hnh 1.3: nh di knh hin vi cc si bn trong t bo keratin

1.2. Keratinase

Keratinase l mt enzyme thuc nhm protease. y l enzyme c kh

nng thu phn cu trc phc tp ca keratin lng. Keratinase c to ra t

nhiu ngun vi sinh vt khc nhau thuc nhm vi sinh vt c, nhm vi sinh

vt nhn tht v nm. Keratinase thuc nhm enzyme protease, c kh nng

thy phn protein keratin khng tan. Cc enzyme ny c to ra trong s qu

trnh phn hy cc c cht nh keratin trong tc, lng, sng, Cc c tnh

v ha sinh v l sinh ca keratinase l rt a dng. Phn ln keratinase hot

ng iu kin ti u l pH trung tnh ti kim v nhit t 300C n

600C. Kerainase c tim nng ng dng ln trong nng nghip, dc hc v

cc lnh vc y sinh hc. Ngoi ra, s dng keratinase trong sinh khi sinh hc



c th gii quyt mt phn no v chuyn i nng lng v ti s dng

cc nhin liu.

Gn y, mt nghin cu cng nhn v nh ngha keratinase l

protease serine do hnh dng cu to c ti 97% ging vi protease kim v

n cng b c ch bi cc thuc c ch ging nh c ch protease serine [18].

Khi lng phn t ty theo tng loi sinh vt c th dao ng t 18 n 35

kDa i vi protease serine c khi lng phn t nh v ln hn 90 kDal i

vi protease serine c khi lng ln.

Hnh 1.4: Cu to phn t ca enzym Keratinase



Tnh cht:

Keratinase thu phn thnh cc axit amin:

Vi tnh cht thu phn ca keratinase, n ph v cu ni disunfua ca

keratin lm t lin kt ca vng xon. Nh vy, sau khi thu phn hp cht

keratin trong lng v c phn hu v chia nh thnh cc axit amin v

peptit ngn. Khi protein keratin mang y cc tnh cht ca protein tan

nh l: Tnh ho tan, kt ta, in ly lng tnh, thu phn v phn ng mu

c trng.

Keratinase ch yu tn cng vo cc lin kt disulfide (S-S), lm suy gim

cc protein dng si fibrin, elastin, collagen v si protein khc.

pH v nhit :

Keratinase chu c nhit t 30oC - 80oC nhng nhit ti u l:

50oC v enzyme ny c n nh khi bo qun -20oC [21]. Keratinase bn

vng trong khong pH t 6,0 - 9,0 nhng pH ti u l 7,5 [21].

Cht km hm:

Khi b sung ng sucrose v lactose lm c ch s tng hp keratinase

[21]. Cht nn c nng v lng c cht qu cao cng s lm c ch s

tng hp keratinase [21]. Khi b sung cc cht c cha cc ion kim loi nng

ho tr hai nh: Cu2+, Ag2+, Hg2+ v Pb2+ th cng lm c ch s hot ng ca

keratinase [22]. Mt s ho cht khc cng c ch phn ng enzyme

keratinase nh: EDTA,[21].

Cht hot ho:

Theo nhiu nghin cu trn th gii th khi b sung cc cht c cha cc

ion kim loi ho tr hai nh: Ca2+, Mg2+, Mn2+ th kch thch s hot ng ca

keratinase [22].

1.3. a dng vi sinh vt c kh nng sinh keratinase cao



Cc keratinase l mt dng protease rt ph bin trong th gii vi sinh vt

v c phn lp t nhiu v tr a l khc nhau c iu kin mi trng

hiu kh v k kh. V vy, vi khun tng hp keratinase c s a dng rt ln

v c tnh ha sinh v l sinh. Cc c tnh ca mt s vi khun tng hp

keratinase c tm tt trong bng 1.1.

Bng 1.1: S a dng vi sinh vt c kh nng sinh keratinase v cc c

tnh ha sinh ca keratianse

Vi sinh vt

Dng xc

tc

Trng

lng

phn t

(kDa)

pH ti

u

Nhit

ti u

(00C)

Chch

dn

Bacillus cereus

DCUW

S e r i n e 8 0 8 . 5 5 0 [ 4 0 ]

Bacillus

licheniformis

K-508

T h i o l 4 2 8 . 5 5 2 [ 4 1 ]

Bacillus

licheniformis

PWD-1

S e r i n e 3 3 7 . 5 5 0 [ 4 2 ]

Bacillus pumilis S e r i n e 6 5 8 . 0 6 5 [ 4 3 ]

Bacillus subtilis

KS-1

S e r i n e 2 5 . 4 7 . 5 [ 4 4 ]

Bacillus subtilis

MTCC (9102)

M e t a l l o 6 9 6 4 0 [ 4 5 ]

Bacillus subtilis

RM-01

S e r i n e 2 0 . 1 9 4 5 [ 4 6 ]

Clostridium

sporogenes

2 8 . 7 8 5 5 [ 4 7 ]

Chryseobacterium

indologenes

TKU014

M e t a l l o P 1 : 5 6 P 1 : 1 0 P 1 : 3 0 5 0 [ 4 8 ]

Fervidobacterim

islandicum AW-1

S e r i n e > 2 0 0 9 1 0 0 [ 4 9 ]

Fervidobacterium

pennavorans

S e r i n e 1 3 0 1 0 8 0 [ 5 0 ]



Kocuria rosea S e r i n e 2 4 0 1 0 4 0 [ 5 1 ]

Microbacterium

sp. kr10

M e t a l l o 4 2 7 . 5 5 0 [ 5 2 ]

Nesternkonia sp.

AL-20

S e r i n e 2 3 1 0 7 0 [ 5 3 ]

Stenotrophomonas

maltophilia

S e r i n e 3 5 . 2 7 . 8 4 0 [ 5 4 ]

Nocardiopsis sp.

TOA-1

S e r i n e 2 0 > 1 2 . 5 6 0 [ 5 5 ]

Streptomyces

sp.S7

S e r i n e -

m e t a l l o

4 4 1 1 4 5 [ 5 6 ]

Streptomyces sp.

strain 16

S e r i n e K I :

2 0 3 . 2

K I : 9 K I : 5 0 [ 5 7 ]

Streptomyces

albidoflavus

S e r i n e 1 8 6 9 . 5 4 0 7 0 [ 5 8 ]

Streptomyces

pactum

S e r i n e 3 0 7 1 0 4 0 7 5 [ 5 9 ]

Streptomyces

gulbagensis DAS

131

4 6 9 4 5 [ 6 0 ]

Streptomyces

thermoviolaceus

4 0 8 5 5 [ 6 1 ]

Thermoanaerobac

ter keratinophilus

S e r i n e 1 3 5 8 8 5 [ 6 2 ]

n nay, keratinase c nghin cu ch yu c ngun gc t vi khun, trong

Bacillus c nghin cu nhiu nht. Nhiu chng B. licheniformis v B.

subtilis c m t c kh nng thy phn keratin [44, 63-66], nhng loi

khc nh B. pumilus v B. cereus cng c kh nng tng hp keratinase [40,

43, 67, 68]. Cc loi khc nhau ca cng mt chi (chng hn, Bacillus) c th

to ra cc keratinase khc nhau (Bng 1.1).

Sinh tng hp keratinase cng c nghin cu x khun, ch yu l t

cc chi Streptomyces. Nhm vi khun ny c phn lp t cc vng t khc

nhau, c kh nng thy phn mt lot cc cht cha keratin nh tc, len v



lng. Gushterova v cng s [69] phn lp hai chng x khun chu nhit

c hot tnh thy phn keratin cao l Streptomyces flavis 2BG v

Microbispora aerata IMBAS-11A. Cc loi chu nhit nh Streptomyces

gulbarguensis [60], Streptomyces thermoviolaceus [61] v Streptomyces

thermonitrificans [70] cng c phn lp t t. Ngoi nhng chng a

nhit, mt s Streptomyces km bn nhit c phn lp nh Streptomyces

pactum DSM 40.530 [59], Streptomyces graminofaciens [39] v Streptomyces

albidoflavus K1-02 [58].

Ngoi cc chng thuc Bacillus sp. v x khun, mt s chng vi khun c

kh nng thy phn keratin din ra nhit cao, pH v kim nhit ha

c cng b nh Fervidobacterium pennavorans [50], Fervidobacterium

islandicum [49], Meiothermus ruber H328 [72], Clostridium sporogenes [47]

v cc chng Thermoanaerobacter sp. [62] c phn lp t cc mi trng

khc nghit nh sui nc nng, l thng hi nhit, v khu vc ni la. Mt

s vi sinh vt hot ng mi trng kim nh Nesternkonia sp. [73] v

Nocardiopsis sp. TOA-1 [54] cng c kh nng thu phn keratin.

c tnh ha sinh ca keratinase

Cc c tnh ca enzyme c to ra t vi khun l rt a dng, ty thuc

vo loi vi sinh vt sn xut ra keratinase. Cc enzyme ny ch yu l enzyme

ngoi bo c tit ra t vi sinh vt. Mt s c im sinh ha ca keratinase

c trnh by bng 1.1. Phn ln cc keratinase c tit ra t vi khun

c pH l kim hoc trung tnh, pH ti u t 7,5-9,0. Tuy nhin, mt s

keratinase khc c ti u hot ng ngoi phm vi ny, pH hot ng

kim ha [74] hoc pH l axt [75,76]. Mt s keratinase n nh trn mt

khong pH rng. V d nh keratinase ca chng Nocardiopsis TOA-1, c

n nh trong khong pH t 1,5-12, trong 24 h 30C [54].



iu kin hot ng ti u ca keratinase rt thay i, thng ph thuc vo

ni v ngun gc phn lp (bng 1.1). Keratinase t chng chu nhit F.

pennavorans c nhit ti u 80C [50] trong khi keratinase t chng

nhit thng Stenotrophomonas maltophila DHHJ c hot ng ti u

40C [55]. Nam v cng s [49] chng minh c F. islandicum AW-1 c

nhit ti u 100C. Trng lng phn t keratinase c xc nh.

Mc d trng lng phn t cc enzyme t 18 kDa (S. albidoflavus [58]) n

240 kDa (K. rosea [51]) nhng phn ln keratinase c trng lng phn t

thp hn 50 kDa (bng 1.1). Phn ln cc keratinase l cc n enzyme, tuy

nhin phn lp a enzyme cng c ghi nhn [57]. Trng lng phn t

cao thng kt hp vi cc keratinase vi cc c tnh ca metalloprotease

hoc chng c ngun gc t chu nhit (bng 1.1).

nh hng ca cc ion kim loi v cc bt hot hot tnh keratinase v

ang c nghin cu. Gn y cc nghin cu ca Gupta v Ramnani [77]

cho thy cc ion kim loi ha tr 2 nh Ca2+, Mg2+ v Mn2+ c xu hng kch

thch hot tnh enzyme keratinase. nh hng ny c th lin quan ti vic

duy tr hot tnh ca enzyme v to ra s n nh phc hp gia c cht v

enzyme. Ngoi ra cc ion kim loi c th bo v cho enzyme chng li bin

tnh v nhit [53, 58]. Ngc li, s chuyn trng thi v cc kim loi nng

khc nh Cu2+, Ag+, Hg2+ v Pb2+ l nguyn nhn ca s bt hot cc enzyme

keratinase. Cc keratinase thng n nh trong s hin din ca dung mi

hu c nh DMSO, Triton X-100.

1.4. Tnh hnh nghin cu

1.4.1 Tnh hnh nghin cu trong nc

Vit Nam l nc sn xut gia cm ch yu quy m nh tiu th ngay

trn th trng a phng hoc cc tnh ln cn, nhng n nay c nhiu

h gia nh bt u sn xut gia cm quy m cng nghip nh cc tri g



lnh khp kn theo dy truyn sn xut ca Cng Ho Lin Bang c. Nhiu

cng ty cng bt u tp trung vo u t ch bin sn xut gia cm nh cng

ty CP d nh xy dng thm 2 nh my ch bin gia cm mi ngoi H

Ni vi cng sut 50.000 con gia cm/tun v mt nh my s c t ti

ng Nai. Theo tng cc thng k, nm 2003 tng gia cm ca Vit Nam ln

ti 250 triu con v cho ra sn lng tht gia cm t ti 371 ngn tn [27].

Do lng to nn 5% trng lng c th gia cm do lng lng v t gia

cm c th l 18,6 ngn tn nm, y c th l ngun nguyn liu ln lm

phn bn hu c v thc n gia sc cho ngnh nng nghip. Nm 1993, Vn

Th Hnh cng cng s nghin cu to protein ha tan t lng v ph thi

bng phng php kim thy phn [3]. Hin nay, ti Phng Vi sinh vt t,

Vin Cng ngh Sinh hc v ang tip tc phn lp cc chng vi khun c

th thu phn lng v. Sau khi tm ra nhng iu kin ti u cho cc chng c

kh nng thu phn tt s tin hnh nh tn chng bng cc phng php ho

sinh (API 20 NE v API 50 CHB) cng nh nh loi bng phng php sinh

hc phn t. Sau khi phn lp v tuyn chn cc chng tt, nhm nghin cu

ang tm ra nhng ng dng hu ch v thit thc nht i vi vt nui v cy

trng.

1.4.2 Tnh hnh nghin cu trn th gii

Hin nay trn th gii, cc kt qu nghin cu v keratinase cho thy

nhiu nhm vi sinh vt khc nhau c kh nng thy phn lng v, chng hn

cc chng thuc cc nhm Bacillus, Chryseobacterium, Actinomycetes, Vibrio

v nm [18, 19, 20]. S dng vi sinh vt v keratinase nhm nng cao cht

lng sn phm dinh dng t bt lng v v ang c quan tm. Bt

lng v thy phn bng Bacillus licheniformis c b sung amino acid lm

thc n cho g to ra s tng trng ca g tng t nh g s dng ngun

thc n u tng [22]. S dng keratinase th t chng Bacillus

licheniformis lm tng kh nng phn gii ngun lng v th cng nh l bt



lng v. Enzyme thy phn ny s lm tng kh nng tiu ha lng v ti

82% v c th thay th ti 7% protein n cho g [24].

Mi nm trn th gii c 24 t g b cht to ra khong 9 t tn lng gia

cm. Thi gian cn thit mt chic lng thc hin qu trnh phn hy t

nhin ca mnh l 5-7 nm, dn n hu qu nhim mi trng.

Mt s nghin cu chng minh c rng vi sinh vt thy phn keratin

c th tn ti trong lng, mng, guc.... Chnh s xut hin ca vi sinh vt ny

lm tng kh nng x l cht thi, gim thiu gy nhim mi trng. c

bit keratinase ca chng vi sinh vt l thnh phn quan trng trong cng

ngh sinh hc lin quan ti ngun cht thi cng nghip lng v v trong chn

nui [24].

i vi phn bn, keratin sau khi b thy phn thnh cc amino acid, cc

peptide ha tan s cung cp dinh dinh dng cho cy trng. Cc dng phn

hu c ny c th c bn qua l vi mt lng nh nhng c tc dng lm

tng qu trnh pht trin ca cy, gim c lng phn bn ho hc vo t

v c kh nng tng cht lng cho sn phm. Nhiu nghin cu trn th gii

cng ch ra rng phun qua l tng hiu qu hn bn qua gc t 8-10 ln.

1.5. Sn xut v ng dng qu trnh thu phn keratin trong lng v

Keratinase c sn xut v bn trn th trng vi nhiu mc ch

khc nhau. Sinh tng hp keratinase thng c cm ng bi keratin. Do ,

c cht keratin (lng g, bt lng v, tc) thng c b sung vo mi

trng nui cy. Tuy nhin, vic b sung c cht keratin khng phi nht thit

cho sn xut keratinase. Cc cht khng phi l keratin, nh bt u nnh

[78], u nnh [79], sa tch kem [54], bt tm [48], gelatin [52], casein v



phomat lng [80], c chng minh l nhng cht cm ng ca sinh tng

hp keratinase.

B sung vo mi trng keratin vi cc ngun carbon hoc cc ngun

nit khc nhau c th lm tng sinh tng hp keratinase. V d, b sung

glucose, sucrose, tinh bt, mt ng, v thm cc ngun nit, chng hn nh

ur, dung dch peptone, tryptone, men chit xut, amoni clorua v nitrat natri

nng cao sn lng enzyme [39, 47, 51, 81]. Tuy nhin mt s vi sinh vt

nht nh li gim sn xut enzyme do b c ch qu trnh trao i cht

[82,83]. Do , hiu qu ca sinh tng hp keratinase c s bin ng ln,

ph thuc vo chng vi sinh vt, cht cm ng v nng ngun carbon v

nit. V vy, thnh phn mi trng phi c xc nh theo tng trng hp

c th. Bn cnh thnh phn mi trng nui cy, cc iu kin khc nh

nhit , pH, v sc kh cng l nhng yu t quan trng c c sn

lng keratinase cao.

Bi tit keratinase thng t c cao nht trong pha log hoc pha sinh

trng cn bng ca t bo. Tuy nhin, chng Serratia sp. HPC 1383 c hot

tnh cao nht trong giai on sinh trng (24 h u tin) trn mi trng bt

lng v, trong khi nhin liu sinh hc ti a c t c 96 h [84].

tng cng kh nng sinh tng hp keratinase v thy phn keratin cc k

thut sinh hc phn t c s dng trong nghin cu pht trin cc chng

t bin v ti t hp. Bng k thut chn dng v biu hin gen ti t hp,

sinh tng hp keratinase ti t hp trong t bo vi sinh vt khng gy hi vi

thng tin di truyn t vi sinh vt c ngun bnh ti sc khe cng c

nghin cu. Gen kerA, m ha keratinse ca chng B. licheniformis, c

biu hin cng vi lng v (lng v l ngun cacbon v nit) trong mi

trng nui cy dn n s biu hin mnh ca protease thy phn keratin.

Ngoi ra, tng nng sut keratinase c nghin cu bng cch tch hp



nhim sc th vi nhiu bn sao ca gen kerA B. licheniformis v B. subtilis

[85].

Phn ln cc bo co nghin cu sn xut keratinase u dch lng, gn

y sn xut cc enzyme thy phn keratin qua quy trnh s dng mi trng

rn cng c nghin cu [86]. Parkash v cng s [87] nghin cu sn

xut keratinase bng cc vi sinh vt ln men tnh.

Keratinase vi sinh vt c coi l cht xc tc sinh hc y ha hn cho

cc mc ch khc nhau, bao gm cc ng dng trong thc n chn nui, phn

bn, cht ty ra, thuc da, v cc ngnh cng nghip dt, ngoi ra cn ng

dng trong dc phm v ng dng y sinh hc [77, 88]. Mc d vy, so vi

cc ngnh cng nghip sn xut enzyme khc th keratinase vn cn trong giai

on non tr. Bng 1.2 a ra cc ng dng hin ti v tim nng ca

keratinase.

Bng1.2: ng dng ca keratinse

L n h v c C c n g d n g

C h n n u i t r n g t r t

- S n x u t t h c n c h n n u i - B s u n g t h c n - S n x u t p h n b n

D c v y s i n h - T h y p h n t h - T n g k h n n g d n t r u y n t h u c - i u t r d a l i u - M p h m

C n g n g h i p

- T h u c d a - B i n i s i ( n g n h d t ) - C h t t y r a - B i n i c h c n n g c a p r o t e i n - M n g s i n h h c ( f i l m , l p p h n g o i , k e o )

- S l n c t h i v q u n l c h t t h i

N n g l n g s i n h h c

- C h u y n h a c h t t h i k e r a t i n c h o c c s n x u t t i p t h e o c a m e t h a n e h o c h y d r o s i n h h c



Cc keratinase c gi tr phn hy protein keratin kh tan. Chnh v vy, s

hiu bit v vi sinh vt sn xut keratinase v cc c tnh ha sinh ca

keratinase s gp phn trong vic qun l ngun cht thi keratin trong nng

nghip mt cch hp l v hiu qu. Ngoi ra, s dng cc kerainase lm tng

ng dn thuc, qu trnh hydroxyl ha, sn xut cc film thy phn bng

sinh hc, v sn xut cc nguyn liu sinh hc, v ang lm tng ln cc

ng dng ca keratinase, c th nh sau:

1.5.1. Tng hm lng cht dinh dng trong thc n gia sc

Khi b sung cc vi khun c phn lp v tuyn chn vo cc loi

thc n th lm cho thc n ca gia sc bo qun c lu hn, khi gia sc n

s d tiu ho hn [3, 24].Trong cng nghip, ph liu lng c bin i

thnh lng b thu phn s dng nhit v p sut cao. Tuy nhin s dng

bt lng thu phn thay th protein c mt s gii hn. Khi thm 6% bt

lng thu phn vo ba n ca g con lm gim trng lng c th ca

chng. Tuy nhin, s dng thu phn bt lng bng vi khun c th to ra sn

phm thay th n 15% protein trong thc n gia cm [26].

Th nghim trn g, ngi ta nhn thy rng feather lysate, c cn bng vi

mt s axit amin, c th thay th u nnh nh l mt ngun protein ti 70%

trong khu phn. Trong cc nghin cu khc, bt lng thng mi c qua

m vi Keratinase to ra mt phn bt lng thu phn. Kh nng tiu ho

amino acid ca bt lng thy phn vi Keratinase g trng thnh l 82%

cao hn so vi bt lng thy phn thng mi, 60%.

Vy c th kt lun rng keratinase c kh nng thu phn v bin i bt

lng v thnh ngun protein c th tiu ho mc ti a l 70% protein khu

phn trong thc n.

1.5.2. To phn bn

Vi tc dng ca cc chng vi khun ny chng c th s dng lm cc

loi phn bn vi sinh nh: phn vi sinh vt c nh m, phn vi sinh vt phn



gii ln, phn vi sinh vt kch thch iu ho sinh trng thc vt, phn vi sinh

vt phn gii xenllulo [2].

Dch mi trng sau khi thu phn lng v c ly tm, tinh ch tch

chit em li tc dng kch thch sinh trng gip cy pht trin tt v chng

chu tt vi cc loi bnh hi cy trng.

1.5.3. To enzyme trong cc ngnh cng nghip

Chng vi khun Bacillus Licheniformis ngoi tc dng thu phn tt hp

cht keratin trong lng v cn c tc dng quan trng trong ngnh sn xut

hai enzyme proteases v amylases [9, 10]. Cc enzyme phn gii protein l

cc enzyme quan trng nht trong cng nghip v cc ng dng rng ri ca

chng trong cng nghip ty ra, sa, dc, thuc da v cng nghip thc

phm. Keratinase trong mi trng lng kch thch tit ra t nht hai protease

kim l subtilisin Carlsberg v subtilisin BPN c kh nng chu nhit (35-

37oC) v n nh trong thi gian di. Hai protease ny thuc nhm subtilisin.

Subtilisin Carlsberg l protease vi sinh vt cng nghip quan trng nht, c

s dng nhiu trong sn xut cc cht ty ra. Subtilisin Carlsberg c tnh c

hiu rng, thy phn nhiu loi lin kt peptide cng nh cc lin kt ester,

nht l nhng lin kt c to thnh t cc amino acid thm. Di tc dng

ca enzyme ny, mt s triglyceride cng c th b thy phn nh tripropionin

v tributyrin. Subtilisin Carlsberg thy phn t 30 n 40% lin kt peptide

ca casein. Subtilisin BPN l mt dy protease ngoi bo v l mt lp

enzyme i din ln nht ca dy protease c cu trc c trng l // v

trung tm hot ng gm Ser 221, His 64, Asp 32. Cu trc khng gian ba

chiu ca chng bn vng trong khong pH rng v ngay c khi c mt ca

dung mi hu c. Hai protease ny u phn hu keratin trong lng thnh cc

peptide ngn v axit amin, nhng cht ny c vi sinh vt s dng lm

ngun cacbon v nit.

1.5.4. Ci to t



Nh chng ta bit h vi sinh vt trong t c tc dng ci to t rt

ln. Ni no cng cha nhiu vi sinh vt th ti t cng ti xp v giu

cht dinh dng. Trong thnh phn ca h vi sinh vt c rt nhiu chng vi

khun m chng ta phn lp v tuyn chn c. Khi bit chnh xc tn

ca tng chng vi khun ta c th b sung vo tng loi t cy trng thch

hp [1]. Nhng chng vi khun c b sung s ci to t, lm tng kt

dnh ca t v lm cho cy trng c pht trin nhanh hn v c nhiu cht

dinh dng cn thit. Ngoi ra nhng vi khun ny cn c th to ra tnh i

khng vi cc vi sinh vt gy bnh cho cy trng.

1.5.5. i vi mi trng sinh thi

Hin nay, vi s pht trin ca nghnh ch bin gia cm, ko theo mt

lng ln rc thi lng v t cc khu git m v trang tri chn nui. Nu

khng c x l th lng rc thi ny s gy ra nhng mi hi thi t kh

H2S lm nh hng n cuc sng ca con ngi v mi trng sng. Chng

ta c th b sung cc chng vi khun c phn lp tuyn chn vo cc

ng rc cc chng vi khun ny phn hu cc hp cht kh tiu hu nh

lng v. y l ngha rt quan trng gip mi trng c trong sch v

gim thiu s nhim mt cch cn thit.

1.5.6. To hp cht mu

Mt s vi sinh vt nh to, nm v vi khun to ra cc cht mu

carotenoid. Trong cng nghip, chng c s dng lm cht mu thc phm.

Cantaxanthin, mt xantophyll carotenoid c s dng nh thc n b sung

cng vi astaxanthin trong cng nghip gia cm nhm ci thin mu ca g,

lng trng g. Cantaxanthin chnh l cht mu c cc chng

Keratinase.rosea tng hp.

1.5.7. ng dng trong cng nghip dc v sn xut thc phm chc

nng



Cc peptide c hot tnh sinh hc t keratin c cha trong cc vt liu nh

len, da, tc v lng l mt trong nhng thnh phn ca thc phm chc nng

c gi r v phong ph t cc ngun t nhin. Cc chui axit amin giu prolin

c hot tnh sinh hc cdng phng chng cc bnh cho con ngi bao

gm: ngn chn s hnh thnh khi u, kim sot tng huyt p v bnh

Alzheimer [24].

PHN II: NGUYN LIU V PHNG PHP 2.1. Nguyn liu

2.1.1. Nguyn liu

Cc mu t v lng v c ly t cc vng t ni git m gia cm

hoc khu cng nghip chn nui tnh Tnh Thi Nguyn. Chng vi khun

Escherichia coli, vector biu hin pET 32a. Chng Bacillus sp. L.N 3.4;

Bacillus sp. .N 1.2 c s dng thuc b chng ging ca Phng Vi sinh

vt t, Vin Cng ngh Sinh hc.

2.1.2. Ho cht

Cc ho cht thng dng c s dng l sn phm ca hng New

England Biolabs, Ferkin Elmer Cetus, Merk/BHD Chemicals (c), Sigma

(M), Peking (Trung Quc), Reachim (Nga), Kanto (Nht Bn)....

2.1.3. Thit b

Cc thit b c s dng t ti Phng Vi sinh vt t v Phng Th

nghim Trng im, Vin Cng ngh Sinh hc Vin Khoa hc v Cng

ngh Vit Nam.

2.1.4. Mi trng nui cy

2.1.4.1. Mi trng Broth Peptone [16]

NaCl 5 g Peptone 10 g



Na2HPO4 3,5 g H2O 1000 ml

KH2PO4 1,5 g pH 7,2 0,2

2.1.4.2. Mi trng I

Mi trng phn lp chng Bacillus licheniformic [12]

NaCl 0,5 g Nm men 0,1 g

NH4Cl 0,5 g Bt lng v 10 g

K2HPO4 0,3 g Thch 20 g

KH2PO4 0,4 g H2O 1000 ml

MgCl2.6H2O 0,1 g pH 8,0

2.1.4.3. Mi trng II

Mi trng phn lp chng Vibrio [17]

2.1.4.4. Mi trng III

Mi trng phn lp chng Chryseobacterium [16]

NaCl 0,5 g Bt lng v 10 g


KH2PO4 0,4 g H2O 1000 ml

pH 7,0

2.1.4.5. Mi trng m Phosphate buffered Saline [17]

NaCl 8,00 g KH2PO4 0,27 g

NaCl 0,5 g Bt lng v 15 g


KH2PO4 0,4 g H2O 1000 ml

pH 7,0



KCl 0,20 g H2O 1000 ml

Na2HPO4 1,44 g pH 7,4

2.1.4.6. Mi trng nui lc lng v [12, 16, 17]

NaCl 0,5 g Lng v 20 g

K2HPO4 0,3 g H2O 1000 ml

KH2PO4 0,4 g pH 7 7,2

2.1.4.7. Mi trng MPA [23]

NaCl 5 g Thch 20 g

Pepton 10 g H2O 1000 ml

Cao Tht 3 g pH 7,2- 7,3

2.2. Phng php nghin cu

2.2.1. Phn lp chng vi khun c kh nng sinh keratinase cao

2.2.1.1. Ly mu v o pH t

Ly mu

Loi b lp dt dy 2-3 cm trn b mt. Sau dng dao hay xng ly

nhng tng t theo su ca lp t nghin cu. Cc mu t v lng ly

c cho vo ti li lng tin hnh phn tch ngay hoc c bo qun 40C.

o pH t

Cn 18,64 gram KCl v 20 gram t cho vo bnh ng 250 ml H2O ct.

Cn 20 gram mu t cho vo 2 bnh tam gic 100ml:

+ Bnh 1 cho 50ml nc ct (o pH b mt)

+ Bnh 2 cho 50ml dung dch KCl (o pH trong t)

Lc cho tan mu vi vn tc 200 vng/ pht trong 10 pht. Sau tnh 24h

ri tin hnh o gi tr pH ca t.

2.2.1.2. Phn lp chng vi khun Chryseobacterium

T cc mu lng thu c, cn 10 gram lng trong mi trng broth

peptone 24h, 30oC. Sau ly 1ml dch nui cho vo 90 ml nc v trng



( pha long 10-1). Dng pipet v trng ht 1 ml mu cho vo ng nghim c

cha sn 9 ml mi trng nc mui sinh l v trng ( pha long l 10-2),

thay pipet tip tc ht 1 ml dung dch va pha long sang ng tip theo. Tip

tc lm nh vy ta s c cc dung dch pha long 10-3, 10-4, 10-5.., ht 0,05

ml dch pha long tri ra a thch cha mi trng III. Mi nng pha

long c lp li 3 ln v c nui 30oC. Sau 24, 48 gi s lng khun

lc c m bng mt thng v tin hnh chn khun lc theo hnh thi

ring.

2.2.1.3. Phn lp chng vi khun Vibrio

T cc mu lng thu c, cn 10 gram lng a vo mi trng m

Phosphate buffered Saline (PBS) lc 20 - 30 pht, 200v/p. Sau ly

1ml dch nui cho vo 90 ml nc v trng ( pha long 10-1). Dng pipet v

trng ht 1 ml mu cho vo ng nghim c cha sn 9 ml mi trng nc

mui sinh l v trng ( pha long l 10-2), thay pipet tip tc ht 1 ml dung

dch va pha long sang ng tip theo. Tip tc lm nh vy ta s c cc

dung dch pha long 10-3, 10-4, 10-5.., ht 0,05 ml dch pha long tri ra a

thch cha mi trng II. Mi nng pha long c lp li 3 ln v c

nui 37oC. Sau 24, 48 gi s lng khun lc c m bng mt thng v

tin hnh chn khun lc theo hnh thi ring.

2.2.1.4. Phn lp chng vi khun Bacillus

T cc mu t thu c ta nghin nh, cn 10 gram cho vo 90 ml nc

v trng ( pha long 10-1) lc 30, 200v/p. Sau dch mu c x l

sc nhit 80oC trong thi gian 10 pht. Dng pipet v trng ht 1 ml mu

c 80oC trong 10 pht cho vo ng nghim c cha sn 9 ml mi

trng nc mui sinh l v trng ( pha long l 10-2), thay pipet tip tc

ht 1ml dung dch va pha long sang ng tip theo. Tip tc lm nh vy ta

s c cc dung dch pha long 10-3, 10-4, 10-5...(pha long 9-10 ln), ht



0,05 ml dch pha long tri ra a thch cha mi trng I. Mi nng pha

long c lp li 3 ln v c nui 37oC. Sau 24, 48 gi s lng khun

lc c m bng mt thng v tin hnh chn khun lc theo hnh thi

ring.

2.2.2. Xc nh kh nng thu phn lng v

2.2.2.1. Nui ging cp 1 vo mi trng MPA

Cch tin hnh:

- Pha mi trng MPA ( nu phn 2.1.4.7).

- Dng que cy ging, h phn u ca que cy ging trn trn ngn la n

cn cho chy rc. Sau a qua a li phn thn ca que cy trn trn

ngn la n cn v trng tuyt i Lm ngui que cy Ly 1 t chng

trong ng nghim a vo cc bnh mi trng, ng thi gi mnh que cy

cho tt c chng ging c cy vo mi trng Ghi tn chng v ngy

cy ngoi v bnh em i nui lc qua m, nhit 30oC vi vn tc

200 vng/pht.

(Lu : Khi ly chng phi tht kho khng c chm vo thnh ng

nghim v khi m np bnh phi h ming bnh mi trng v nt bng qua

ngn la n cn trnh nhim khun).

Cy ging cp 1 vo bnh mi trng nui lc c b sung lng v

Cch tin hnh:

- Pha mi trng nui lc lng v ( nu phn 2.1.4.6).

- Dng pipet thu tinh ly 0,5 ml ging cp 1 cho vo bnh mi trng (mi

bnh 1% ging). Mi chng ging cy vo 1 bnh. Sau em i nui lc

cng vi bnh i chng 30oC - 35oC, vi tc 200 vng/pht, trong

khong thi gian l 3-5 ngy.

- Pha long v cy tri ging cp 1, theo di cc chng ging qua cc ngy

nui cy.

Nhn xt v o pH ca dch nui tng chng sau khi nui cy



- o pH, nhn xt ca tng chng nui cy.

- Xc nh trng lng lng cn li sau thi gian nui lc (Lm kh lng v

cn khi lng cn li sau nui cy lc) xc nh t l % v kh nng thu

phn lng v ca cc chng vi khun theo cng thc:

A (%) = %100

B

CB

m

mm

A (%): T l phn trm v s thu phn ca cc chng

mB: Trng lng lng v ban u.

mC: Trng lng lng v cn li sau thi gian nui cy lc.

Chn lc cc chng vi khun c kh nng thy phn keratin mnh nht

(sinh keratinase cao).

2.2.2.2. Xc nh mt t bo

Mt t bo vi khun sau khi pha long v cy tri trn mi trng c

tnh bng cng thc:

Y = a x 20 x 10n

Y: S khun lc thu c trn 1 ml dch.

a: S khun lc thu c trn a thch.

20: (1000 l/50 l)

10n: pha long mu.

2.2.3. La chn mi trng thch hp cho cc chng vi khun c kh

nng sinh keratinase cao

Cc chng vi khun c tuyn chn l nhng vi khun c kh nng

thu phn lng v tt nht. Sau em i th nghim trn cc mi trng

khc nhau vi mc ch l tm ra mi trng thch hp v c gi tr kinh t.

Chun b mi trng cy chng bao gm: Mi trng giu dinh dng

(mi trng MPA), mi trng dinh dng bnh thng (mi trng khong)

v mi trng ngho dinh dng (mi trng nc).

- Nui ging cp 1vo mi trng MPA



Cch tin hnh: cc bnh mi trng lm nh phn phng php nghin cu

(2.2.2.1).

- Cy ging cp 1 vo cc bnh th mi trng nui lc c b sung lng v

Cch tin hnh: lm nh phn phng php nghin cu (2.2.2.1).

- Sau :

+ Xc nh mt ca chng vi khun.

+ o pH dch nui

+ Nhn xt s thu phn ca tng chng trn mi mi trng sau nui

cy Cn khi lng lng cn li sau 4 ngy nui cy.

2.2.4. nh hng ca nhit v pH ln kh nng sinh trng ca vi

khun thy phn lng v

Cc chng vi khun chn lc l c kh nng thu phn lng v tt nht,

c th nghim trong cng mi trng lng v trn cc khung nhit khc

nhau vi mc ch l tm ra nhit thch hp. Khi tm c nhit thch

hp vi tng chng th gip cc chng c kh nng thu phn lng v tt

nht.

Cc chng c th trn cc khung nhit l: 25oC, 30oC, 35oC v 40oC.

Chun b ging cp 1, cy ging cp 1 vo cc bnh mi trng lm nh phn

phng php nghin cu (2.2.2.1). Sau , pha long m mt ging

cp 1 sau 3 ngy nui lc. o pH, nhn xt s thu phn ca tng chng trn

mi nhit sau tng ngy nui cy. Cn khi lng lng cn li ca tng

chng trn cc nhit sau 3 ngy nui cy.

2.2.5. Xc nh c im hnh thi ca cc chng vi khun

Cc chng vi khun c ria sch trc 24 h, a ln lam knh nh sn

1 git mui sinh l. nh tan sinh khi ri y lam men ln. Sau , cc

chng vi khun c em ln knh hin vi nhn bit v hnh dng, kch

thc t bo.



2.2.6. Xc nh gram m v gram dng ca vi khun

2.2.6.1. Phng php Hucker ci tin

Vt liu v ho cht:

Dung dch tm kt tinh (crystal violet)

- Cn 2g tm kt tinh ho tan trong 20 ml etanol 95% v 0,8 g amon oxalat ho

tan trong 80 ml nc ct. Sau trn hai dch li vi nhau, gi 48 gi ri lc.

Bo qun trong l ti, s dng vi thng.

Dung dch Iod:

- Ho tan 1 g Iod (Iodine) trong 3-5 ml nc ct, thm 2 g KI (kali iodie)

khuy cho tan ht, cho 300 ml nc ct. Bo qun trong l ti.

Dung dch ty mu:

Etanol 95% hoc trn 70 ml etanol 95% vi 30 ml aceton.

Dung dch nhum b sung:

- Chun b sn dung dch safannin 2,5% trc khi dng pha vi nc ct theo

t l 1: 5, c c dung dch 0,5%.

Cc bc tin hnh:

- Chun b vt bi: Dng que cy v trng ly mt t vi khun t thch (sau

khi cy 24 gi) ho vo git nc ct gia phin knh, lm kh trong khng

kh.

- C nh t bo:

+ H nhanh vt bi qua ngn la n cn 2-3 ln.

+ Nhum bng dung dch tm kt tinh trong mt pht, ra nc v thm

kh.

+ Nhum li bng dung dch Iod trong mt pht, ra nc v thm kh.

+ Nh dch ty mu, gi khong 30 giy (cho n khi va thy mt

mu) ra nc v thm kh.

+ Nhum b sung bng dung dch Safrannin trong 2-3 pht, ra nc,

kh trong khng kh.



+ Soi knh: Dng vt knh 100X

Kt qu:

+ Vi khun gram dng bt mu tm.

+ Vi khun gram m bt mu .

2.2.6.2. Xc nh bng cch kim tra kt dnh

Ly mt vng que cy sinh khi t bo c ria sch sau 24 gi cho ln

lam knh c 1 git KOH 3%. nh tan sinh khi, trong khi lm tan sinh

khi th nhc que cy kim tra kt dnh. Kt qu:

+ Vi cc chng m kt dnh nht th kt lun chng vi khun l

gram m (G-).

+ Vi chng m kt dnh khng nht th kt lun chng vi khun l

gram dng (G+).

2.2.7. Phng php phn loi vi khun theo c tnh sinh ho

Chng phn lp c chn lc v tin hnh phn loi bng kit chun sinh

ho API 50 CHB v 20 NE, qu trnh phn loi c tin hnh theo ch dn

ca nh cung cp.

Phng php th API 50 CHB dng xc nh nhm vi khun thuc

Gram dng (G+)

y l phng php dng 49 ngun carbonhydrat ln men (API 50

CHB) v nhn bit vi khun Bacillus trong c cc phn ng vi cc cht

bit t trc, th cc phn ng v tra bng i chiu vi kho phn loi tm

ra tn loi ca chng. Ch dng mt khun lc sch th phn ng. 49 php

th sinh ho API 50 CHB c trnh by bng 2.1.

Bng 2.1: 49 php th ln men API 50 CHB

STT Php th C cht STT Php th

C cht

0 CTL Control 25 ESC Esculine

1 GFL Glycerol 26 SAL Salicine

2 ERY Erythritol 27 CEL Cellobiose



3 DARA D-Arabinose 28 MAL Maltose

4 LARA L-Arabinose 29 LAC Lactose

5 RIB Ribose 30 MEL Melibiose

6 DXYL D-Xylose 31 SAC Saccharose

7 LXYL L-Xylose 32 TRE Trehalose

8 ADO Adonitol 33 INU Inuline

9 MXL Methyl-xyloside 34 MLZ Melezitose

10 GAL Galactose 35 DRAF D-Rafinose

11 DGLU D-Glucose 36 AMI Amidon

12 DFRU D-Fructose 37 GLY Glycogene

13 DMAN D-Mannose 38 XLT Xylitol

14 LSBE L-Sorbose 39 BGEN -Gentiobiose

15 RHA Rhamnose 40 DTUR D-Turanose

16 DUL Dulcitol 41 DLYX D-Lyxose

17 INO Inositol 42 DTAG D-Tagatose

18 MAN Mannitol 43 DFUC D-Fucose

19 SOR Sorbitol 44 LFUC L-Fucose

20 MDM Methyl-D-mannoside

45 DARA D-Arabitol

21 MMG Methyl-D-glucoside

46 LARA L-Arabitol

22 NAG N-Acetyl

glucosamine

47 GNT Gluconate

23 AMY Amygdaline 48 2CG 2 ceto-gluconate

24 ARB Arbutine 49 5CG 5 ceto-gluconate

Cch tin hnh:

- Lm m phin th bng nc ct v trng.

- Ho tan 1 khun lc vo 1 ml dung dch mui sinh l sao cho c (OD)

t khong 0,3.

-Ly 0,3 ml dch trn cho vo 5 ml dung dch mui sinh l v ly 0,6 ml cho

vo mi trng 50 CHB.

- Dng pipet cy huyn ph vi khun t mi trng 50 CHB/E vo 50

ging ca b kit 50 CHB.



- Trong qu trnh tin hnh khng cc bt kh hnh thnh trong cc ging

th.

- Ph parafin ln cc ging ca b kit 50 CHB.

- t kit th vo t m 30oC, c kt qu sau 24 gi v 48 gi.

- Tra bng i chiu vi kho phn loi tm ra tn loi ca chng.

Phng php th API 20NE dng cho cc trc khun gram m (G-), khng

c nhu cu dinh dng c bit

y l phng php dng 20 php th ha sinh nhn bit vi khun

gram m trong c cc phn ng vi cc cht bit t trc, th cc phn

ng v tra bng i chiu vi kha phn loi(bng ch s Analytical Profile

Index, 5th

edision, BioMerieux S.A, 1992) tm ra tn loi ca chng. Ch

dng 1 khun lc sch phn ng. 20 php th ha sinh c trnh by

bng 2.2.

Bng 2.2: 20 php th sinh ha API 20 NE

STT Php th C cht Phn ng/Enzyme

1 NO3 KNO3

Kh nitrat thnh nitrit/ khi thm Zn th

kh nitrat thnh nito

2 TRP Tryptophan To indol

3 GLU Glucose Ln men Glucose

4 ADH Arginin Tng hp argnin

dihidrolaza

5 URE Ure Tng hp ureaza

6 ESC Esculin Tng hp esculin

7 GEL Gelatin Thy phn gelatin

8 PNPG p-nitrophenyl -D-galactosepyranosit

Tng hp - galactosidaza

9 GLU Glucose ng ha

10 AGA Arabinose ng ha

11 MNE Mannose ng ha

12 MAN Mannitol ng ha

13 NAG N- axetyl- glucosamin ng ha

14 MAN Malat ng ha

15 GNT Gluconat ng ha



16 CAP Caprat ng ha

17 ADI Adipat ng ha

18 MNT Maltose ng ha

19 CIT Citrat ng ha

20 PAC Phenyl-axetat ng ha

Cch tin hnh:

- Lm m phin th bng nc ct v trng.

- Ho tan 1 khun lc vo 2,5 ml dung dch mui sinh l sao cho c (OD)

t khong 0,3.

- Dng pipet cy huyn ph vi khun vo 8 ging u tin (t NO3 n

PNPD).

- Ly 0,2 ml dch t bo trn cho vo mi trng AUX trn u. Dng dch

ny nh y vo cc ging cn li.

- Trong qu trnh tin hnh khng c cc bt kh hnh thnh trong cc ging

th.

- Ph parafin ln cc ging 3, 4, 5.

- t kit th vo t m 30oC, c kt qu sau 24 gi v 48 gi.

- Tra bng i chiu vi kho phn loi tm ra tn loi ca chng.

2.2.8. Chn dng gene keratinase vo vector biu hin pET32a

2.2.8.1. Tch chit DNA tng s ca chng vi khun

DNA tng s c tch chit theo phng php Masterson v cng s

[32], bao gm cc bc nh sau:

- Bc 1: ly 5 ml mi trng LB lng cho vo ng nghim, sau ly t bo

VK cho vo ng nghim nui lc qua m nhit 30 - 370C ti gn pha

Log

- Bc 2: ht khong 2 ml dch VK trn vo ng eppendort

- Bc 3: Ly tm 12000 v/p, 10 pht thu cn

- Bc 4: Ha tan cn trong 567 l TE (10:1), votex

- Bc 5: B sung thm 40 l SDS 10%, mix tan cn



- Bc 6: B sung thm 3lproteaza K, 370C/1gi

- Bc 7: B sung thm 280 l NaCl 6 M, trn u, 650C/10 pht

- Bc 8: Chit bng Chloroform:izoamyl (24:1), votex ly tm 12000 v/p,

10thu dch ni

- Bc 9: Ta DNA bng Ethanol 100% -200C qua m (t nht 4 gi)


- Bc 11: Ra cn bng Ethanol 70%


- Bc 13: Sy kh b sung 50 l nc kh ion v trng ly 4 l em

in di kim tra trn gel agarose 0,8%.

in di trn gel agarose

kim tra kt qu tch DNA, sn phm tch chit c tin hnh in di

trn gel agarose. Ha tan 0,8% agarose trong 100 ml dung dch m TAE 1X,

un si cho gel agarose tan hon ton. nhit h xung khong 500C - 600C,

dung dch agarose vo khun gel in di ci sn rng lc thch hp.

Sau 30-60 pht, khi gel ng cng t gel vo b in di. m TAE 1X

ngp cch mt gel t 1-2 mm, nh nhng g b rng lc ra. Mu DNA c

trn vi 3 l m mu (20% glyxerol; 0,1M Tris-HCL pH 8,0; 0,01 M EDTA

pH 8,0; 0,25% bromphenol blue) tra mu vo cc ging nh trn gel, chy

in di cng vi DNA thang chun (DNA thang chun 1 kb) xc nh kch

thc phn t. Tin hnh in di vi dng in 1 chiu c hiu in th 100V,

cng dng in 60-80 mA, trong 30 pht.

Nhum DNA bng dung dch Ethidium Bromide (EtBr)

Bn gel c ly nh nhng khi khun v ngm vo dung dch EtBr nng

0,5 g/ml trong thi gian t 10 15 pht c lc nh trn my lc ri ra

bn gel bng nc ct. Quan st bn gel di nh sng t ngoi (UV) vi

bc sng 254 nm trn my soi DNA, chp nh trn my GEL-DOC.



2.2.8.2. Nhn bn on DNA bng PCR

K thut PCR do Mullis v cng s pht minh ra nm 1985. K thut ny

cho php nhn 1 on DNA mong mun t h gene ca c th sinh vt thnh

nhiu bn sao.

Nguyn tc phn ng PCR l s dng enzyme ADN polymerase chu nhit

tng hp trong ng nghim cc on DNA mi t mch khun trong mi

trng c dNTP v cha cc cp mi c hiu.

Nhn on gene m ha 16S rRNA

on gene m ha 16S rRNA c nhn ln t ADN tng s nh phn ng

PCR s dng cp mi c hiu: 16SF 5-CGG AAT TCA TTG CTG GAC

CTG- 3; 16SR 5-GTC CTA GAG CTT TGT CTT TAG G-3 ca gene 16S

rARN t vi khun, phn ng PCR cho sn phm l on gene di khong1,5

kb.

Phn ng nhn bn on gene 16S rARN c tin hnh vi tng th tch 25

l/mu, bao gm cc thnh phn sau y:

Thnh phn phn ng PCR Th tch (l)

Nc 14.7

Buffer 5

dNTP(10pmol) 2

Mi xui(10pmol) 1

Mi ngc(10pmol) 1

Khun DNA 1

Enzyme 0.3

Tng 25



Chu trnh nhit

Sau khi phn ng kt thc, ly 8 l sn phm PCR in di kim tra trn gel

agarose 0,8%. Sn phm PCR c tinh sch c trnh t gene Phn

tch s liu nh tn chng vi khun.

Nhn on gene keratinase

- on gene keratinase c nhn ln t ADN tng s nh phn ng PCR s

dng cp mi:

- Primer-F: 5-CGT GGG ATC CAA AAA ATT GTG GAT CAG C- 3

- Primer-R: 5- TTA TTC TCG AGC TGC TTG TAC GTT GAT - 3

- Thnh phn phn ng PCR gm:

Thnh phn phn ng PCR Th tch (l)

Nc 39,7

Buffer 5

dNTP(10pmol) 2

Mi xui(10pmol) 1

Mi ngc(10pmol) 1

Khun DNA 1

Enzyme 0,3

Tng 50

Chu trnh nhit ging phn 2.2.8.2. Sau khi phn ng kt thc, ly 5l sn

phm PCR in di kim tra trn gel agarose 0,8%.



Thi gel tinh sch gene

Phng php thi gel tinh sch c s dng theo phng php ca

William v ng tc gi (1990) v GenJETTM Genomic DNA purification Kit

- Bc 1: in di sn phm PCR

- Bc 2: Ct gel cho vo ng eppendort, xc nh trng lng ming gel:

+ Cn ng eppendort mi

+ Cn ng eppendort cha ming gel xc nh trng lng ming gel

- Bc 3: B sung 400 l binding buffer 100 mg gel (0,8 %)

- Bc 4: cho tan gel hon ton nhit 50-600C, 10 pht

- Bc 5: Cho gel ln ct ly tm 12000 v/p trong 2 pht phn dch

- Bc 6: B sung 500l Wash solusion (ethanol) ly tm 12000 v/p trong 2

pht phn dch

- Bc 7: B sung 30 50 l elution buffer sau nhit 5-100C trong

nhit phng ly tm

- Bc 8: Ly 5 l in di sn phm kim tra tinh sch ca gene

Gn sn phm PCR thi gel tinh sch vo vector pET32a

Phng php tch dng gene c s dng theo Sambrook v cs (2001)

Phn ng ct ni

Mc ch ca phng php ny l a mt on DNA ngoi lai vo mt

vector thch hp nh enzyme T4 DNA ligase. Enzyme ny xc tc cho qu trnh

hnh thnh lin kt ni hai on DNA.

Cch tin hnh:

- Dng 2 enzyme ct Xho I; BamH I, ct m vng vector v ct on gene

keratinase. Thnh phn phn ng gm:

Thnh phn Th tch( l)

Nc 1

Buffer 2



Xho I 1

BamH I 1

DNA 15

Tng 20

- Tinh sch on gene keratinase v vector sau khi x l bng enzyme ct

- Lai on gene keratine trn vo vector pET32a. Thnh phn phn ng ni:

Thnh phn Th tch (l)

H2O 0,5

Buffer 2

Vector 5

DNA 12

Enzyme ligase 0,5

Tng 20

- Hn hp phn ng trn c qua m 160C

Bin np vo vi khun E.coli DH10B

Chun b t bo kh bin

- Bc 1: Cy chng E.coli DH10B ln mi trng LB c, nui qua m

370C

- Bc 2: Ly 1 khun lc n cy vo 3 ml mi trng LB lng, lc qua m

370C

- Bc 3: Cy chuyn 0,8 ml dch nui sang mi trng LB lng, nui lc tip

khong 3 gi o OD t t 0,6 0,8

- Bc 4: Chuyn dch trn sang ng ly tm gi trn , 30 gi lnh

trn 15 pht



- Bc 5: Ly tm vi tc 4200 v/p, 10 pht, 40C loi b dch ly tm, gi

li cn

- Bc 6: Ha tan cn nh nhng trong 20-30 ml 0,1M CaCl2, gi trn 40

pht

- Bc 7: Ly tm vi tc 4200 v/p, 10 pht, 40C thu cn ha tan cn

nh nhng trong 0,7-1,5 ml 0,1M CaCl2

- Bc 8: B sung 800 l 0,1M CaCl2 v 400 l glyxerol chia u vo cc

ng eppendort (50-100 l) gi -800C

- Bc 9: Bin np

Quy trnh bin np

- Ly t bo kh bin DH10B gi -200C ra trn 30 pht

- B sung 5 l DNA plasmid vo ng t bo kh bin mix nh, trn

15-20 pht

- Sc nhit 420C, trong 1 pht trn 5 pht

- B sung 0.5 ml mi trng LB lng, nui lc 370C, trong 1 gi

- Cy tri 50 l trn a pettri cha LB c c b sung thm khng sinh

ampicillin (50 l/ml), nui 370C qua m theo di khun lc mc la

chon khun lc trng xc nh kh nng cha plasmid ti t hp.

Tch chit DNA plasmid ti t hp

- Bc 1: Nui chng bin np E.coli DH10B trong mi trng LB lng c

cha khng sinh (nui lc 200 v/p 370C qua m)

- Bc 2: Chuyn dch nui vo ng eppendort Ly tm 6000 v/p, 6 pht,

40C thu t bo

- Bc 3: Loi b dch, gi phn ta y ng eppendort

- Bc 4: B sung 150 l dung dch SOL I, Votex tan t bo

- Bc 5: B sung 150 l dung dch SOL II, o u trn

- Bc 6: B sung 150 l dung dch SOL III, o nh trn



- Bc 7: Ly tm 12000 v/p, 40C, 15 pht ht dch trong sang ng

eppendort

- Bc 8: B sung Chloroform : Isoamylalchol (24:1)lc mnh vi ln

Ly tm 12000 v/p, 40C, 15 pht to phn lp ri ht ly phn dch ni sang

ng eppendort mi

- Bc 9: B sung 2 th tch Ethanol 100% , o u ta DNA plasmid -

200C trong 3 gi Ly tm 12000 v/p, 40C, 15 pht, loi b dch ni thu cn

DNA

- Bc 10: B sung 1ml Ethanol 70% Ly tm 12000 v/p, 40C, 15 pht, loi

b dch ni thu cn DNA

- Bc 11: Lm kh ha cn DNA trong TE cha RNAase (nng 20

l/ml), 370C trong 1gi loi RNA

- Bc 12: in di trn gel agarose 0,8% kim traTinh sch DNA

plasmid.

Ct kim tra sn phm tch DNA plasmid sau bin np

xc nh plasmid la chn c mang on DNA cn gn hay khng,

ta s dng 2 enzyme ct thch hp c c im ct trn on DNA chn vo.

Phn ng c tin hnh vi thnh phn sau:

Thnh phn Th tch (l)

H2O 9

Buffer 5

Xho I 0,5

BamH I 0,5

plasmid

DNA

10



Tng 25

Sau hn hp phn ng c trn u, 370C trong 3 h hoc qua m

in di kim tra trn gel agarose 0,8%.

2.2.9. Phn tch hot tnh keratinase ngoi bo

Th hot tnh keratinase bng azokeratin

- Bc 1: Nui lc chng vi khun trong 3 ml mi trng c b sung lng v

370C, 150 v/p, qua m

- Bc 2: Chuyn sang nui lng ln trong 100 ml mi trng, 370C, 72

gi, 150 v/p

- Bc 3: Ly tm 10000 v/p, 10 pht thu dch ni s dng lm enzyme th

- Bc 4: Trn hn hp phn ng: 200 l enzyme th + 1.6 ml (buffer

glycine/NaOH + azokeratin)

- Bc 5: hn hp phn ng 600C, 15 pht. Sau ngng phn ng, b

sung

1.8 ml TCA (trichloroaxetic axit) vo hn hp phn ng nng cui cng

l 100 g/l

- Bc 6: Ly tm 10000 v/p, 10 pht, o quang ph bc sng 440 nm

Trong , c 0.01 gi tr hp th nh sng bc sng 440 nm tng ng vi

1U hot tnh enzyme 600C / 15 pht

nh lng Protein bng phng php Bradford

Xy dng ng chun bng cch o OD ti bc sng 595 nm ca

BSA chun cc nng khc nhau



Bc 1: Pha long Stock BSA 10 mg/ml ra 0.1 mg/ml, pha long 100 ln

Cch lm: Ht 5 l BSA vo ng eppendorf 1,5 ml sau b sung 495 l

nc kh ion

Bc 2: Pha dung dch mu (Coomasie brilliant blue Reagent 1X)

Cch lm: Pha 3 ml dung dch Coomasie brilliant blue ( c EtOH) vi 3 ml

dung dch axit photphoric 85%

Bc 3: Chun b 5 ng eppendorf 1,5 ml vi cc nng protein chun BSA

nh sau:

STT Nng BSA (g/l)

BSA 0,1

mg/ml (l)

H2O

(l)

Brardford

reagent (l) Tng th tch (l)

0 0 0 200 1000 1200

1 0.01 20 180 1000 1200

2 0.02 40 160 1000 1200

3 0.03 60 140 1000 1200

4 0.04 80 120 1000 1200

5 0.05 100 100 1000 1200

Bc 4: Trn nh u hn hp phn ng trn, nhit phng t nht 15

pht (ti a khng qu 1gi)

Bc 5: o gi tr OD bc sng 595nm, ghi li kt qu ca cc ng. Sau

lp ng chun y = ax + b vi trc honh l nng BSA v trc tung l

gi tr OD/595 nm ng vi mi ng, bng phn mm Excel.

Xc nh nng protein trong mu nghin cu



Bc 1: B sung 80 l mu vo 120 l nc kh ion v 1000 l Brardford 1X

Bc 2: Trn u hn hp phn ng, nhit phng ti thiu 15 pht

(ti a khng qu 1 gi).

Bc 3: em o trn my quang ph bc sng 595 nm, o hp th

ca cc dung dch protein, ri ghi li kt qu hp th protein sau tnh ton

nng Protein da trn ng chun BSA bng phn mm Excel.

Cng thc tnh:

a, b l h s ng chun protein

D: pha long (D= 2.5 ln)

PHN III: KT QU V THO LUN

3.1. Kt qu ly mu cc a im

Mu t, lng c ly ni chn nui v git m gia cm ti cc a

im khc nhau (Bng 3.1), gi 40C.

Bng 3.1 a im ly mu

STT Tn vit tt a im ly mu

1 L.GT Lng g ti khu chn nui h gia nh Gang Thp

2 .GT t, phn g ti khu chn nui h gia nh Gang Thp

3 L.SC Lng g ti khu chn nui h gia nh Sng Cng

4 .SC t, phn g ti khu chn nui h gia nh Sng Cng

L.GT: Lng Gang Thp .GT: t Gang Thp

L.SC: Lng Sng Cng .SC: t Sng Cng



3.2. Gi tr pH ca cc mu t

Gi tr pH l mt ch tiu nh gi tnh cht ha l ca t. Da vo

phng php xc nh gi tr pH ca t (Bng 3.2) ta c c kt qu nh

sau:

Bng 3.2: Gi tr pH ca cc mu t

Mu pH (H2O ) pH (KCl )

.GT 6,5 5,5

.SC 6,5 5,5

.GT: t Gang Thp .SC: t Sng Cng

bng 4 cho thy gi tr pH ca cc mu t khng thy chnh lch nhiu,

hu ht l trung tnh hoc axit yu. pH ny l rt thch hp cho nhiu loi vi

khun pht trin. Cc vi khun thu phn lng v gia cm thng pht trin

trn mi trng c pH t 7,0 - 7,2.

3.3. Phn lp v tuyn chn cc chng vi sinh vt c kh thy phn

keratin

T 4 mu t v lng thu thp ti cc khu chn nui v ni git m gia

cm Thi Nguyn, phn lp c 20 chng vi khun. Trong c 6

chng c hot tnh thy phn lng v cao t t l khong t 71,65% -

81,55%, 3 chng khng c hot tnh, cn li 11 chng c t l thy phn

khong t 50 % (Bng 3.3).

Qua nghin cu th nghim nui lc, c th thy rng vic c nui lc

gip vi khun sinh trng, pht trin nhanh v mnh hn. Nhn vo kh nng

thu phn lng v ca vi khun qua tng ngy theo di ta c cc kt lun:

Qua qu trnh thu mu, phn lp v chn lc c cc chng vi khun

c kh nng sinh keratinase cao trn mi trng nui cy. Lng lng v cn

li cho thy mc thy phn ca cc chng l rt khc nhau theo tng ngy

nui cy, v kh nng thy phn ca cc chng vi khun t cao nht vo



ngy th 4. Trong , c 4 chng phn hy keratin mnh nht l .GT.2.2;

L.SC.1.2; L.GT.2.1 v .SC.1.1 vi t l l 75,45% 81,55% (bng 3.3). S

nh gi kh nng thy phn lng v, c xc nh bng cng thc nu

phn phng php. So snh vi cc phng php nh gi kh nng thy

phn lng v trn th gii th hu ht s dng c cht Azokeratin, tuy mang

li hiu qu chnh xc nhng tn cng, gi thnh rt cao [39,42,52], phng

php m chng ti p dng l phng php sinh hc. y l phng php

mi, d lm, n gin, khng tn km. Song song vi s nh gi kh nng

thy phn lng v, th pH ca cc chng vi khun tng dn sau tng ngy nui

cy, c bit l cc chng vi khun sinh trng v pht trin mnh. Ngy u

tin l pH = 7,0 - 7,5; ri tng dn ln n pH = 8 - 8,5 v thng dng li

pH = 9,2 - 9,5.

Nhng chng vi khun sinh trng pht trin tt, th sau ngy u tin s

dng cht dinh dng c trong mi trng, ngy th hai s tin hnh thy

phn lng v. Khi nhng si lng v tan ht vo mi trng th nhng

chng vi khun ny s bt u thu phn cung lng. Theo di tng ngy,

chng ti nhn thy s bin i v kh nng thy phn ca vi khun xy ra rt

r rng.

Nhng chng vi khun sinh trng v pht trin yu th vic thu phn lng

v khng xy ra hoc xy ra rt chm, pH ca cc chng ny khng tng m

lun dng li pH = 7,0 - 7,3.

Bng 3.3: T l thu phn lng v ca cc chng tuyn chn

STT Tn chng Trng lng

lng v ban

u (g)

Trng lng

lng v cn li

(g)

T l thu phn

(%)

1 C 0,2 0,2 0

2 .GT.1.1 0,2 0,0837 0,0 1 58,15

3 .GT.1.2 0,2 0,0567 0,014 71,65

4 .GT.1.3 0,2 0,2 0

5 .GT.1.4 0,2 0,0663 0,011 66,85



6 .GT.1.5 0,2 0,0721 0,02 63,95

7 L.SC.1.2 0,2 0,0415 0,012 79,25

8 .GT.2.1 0,2 0,0952 0,016 52,40

9 .GT.2.2 0,2 0,0391 0,021 80,45

10 .GT.2.3 0,2 0,2 0

11 .GT.2.4 0,2 0,0650 0,015 67.50

12 L.GT.2.1 0,2 0,04790,031 75,45

13 L.GT.1.2 0,2 0,07670,031 61,65

14 L.SC.1.1 0,2 0,03710,031 81,55

15 .SC.1.2 0,2 0,08720,035 56,40

16 .SC.1.3 0,2 0,03710,031 63,45

17 L.SC.1.4 0,2 0,09260,037 53,70

18 .SC.1.5 0,2 0,08960,046 55,20

19 .SC.1.1 0,2 0,05540,021 72,30

20 L.SC.1.4 0,2 0,06790,031 66,05

21 L.SC.2.1 0,2 0,08560,045 57,20

T kt qu ny chng ti chn ra 4 chng L.SC.1.2; .SC.1.1; L.GT.2.1;

.GT.2.2 la chn mi trng thch hp (Hnh 3.1).

Hnh 3.1: Kh nng thy phn lng v ca 4 chng vi khun chn lc ( Sau 4 ngy nui cy lc)

3.4. La chn mi trng thch hp ca cc chng c chn lc

Mi vi khun c tc ng rt ring v c trng ln tng c cht trong

nhng iu kin nht nh. V vy vic la chn mi trng thch hp cho

tng chng vi khun l rt cn thit. C nhiu chng vi khun sinh trng tt

trn mi trng giu dinh dng ng thi to ra hot tnh enzyme ti a.

Ngc li, c nhng chng vi khun sinh trng tt trn mi trng ngho

dinh dng nhng vn c th to ra cc hot tnh enzyme ti a. C 4 chng



sau khi chn lc c tin hnh nui th nghim v nh gi kh nng thy

phn lng v trn 3 mi trng khc nhau (mi trng giu dinh dng-MT1;

mi trng dinh dng bnh thng -MT2; mi trng ngho dinh dng-

MT3). Kt qu cho thy s khc bit r rt v s sinh trng, mt t bo,

pH cng nh kh nng thy phn lng v ca cc chng vi khun trn cc mi

trng khc nhau (Hnh 8-9). Nhn chung pH dch nui ca cc chng u

tng nhanh trong 2 ngy u nui cy vi khong pH t 7,0 - 8,5, bt u

chng v dng li vo ngy th 4 pH trong khong t 9,0 9,5.

Da vo kh nng thu phn ca cc chng c t l cao sau thi gian nui

lc th nghim, thy rng mi trng giu dinh dng (MT1) c 4 chng

chn lc u sinh trng tt, mt t bo cao khong 7 x 10 6 CFU/ml.

Tuy nhin kh nng thy phn lng v li khng cao, ch t 59 69% sau 4

ngy nui cy lc, mt khc mi trng ngho dinh dng kh nng thy

phn lng v th hin khng r rng. S sinh trng ca cc chng tng

chm, s lng tng rt t trn mi trng ngho dinh dng mi trng

nc. V trn mi trng ny khng c cht inh dng cn thit nn cc

chng ny khng th pht trin v tng v s lng c.

S thu phn ca cc chng din ra mnh nht trn mi trng dinh dng

bnh thng (MT2), hay mi trng lng v. Ti mi trng ny cht dinh

dng ch yu dng vi khun pht trin l mui. Sau 2 ngy nui lc mt

chng vi khun trn mi trng ny tng ti a. V cc chng vi khun ny

bt u c s thu phn lng v. Sau 4 ngy nui cy th trn mi trng ny

trng lng lng cn li l rt t. S lng cn li ch t 0,0479 g n 0,0371

g. Nh vy, theo cng thc t l phn trm thy phn (2.2.2.1), sau 4 ngy

nui lc th cc chng ny thu phn c t 75,28% - 81,11%. Vy kh

nng thy phn lng v ca 4 chng vi khun chn lc t t 75 81% sau 4

ngy nui cy lc. Vi kt qu trn hon ton ph hp vi nghin cu ca cc

nhm nghin cu khc khi s dng mi trng dinh dng bnh thng c b



sung thm bt lng v nui cy chng vi khun Flavobacterium [61], Vibrio

[48] Chryseobacterium [7,8] vi mc ch thu hi keratnase. Vy vi kt qu

ny mi trng dinh dng bnh thng (MT2) l mi trng thch hp cho

cc chng vi khun thy phn lng v, ng thi c th c la chn cho

cc nghin cu tip theo thu hi keratinase t 4 chng vi khun c chn

lc. y cng chnh l mi trng vi hm lng dinh dng ch yu l cc

mui, nn gim c chi ph nghin cu.

Nh vy, qua kt qu v s sinh trng v phn hu ca cc chng trn

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