B GIO DC V O TO TRNG I HC K THUT CNG NGH TP.HCM KHOA MI TRNG
& CNG NGH SINH HC
TIU LUN: NGHIN
CU QUY TRNH SN XUT
KHNG SINH PENICILLIN BNG ENZYME C NH PENICILLIN ACYLASE.
Lp: 06DSH Gio vin hng dn: Ths. Phan Vn Dn Nhm 3: 1. Trn ng H 2.
Trn Nht Linh 3. ng Th L Phng 4. Nguyn Kiu Trang 5. Nguyn Duyn Hong
M
MC LCPHN I: GII THIU PHN II: NI DUNG I/ Gii thiu v enzyme c nh
1. ngha ca enzyme c nh 2. nh ngha enzyme c nh 3. Cc phng php iu ch
enzyme c nh3.1 3.2
Phng php gn enzyme bng lin kt cng ho tr Phng php gi enzyme trong
khun gel
3.3 C nh enzyme bng phng php hp th trn cc cht mang hoc khng c in
tch.4 Mt s c tnh ca enzyme c nh. 5 Mt s ng dng ca enzyme c nh trong
sn xut 5.1 5.2 5.3 5.4
S dng aminoacylase c nh sn xut axitamin. Sn xut L-axit aspartic
bng enzyme asperza c nh. Thu phn lactoza bng enzyme lactaza c nh.
Sn xut nhm penicilin-axit 6 amino penicillinic (6-APA) bng
enzyme c nh penicillin acylase. II/ Gii thiu v khng sinh Lch s
hnh thnh Cu to ca penicillin Tnh cht ha l Cc yu t nh hng4.1
1.
2.
3.
4.
ng hc ca enzyme penicillin acylase Bn cht v tnh cht ha hc ca cht
mang-6-
4.2
4.3 4.4 1. 2. 2.1
S khuch tn ca c cht, sn phm v cc phn t khc nh hng pH
III/ Quy trnh sn xut K thut sn xut enzyme penicillin acylase K
thut sn xut khng sinh bn tng hp Sn xut penicillin G t nguyn liu t
nhin 2.1.1 iu kin ln men: 2.1.2 Thnh phn mi trng ln men 2.1.3 Quy
trnh ln men: 2.1.4 X l dch ln men v tinh ch thu nhn penicillin t
nhin2.2
Sn xut penicillin bn tng hp t penicillin G t nhin: 3. Cc sn phm
khng sinh bn tng hp Phn III : KT LUN
-7-
PHN I: M U Cc khng sinh l mt nhm thit yu trong y hc hin i. Nh
khng sinh m y hc c th cha c cc bnh nguy him nh dch hch, t, thng hn,
v iu tr hiu qu nhiu loi bnh do vi khun gy ra. i vi nc ngho, Cc thuc
khng sinh gi mt v tr quan trng v cc nc ny do v sinh yu km v mc sng
cn thp nn thng xy ra cc v dch nhim khun h hp, kit l... Hin nay trn
th gii pht hin trn 8000 khng sinh v mi nm c vi trm khng sinh mi c
pht hin. Trong tng lai s c nhiu khng sinh mi c pht hin t qu trnh bn
tng hp bng cch s dng enzyme c nh. Vic s dng enzyme c nh lm cht xc
tc sinh hc cho qu sn xut khng sinh bn tng hp va c ngha v mt kinh t
va gii quyt nhiu vn x hi, cc vn mi trng. Xut pht t thc t , chng ti
thc hin ti Nghin cu qu trnh sn xut khng sinh bn tng hp bng enzyme
penicillin acylase.
-8-
PHN II: NI DUNG I/ Gii thiu v enzyme c nh 1. ngha ca enzyme c nh
Trong nhng nm cui ca th k 20 bt u ph bin k thut c nh enzyme. Enzyme
ho tan sau khi s dng thng ln vo cng vi sn phm khng tch ra c. Nu tch
ra th enzyme b mt hot tnh, v vy vi mt lng enzyme nht nh ch c th s
dng c mt ln. S dng enzyme c nh c mt s u im sau: Mt lng enzyme c th
s dng lp i lp li nhiu ln trong mt thi gian di. Enzyme khng ln vo
trong sn phm, do trnh c nh hng khng tt n lng sn phm; Dng enzyme c
nh c th dng nhanh chng phn ng khi cn thit bng cch tch n ra khi c
cht. 2. nh ngha enzyme c nh Enzyme c nh l enzyme c s dng nhiu ln lp
li. y l mt hng cng ngh rt trin vng. Cho n nay c 2 cng ngh s dng
enzyme c nh trong sn xut cng nghip: glucose isomerase c nh trong sn
xut siro frustose v penicillin acylase (amidase) c nh trong sn xut
penicillin. L enzyme c s tham gia hot ng trong mt khng gian b
giihn. S gii hn hot ng vn linh hot ca enzyme bng cch gn n vo mt pha
cch ly, tch n khi pha lng t do v n vn c kh nng tip xc c vi cc phn t
c cht. Pha gn enzyme thng khng tan trong nc nhng cng c th l cc
polyme a nc. Trong trin khai cng ngh s dng enzyme c nh ngi ta quan
tm mt s vn : To thit b hot ng c hiu sut hot ng v mc hot ng t ha
cao, s dng nn cha enzyme c nh hot ng m c hn.-9-
Tp trung trin khai hon thin qu trnh kim sot v cng ngh sn xut lin
tc. Do vy khng phi bt c enzyme no cng tm cch s dng dng c nh, ch nn
s dng cng ngh enzyne mt cch c hiu qu nht. Xu hng hin nay l tp trung
trin khai cng ngh s dng enzyme c nh t tin, c gi tr cao, xc tc trn c
cht c MW nh, d kim sot s ly nhim v sn phm khng cha enzyme. Enzyme
khng tan c nghin cu v ng dng t nhng nm 1950. ch to enzyme c nh c th
dng phng php hp ph, lin kt ho tr gn kt enzyme gi l cht mang
(enzyme), hin nay ngi ta dng xenluloza, tinh bt, sephadex, agaroza,
alghinat, canxi, gel polycylamit, bt thu tinh, nilon Ch phm enzyme
khng tan c th dng bt, ht, phin mng mng. Trong mt s trng hp khng cn
thit phi gn enzyme vo cht mang m c th gi n bn trong mng li polyme
bao quy ly phn t enzyme. Mng li c mt nh s khng cho php enzyme thot
ra khi nhng vn ln sn phm v c cht to ra d dng. 3. Cc phng php iu ch
enzyme c nh Theo k thut ch to enzyme c nh, enzyme c gn vo mt vt
mang no . Nhng vt mang nh vy c th l cellulose v cc dn xut ca n, cc
ht knh xp, gel polyacrylamide, sephadex, collodium, tinh bt, cc loi
allumosilicate v oxide kim loi.. V nguyn tc, c 3 phng php iu ch cc
enzyme c nh : - Gn bng lin kt ng ho tr phn t enzyme vo cht mang
khng ho tan hoc gn cc enzyme vi nhau to nn i phn t khng ho tan
- 10
- nh enzyme ln b mt cht mang hoc vo trong lng khun gel c kch thc
l kh nh gi enzyme, cn cc cht khc qua li t do
0
- Hp ph enzyme ln trn cc cht mang khng ho tan c mang
hoc khng mang in tch
- 11
3.1
Phng php gn enzyme bng lin kt cng ho tr
a s enzyme c nh thu c bng phng php ny. Vi phng php ny c th thu
enzyme c nh bng hai cch
Kt hp phn t protein enzyme vo cht mang khng ho tan Cc cht mang
gn enzyme phi tho mn nhng yu cu sau :
-
C ho tan thp v bn vng i vi cc tc ng c hc v ho hc. Khng gy tc ng
km hm n hot tnh enzyme. Khng hp ph khi chn lc i vi cc protein khc.
Cht mang tt hn c l c tnh ho nc, v cht mang k nc c th
gy tc dng c ch enzyme lin kt. Vic gn enzyme s c hiu qu hn khi in
tch ca enzyme v ca
cht mang c du ngc nhau. Cc cht mang loi ny thng l polypeptide,cc
dn xut ca cellulose v Dextran (DEAE-cellulose, CM-cellulose,
DEAE-sephadex, CM,sephadex),agarose v cc polimer tng hp khc nh
polyacrilamide, polysyrol, polystytol, polyamide v cc cht v c nh
silicagen, bentonit,.v..v.. Cht mang ph bin hn c l dextran c lin kt
ngang (Sephadex ) v garose ht (sepharose). Cc hai loi cht ny u c cu
trc l xp khin cho cc phn t ln c th xm nhp vo gel mt cch d dng.
Poliacrylamide cng l cht mang rt tin li vi c bn vng c hc v ho hc
cao. Cht mang v c thng rt bn vi nhit, c hc, vi dung mi hu c v cc vi
sinh vt, nht l khng b bin i cu hnh ca khun khi thay i tnh cht ca mi
trng chung quanh. Nhiu dn liu cho thy rng enzyme nh vi khun v c khi
bo qun thng bn vng hn enzyme gn vi khun polymer hu c.
- 12
Tham gia to thnh cc lin kt dng ho tr c th c cc nhm chc ca phn t
protein enzyme nh nhm - v - carboxyl COOH ca acid asparaginic v aid
glutamic, nhm -NH2 ca lysine, nhm -SH ca cysteine, vng phenol ca
tyrosine, nhn imidasol ca histidine, cc nhm OH ca serine,
threonine, nhm guanidine ca arginine v imidasol ca tryptophan. Qu
trnh kt hp enzyme s xy ra mt cch trc tip khi cht mang c cha cc nhm
chc c kh nng lin kt trc tip ni trn trong phn t protein enzyme. V d
gc anhydride maleic lin kt vi nhm -NH2 ca lysine. Trong cc trng hp
khc nhau s lin kt gia cht mang v protein enzyme ch xy ra sau khi
cht mang c hot ho. Vic hot ho s b cht mang c th c thc hin bng nhiu
cch. V d, cc nhm hydroxyl ca dextran hoc polyoside khc c th c hot
ho bng bromur cyan ( BrCN). Sau cht mang c hot ho mi lin kt vi
enzyme
Cc cht mang c cha nhm amin nh aminobenzoylcelluse,
polyaminostyrol c th c hot ho bng phn ng diazo.
- 13
V d, polyaminostyrol trc ht c diazo ho bng natri nitrit trong
dung dch mi trng acid thnh mui diazo, sau mi kt hp vi enzyme:
Phn ng kt hp enzyme c tin hnh nhanh chng trong iu kin nhit d
thng v dung dch nc trung tnh. Nu cht mang c cha nhm amin c th hot
ho bng cch cho tc dng vi phosgen hoc tiophosgen to thnh dn xut
isosianat hoc izothyosianat. Cc nhm isosianat hoc izothyosianat pH
trung tnh s lin kt d dng vi nhm NH2 ca lyzine.
Bng phng php ny ngi ta iu ch c cc dn xut enzyme c nh nh trypsin,
chimotrypsin, -,-amylase, glucoamylase. Nu cht mang c cha nhm COOH
nh CM-cellulose hoc nha tng hp th cn c hot ho trc khi gn kt vi
enzyme bng cc phng php axit, carbodiimit- 14
V phn ng xy ra trong mi trng acid yu (pH=5) nn phng php ny c bit
thun li vi cc enzyme c tnh acid nh pepsin. Kt hp ng ho tr gia cc
phn t enzyme vi cht mang. C nh enzyme bng cch lin kt ng ho tr c thc
hin bng cch khc nhau : 1/ Lin kt ng hoa tr c to ra gia cc nhm phn
ng ca vt mang v enzyme; trong mt s trng hp vt mang cn c hot ho s b;
2/ Cc phn t enzyme sau khi c hp th trn cht mang hoc nm trong lng
phn t cht mang lin kt vi nhau nh tnh lng chc hoc a chc ca cht phn
ng. Ngi ta thng dng aldehyde glutaric gn cc nhm amin ca
- 15
lysine cc phn t enzyme. V d,trypsine c nh trysin c iu ch bng cch
dng aldehyde glutaric 2! lin kt cc phn t enzyme v gn chng vo
aminoethylcelluse.3.2 Phng php gi enzyme trong khun gel
gi enzyme vo khun gel ngi ta tin hnh trng hp ho hc cc gel khi c
mt ng thi enzyme. Sau khi hon thnh qu trnh trng hp enzyme b gi chc
trong gel. Gel c enzyme c th nghin nh bng cch p qua ry c l nh v sy
kh nhit thp.Dn xut enzyme loi ny ln u tin do Bernfeld (1993)thu c
bng cch trng hp acrylamide vi N,N-methylenbisacrylamide. Phng php
ny thng dng do cht trng hp thu c d to thnh dng ht v tu thuc vo iu
kin tin hnh m gel c th c xp khc nhau. Sau y l mt s phng php
gienzyme. Cc gel c th c hnh thnh t cc polymer tng hp nh acrylamide,
hydroxyethyl-2-metacrylate, to phc cng cua bng
ethyleneglycol-dimetacrylate, polyvinyl, polyuretan. Enzyme c ho
tan hoc phn tn trong mt dung dch monomer v sau trng hp trong s c mt
ca mt hay nhiu tc nhn to phc cng cua. Gel c th ct thnh mng t trn mt
gi th rn, hoc cng c th nghin thnh bt sau khi loi tr nc. Alginate v
caraghenan ly t rong bin thng c kh nng tao gel rt tt v thun li bao
gi cc enzyme hoc t bo nguyn vn. Cc enzyme cng c th b nht trong cc l
nh ca cc si tng hp. Vi cch ny ngi ta cho dch lng chy bn trong si ,
do hn ch c s phn cc b mt v s bt lp thng gp vi lp mng. Mt nh tng ca
cellulose triacetate trong methylene chlorua v enzyme trong dung
dch m c cha glycerol c p qua mt khun lc di p sut nito. Cc si i ra
khi khun c nhng vo mt ci b ng t c cha toluel, sau c lm kh trong chn
khng. Cc si ny bn vi acid yu hoc kim yu, lc ion cao v chu c mt s
dung mi hu c. Tuy nhin phng php ny ch thch hp i vi nhng enzyme kh b
mt hot tnh trong cc dung mi khng ho ln vi nc.- 16
Phng php gi enzyme trong cc bao vi th (microcapsul). Enzyme c gi
trong cc bao vi th c mng bn thm c to ra t cc polymer c kch thc l nh
ngn cn s khuch tn ca enzyme ra ngoi v ln c cht i vo v sn phm phn ng
i ra d dng. C nhiu phng php gi enzyme trong microcapsul. Sau y l mt
trong cc phng php . Cho mt dung dch long cha enzyme v monomer a nc
trong mt dung mi hu c khng ho ln trong nc to nh tng , sau thm mt
monomer k nc gy phn ng trng hp to ra mt mng bao quanh nhng git lng
nh. Thng phi thm vo mt tc nhn hot ng b mt lm bn nh tng cng nh iu
chnh cc capsul c khch thc mong mun t 1 n 100 m Phng php tin polymer
gi cc cht xc tc sinh hc. C nh enzyme bng phng php ny c nhng u im nh
sau: Qu trnh gi rt n gin trong nhng iu kin nh nhng. Cc cht tin trng
hp khng cha cc monomer vn c th gy nh
hng xu n enzyme c gi. Cu trc li ca gel c th c iu chnh bng cch
dng cc tin
polymer c chiu di chui bt k. Cc tnh cht ho cht l ho ca gel c th
dnh hng trc bng cch
chn cc tin cht polymer thch hp vn c tng hp ho hc trong iu kin
vng mt enzyme. Di y l mt v d v phng php tin polymer. l phng php to
enzyme lin kt cho gia cc tin polymer bng cch chiu tia cc tm gi
enzyme. Khi chiu tia cc tm gn ln dung dch c cha cht tin polymer v
enzyme th s to ra cc lin kt cho gia cc gc ca tin polymer v mt gel c
hnh thnh bao ly enzyme. S gi enzyme bng phng php ny c th tin hnh iu
kin nh nhng, trnh c cc thay i pH qu kim hoc qu acid, thi gian li rt
nhanh, ch trong vng t 3-5 pht. Vi phng php ny c th gi enzyme, t bo
vi sinh vt hoc cc bo quan
- 17
3.3
C nh enzyme bng phng php hp th trn cc cht mang hoc khng c in
tch.
Vi phng php ny enzyme c hp th trn cc cht co hot tnh b mt nh than
hot tnh, cellulose, tinh bt ,dextran, collagen,v mt s nha trao i
ion, silicagen, thu tinh. Trong trng hp cht mang khng c l enzyme c
nh vo cht mang thnh tng lp trn b mt, khi cht mang c l th cc phn t
enzyme s xm nhp vo trong cc l. 4. Mt s c tnh ca enzyme c nh Vic c
nh lm thay i ng k nhiu tnh cht ca enzyme nh bn, tnh c hiu, c im ng
hc. Thng thng vic c nh lm tng ng k bn ca enzyme. V d , c nh
protease lm tng bn vi nhit gp chc nghn ln. Sau khi c c nh khng nhng
bn vi nhit ca enzyme tng ln m phm vi nhit ti thch ca enzyme cng c m
rng. Nhit cao lm cho enzyme b bin tnh, cu trc bc ba ca enzyme b ph
v v enzyme b mt hot tnh. Khi enzyme c c nh nh nhiu lin kt gn vi cht
mang qu trnh bin tnh s b ngn cn, lm cho enzyem chu c nhit cao.
Trong trng hp i vi cc enzyme thu phn protein, v d papain v trysin s
c nh lm ngn cn mt phn hoc hon ton hin tng t phn. iu ny rt c ngha
thc t v n gip lm tng thi gian lm vic ca enzyme. Mt trong cc nguyn
nhn lm bin i tnh cht ca enzyme khi chng c c nh trn cc cht mang c in
tch. V d mt cht trao i anion mang in tch m c bao quanh bi mt mi
trng giu proton, v v th pH trong cc lp nm k st vi cho ng cong ph
thuc pH ca hot tnh enzyme c nh trn cc cht mang anionit,cationit v
trung tnh s khc nhau. 5. Mt s ng dng ca enzyme trong sn xut 5.1 S
dng aminoacylase c nh sn xut axitamin Trong qu trnh tng hp ho hc
hay ln men ( sinh tng hp) sn xut cc axit amin nhng nhc im ln nht ca
phng php ny cho ra cc sn phm- 18
raxemic, tc l hn hp ca 2 dng ng phn quang hc D v L trong ch c
dng L mi c hot tnh sinh hc cao ,c ngha trong khoa hc ho sinh. Nu s
dng phng php ho hc hay kt hp vi sinh tng hp ( ln men 2 pha ) s tn
km, khng kh thi. T nm 1969 hng tanabe Seizaku ( Nht Bn ) s dng
enzyme c nh amino acylaza ( AACD: enzyme ng phn ho chuyn t dng D
sang dng L ca axit amin ) chuyn ho hn hp D,L axit amin. Trong
enzyme aminoacylase c c nh trn DEAE. Sephadex bng lin kt ion vi thi
gian bn hu l 65 ngy 50oC. 5.2 Sn xut L-axit aspartic bng enzyme
asperza c nh
L c cht trung gian ca rt nhiu qu trnh chuyn ho ho sinh tng hp cc
axit amin khc nhau rt quan trng trong dinh dng ng vt v ch bin thc
phm (sn xut axit l-vacin, tng hp axit -xetoglutamic( tin cht chuyn
ho thnh axit l-glutamic )). C ch ho sinh ca s to thnh axut aspartic
l qu trnh to lin kt ng ho tr gia NH3 vi axit fumaric bi enzyme
aspartic.
T nm 1973, hng TANABE SEIZAKY s dng t bo c cha enzyme aspartaza
(ni vi khun Brevibacterium flavum nui cy trn mi trng r ng giu
biotin ) v gi n trong gel polyacrylamit vi bn chu k hot ng l 120
ngy 37C. 5.3 Thu phn lactoza bng enzyme lactaza c nh: Lactoza l
disaccarit c trong sa nn c gi l ng sa. Loi ng ny c ngt ngo thp (
bng 30% vi ng saccaroza cng nng ), ho tan
- 19
km ( gy nn hin tng sn ng trong sa), mt b phn ngi s dng sa khng c
kh nng tiu ho hp th c sa ny. Mt khc ng sa hu nh c thi cng vi sa nu
thu phn lactoza to thnh 2 monosaccarit cu thnh n l glucoza v
galactose s mang li hiu qu cao. Lc ny sa s c cht lng cao hn, loi b
hin tng sn sa, nng cao tiu ho, cc monosaccarit s c vi sinh vt s dng
khi ln men sa (cc sn phm sa chua v phomat ). 5.4 Sn xut nhm
penicilin-axit 6 amino penicillinic (6-APA) bng enzyme c nh
penicillinamidaza Penicillin l mt nhm cht c tnh khng sinh, cu to
chung l: Vi R l gc axyl th ta c penicillin G- l penicillin thng mi
v sinh hot ph bin nht hin nay. Enzyme penicillinamidaza xc tc thu
phn benzyl penicillin (penicillin to thnh nhm penicillin 6-APA v
axit phenyl axetic. 6-APA c s dng rng ri trong cng nghip dc phm sn
xut cc loi khng sinh h penicillin. Hin nay ton b 6-APA ca th gii u
c sn xut bng phng php s dng enzyme penicillinamidaza c nh. y l
enzyme ni bo khi sinh tng hp bi vi khun Esterichia coli, bacterium
faecalic, alacaligus vi moi trng cazein thu phn, cao ng, glucoza,
axit phelnylaxetic lm cht cm ng. Phng php gi enzyme ca hng SNAM
Progetti (Italy) nh sau :10 lt dung dch penicillinamidaza pH=8 trn
vi 5kg triaxetic xenlulose trong 71.4 kg clorimetylin 4C, lc k cho
n khi to gel c cc si. mi kg si thnh phm c nhi trong cc ct kch thc
43 x 14 cm. 26 lit penicillin G( mui kali ) cho chy lin tc qua ct
pH=8.2 cho n khi t mc chuyn ho 97% 6 APA Cng ngh ca hng TANABE
SEIZAKU : s dng bioreactor t bo c nh cha penicillinamidaza trong
gel polyacriamit nh sau : dung dch penicillin G
- 20
0.65M pH = 8.5 cho chy qua ct vi tc 0.12-0.14 th tch ct/h. Hiu
sut phn ng t 80%. II/ Cht khng sinh penicillin bn tng hp: 1. Lch s
hnh thnh: Cc penicillin l i din tiu biu cho cc khng sinh c ngun gc
t nm mc. Ngoi ra cn c mt vi khng sinh khc nh griseofulvin,
trichotrxin, fumagillin cng c sinh ra t cc chng nm mc khc nhau.
Penicillin G l cht khng sinh tiu biu c Alexander Flemming tm ra u
tin vo nm 1928 trong qu trnh nghin cu v t cu khun. ng nhn thy trn
hp petri nui cy t cu khun c nhim nm mc Pinicillin notatum v to vng
v khun. Flemming gi cht c ch to vng v khun ny l penicillin. Sau , M
trin khai ln men thnh cng penicillin theo phng php ln men b mt
(1931). Tuy nhin, cng trong khong thi gian mi n lc nhm tch v tinh
ch penicillin t dch ln men u tht bi do khng bo v c hot tnh khng
sinh ca ch phm tinh ch v do vn penicillin tm thi b lng qun. Nm 1938
Oxford, khi tm li cc ti liu khoa hc cng b, Ernst Boris Chain quan
tm n pht minh ca Fleming v ng ngh Howara Walter Florey cho tip tc
trin khai nghin cu ny. Ngy 25/05/1940 penicillin c th nghim rt thnh
cng trn chut. Nm 1942, tuyn chn c chng cng nghip Penicillium
chrysogenum NRRL 1951 (1943) v sau c bin chng P. Chrysogenum Wis Q
- 176 (chng ny c xem l chng gc ca hu ht cc chng cng nghip ang s dng
hin nay trn ton th gii ); thnh cng trong vic iu chnh ng hng qu trnh
ln men ln men sn xut penicillin G (bng s dng tin cht
Phenylacetic).... Nm 1943, penicillin c sn xut qui m cng nghip M ph
v cc bnh nhim trng cho thng binh trong chin tranh trong th gii th
II.- 21
Penicillin cng l cht khng sinh u tin c sn xut ln men chm qui m
cng nghip (1947). Nm 1941, penicillin G l khng sinh c hiu qu cao
chng li hu ht cc chng Staphylococcus aureus. Tuy nhin n u nm 1947
phn ln cc vi khun phn lp trong bnh vin u khng li penicillin. gii
quyt vn ny ngi ta n lc tm ra cc penicillin mi c tc dng u tr tt hn.
Cng ngh sn xut penicillin bn tng hp bt u c nghin cu.2. Cu to ca
penicillin:
Penicillin gm nhiu loi, chng c cu to gn ging nhau, bao gm mt vng
thiazolidine, mt vng -lactam, mt nhm amino gn vi CO2 v mt mch bn (
R). Tt c cc penicillin u l dn sut ca acid 6-aminopenicillanic. S
thay th R to nhiu acid amin khc nhau. Hu ht cc peniciliin u c phn
phi di dng mui natri hoc mui kali v c ngha trong iu tr cng nh trong
thng mi nh penicillin G, penicillin V
Cu to chung ca phn t penicillin3. Tnh cht ha l:
Tnh cht vt l: Cc penicillin di dng mui hoc dng acid l nhng bt
trng khng mi khi tinh khit.
- 22
Ph UV: a s cc nhm R acyl ha trn 6-APA u l vng thom7nen6 cho ph
hp thu vng UV c th ng dng c. Ph IR: vng 1600 1800 cm-1 c cc nh c
trng cc nhm sau : Nhm lactam gia 1760 v 1730 cm-1. Chc carboxyl
khong gia 1600 cm-1. Nhm chc amid ngoi vng gia 1700 v 1650
cm-1.
Tnh cht ha hc: Cc penicillin c kh nng to mui natri v kali tan
trong nc, trong khi cc mui kim loi nng ( vi d mui Cu2+) th khng tan
hoc kch thch s phn hy. Cc penicillin cng c kh nng to mui vi cc
amin: To cc penicillin thy gii chm ( tc ng tr) nh procain
penicillin ( tc
ng ko di t 24 48h), benethamin penicillin ( tc ng ko di th 3 7
ngy), benzathin penicillin ( tc ng ko di 2 4 tun). Mt s c tnh base
v d cc aminosid, cc alkaloid khi trn chung vi penicillin trong cng
mt ng tim s gy ra kt ta. Cc penicillin cng c kh nng to ra cc este,
s l nhng tin cht c kh
nng phng thch cc khng sinh ny trong invivo. Tnh khng bn ca vng
beta lactam: S phn hy trong mi trng kim: pH = 8 s c s tn cng ca ion
OHtrn carbonyl lactam gy ra s m vng theo qui lut chung, cui cng s c
s to thnh acid penicilloic, nhng s decarboxyl c th xy ra tip theo
to acid peniloic. Nu trong mi trng c s hin din ca nhng mui kim loi
nng ( Zn2+, Cd2+, Pb2+ hoc Hg2+) s lm cho acid peniciloic b phn hy
thnh carbinolamin khng bn, cht ny s tip tc b phn hy to
D-penicillamin v acid penaldic. Acid penaloic n lt n c th b
decarboxyl ha tr thnh penicillo-aldehyd. S alcol phn v amino phn:
vng beta lactam nhy vi mt s tc nhn i nhn khc vi xc tc ca cc ion kim
loi nng: Cu2+, Zn2+, Sn2+.- 23
S phn hy trong mi trng acid: di s hin din ca ion H+, s tn cng i
in t trn nguyn t S, kch thch s m vng lactam v vng thiazolidin, tip
theo l s ti sp xp to thnh cu trc oxazolic cua acid penicillenic.
Cui cng, nu mi trng qu acid c th to thnh acid penillic. Ngoi ra vng
lactam c th b m bi lactamase tit ra t vi khun.4. Cc yu t nh hng
:
Trong qu trnh nghin cu penicillin G, ngi ta tm thy c enzyme
penicillin amidase c kh nng thy phn penicillin G (hay V) thay th cc
ha cht ng thi thc y phn ng xy ra nhanh hn trong sn xut khng sinh
penicillin bn tng hp. Do vy tng hiu qu sn xut cn nghin cu cc yu t
nh hng n enzyme thy phn 4.1 ng hc ca enzyme penicillin acylase: Qu
trnh c nh c th gy nh hng ln n tnh n nh ca enzyme. Nu qu trnh c nh a
ra bt k mt yu t no lm bin dng i vi enzyme, n s thc y s bt hot ca
enzyme di cc iu kin gy bin tnh nh nhit , pHS kt hp gia gi th v
enzyme cng t nhin th s n nh ca enzyme cng ln, cng bo v tnh cht vt l
ca enzyme, cng t b bin tnh. Mc n nh ca enzyme c xc nh bi nng gel v
s lng cc tng tc cng ha tr . Cc hng s ng hc ng hc K m, Vmaxb bin tnh
bi qu trnh c nh l do thay i cu trc bn trong v gii hn vo v tr hot ng
ca enzyme, hay do nhng thay i ca phn t khch tn trong mi trng. 4.2
Bn cht v tnh cht ha hc ca cht mang: Cc tnh cht ha hc ca cht mang nh
tnh ha tan, tnh bn c hc, trng, in tch, tnh ho nc hay k nc u nh hng
n lng enzyme c lin kt , tnh bn, dn xut enzyme penicillin, ti kh nng
ca cc cht mang hp th khng c trng cc cht t mi trng phn ng. Cht mang
cng c tc dng hn ch s bin tnh ca penicillin trong dung mi c kh nng
lm t mi lin kt hydrogen v lin kt k nc.- 24
Dung dch m s dng trong trong sn xut enzym penicillin acylase ny
l sephadex l mt loi dn xut ca dextran- mt loi polysacchride phn t
ln ca mt s loi vi sinh vt Leuconostoc sinh trng v pht trin trong mi
trng c saccharose. Phn t dextran c cu to t cc chui, trong cc gc
glucose ni vi nhau bng lin kt glycoside 1:6. Trng lng phn t ca
dextran t n mt triu hoc cao hn. Sexphadex c thu nhn t dextran bng
phng php x l ho hc to ra cc lin kt ngang, lm cho sn phm khng ho tan
trong nc nhng c th trng phng trong nc. Ngy nay trn th gii ngi ta sn
xut nhiu loi sephadex vi kch thc ht v phn nhnh khc nhau v do c kh
nng tch chit khc nhau. Nhng sn phm ny c k hiu bng cc con s : G-10,
G-25,G-50,G-75,G-100,G-200 v.v Cht mang polyacrylamide: L polymer
rt ng nht, trng tt,kch thc ca l gel c th iu chnh c v din tch b mt
tip xc ln. Polyacrylate v polyacrylate thng c np vo ct, v c rt nhiu
dng khc nhau nh polymer c thiol,amine, hoc aldehyde. Thng th tng kh
nng ng mch ca gel polyacrylamide,cn phi b sung thm
N,N-methyllenbisacrylamide lm cht ng mch (crosslinking agent). 4.3
S khuch tn ca c cht, sn phm v cc phn t khc: Tc khuch tn ca c cht, c
cht v cc cht hp ph khc ph thuc vo: Kch thc l gel ca cht mang. Trng
lng phn t ca c cht. S chnh lch nhit gia vng mi trng vi m xung quanh
enzyme penicillin amidase v dung dch t do. Nhit ti u cho enzyme hot
ng 600C.
- 25
Trong kch tht ca cht mang v trng lng phn t ng vai tr quan trng.
nh hng pH: in tch ca cht mang c nh hng n pH ti u ca enzyme, pH ti u
ca penicillin acylase sau gn ln cht mang. pH thch hp cho enzyme 7.5
- 8.0 III/ Quy trnh sn xut:1. K thut sn xut enzyme penicillin
acylase:
Acylases Penicillin c tm thy trong cc vi sinh vt nh vi khun, nm
men, v nm, c s dng xc tc cc thy phn t nhin ca penicillin.
Penicillin ngoi bo c chit xut t: x khun, nm men, nm mc. Enzyme ny c
kh nng thy phn cc penicillin K, F, V nhanh, cn i vi penicillin G li
thy phn rt chm ( thp hn khong 100 ln) Penicillin ni bo ch yu c
trong vi khun c tc dng thy phn nhanh phn t penicillin G, ct t dy ni
peptid mch bn 6-APA. Enzyme ni bo v enzyme ngoi bo c pH hot ng khc
nhau: ni bo hot ng mnh pH=7.3-8.5; cn enzyme ngoi bo hot ng mnh
pH-9.0. Vic s dng loi enzyme ny l mt trong nhng thnh cng sm nht ca
cc qu trnh lin quan n cc enzym c nh v c th c ti s dng trn 100 ln.
Cc enzyme acylase thy gii mt s amides, c tng qut bng cng thc di
y:
R-CO-NHR' R-CO-NHR 'Qu trnh thy phn ch yu da vo gc R (acyl) , cn
gc R , khng c tc dng thy phn.- 26
Quy trnh sn xut:
E.coli
NCIM 2350 NCIM 2350 v t bo, chit ra bng dd m, pH =8 PhaDch chit
th
Kt ta amioniumsunfat 70% bo ha loi b acia nucleic
Enzyme
thLc qua gel Sephadex G-200
enzyme tinh schin di trn gel polyacylamide + SAS
Kim tra tinh schezyme
Enzyme c nh
- 27
Gii thch quy trnh: C nhiu vi sinh vt c kh nng sinh enzyme
penicillin acylase. Trong cng nghip hay dng E.coli c nui dng trong
mi trng dinh dng c thnh phn mi trng dinh dng nh sau: Cao ng: Pepton
: Glucose: Acid penylacetic Nhit Thi gian 2% 0.5% 2% 0.15% 300
24h
Enzyme penicillin acylase c sn xut t E.coli NCIM 2350. Trc ht ta
ph v t bo bng my nghin v b sung thm dung dch m phosphate 0.03M pH =
8 chit rt dch chit th c cha enzyme. Dch chit thu c thng cha rt nhiu
protein cc hp cht khng mong mun, nng enzyme rt thp. Do vy ta cn b
sung thm cc tc nhn gy kt ta. ta enzyme t dch chit th bng amonium
sulfate 70% v mui ny c ha tan rt cao ( c th t 720g/l, t0=250C) t lm
mt hot tnh ca enzyme, ngoi ra c th lm bn enzyme.Sau em ly tm ....ha
tan ta tr li bng dung dch m pH=8.Ta thu c enzyme th. tch tng phn v
tin ti tinh sch hon ton enzyme, ngi ta dng phng php sc k lc gel.
Nguyn tt ca phng php ny da vo mc dch chuyn khc nhau ca cc phn t c
kch tht khc nhau.Gel s dng Sephadex G-200.Cc phn t i qua h thng cc
ht gel, nu c kch tht nh hn kch tht ca cc l gel c th chui vo trong
ht gel. Do thi gian i qua ct s lu hn cc phn t c kch thut ln hn. Cn
cc ht c kch tht ln s i qua khong trng cc l gel. Cc phn t c kch tht
ln s chui ra trc cc
- 28
phn t c kch tht nh s chui ra sau. Nh vy cho php ta phn chia ra
cc i phn t c kch tht khc nhau. S dng k thut in di gel
polyacrylamide (PAGE) bin tnh s dng gel v m c cha sodium dodecyl
sulfate ( SAS). Cc phn t enzyme trong mi trng c cha SAS s b dui
thng ra v tr nn tch in m, v vy s dch chuyn v cc dng trong in trng.
Tc dch chuyn ph thuc khi lng phn t ca cc enzyme, cc enzyme c khi
lng cng nh th chy cng nhanh, cc emzyme c khi lng phn t nh th chy
chm hn. Sau qu trnh in di , gel c nhum bng coomassie. in di trn gel
polyacylamide c cha SAS thng tin hnh vi cc enzyme chun c khi lng
phn t bit , v th d dng xc nh khi lng ca phn t ca enzyme penicillin
acylase. Phng php in di trn cht mang polyacrylamide (PAGE) Gel to
bi s polymer ha acrylamide ha acrylamide c mt s u im sau: c phn tch
tt i vi cc phn t protein v nucleic acid c MW trung bnh (ti 106Da),
tch c phn t c kch thc tng i ln. Tng tc gia phn t di chuyn v nn gel
l nh v nn gel kh bn v mt tnh vt l. Khc vi phng php lc gel, phng php
in di va c tnh cht tch phn t nh xp ca cht nn,li va phn tch theo in
nng m chng c. Trong phuong2w php lc gel phn t ln chy nhanh hn phn t
nh,cn trong in di li ngc li. Gel polyacrylamide to bi qu trnh
polymer ha acrylamide vi cht to lin kt ngang
N,N-methylene-b-acrylenide. Qu trnh trn c kim sot bi sot bi h xc tc
khi ng,ammonium pulfate-n,n,N,N-tetramethylene diamine (TEMED),
riboflavin xc tc polymer ha ph thuc UV. Gel tch protein thng l loi
gel 7.5% polyacrylamide,cho php tch protein c MW t 10.000-1.000.000
Da, tuy nhin tt hn c l trong khong t 30.000-300.000Da. Nguyn tc ch
yu l nng acrylamide cng thp gel to thnh cng xp,cng cho php phn t ln
hn.
- 29
Phng php in di gel polyacrylamide sodium dodecyl sulfate
(SAS-PAGE) (SAS-PAGE). Phng php SAS-PAGE ch yu xc nh MW phn t
protein. Khi in di phn t protein trong SAS v mercaptoethanol. Do s
di chuyn ca phc SAS-protein lc ny ch yu ph thuc vo kch thc ca phn t
protein, phn t ln di chuyn chm hn phn t nh. Kt qu thc nghim cho thy
c tng quan tuyn tnh gia log MW v tc in di. Kt qu in di SAS cho thy
s hin din ca hai thnh phn ca di polipeptid c khi lng 20000 v
700000. Kt qu: Kt hp vi s liu v khi lng phn t t nhin qua sc k lc
gel v in di SDS cho thy: Trong E.coli acylase penicillin c hai chui
monomer (A v B), trong bao gm 209 v 557 axit amin.Hai chui phn t ny
cun vo nhau cht ch to thnh mt hnh chp.Kt thc qu trnh nui cy chit
xut ly penicillin acylase ri c nh enzyme trn polyacrylamide. 2. K
thut sn xut khng sinh: Cng ngh ln men sn xut penicillin mang nt c
th ring ca tng c s sn xut v cc thng tin ny rt hn ch cung cp cng
khai, ngay mi bng sng ch thng cng ch gii hn nhng cng on nht nh; v
vy rt kh a ra c cng ngh tng qut chung. Theo cng ngh ln men ca hng
GistBrocades (H Lan), ton b dy chuyn sn xut thuc khng sinh
penicillin c th phn chia lm bn cng on chnh nh sau: Ln men sn xut
penicillin t nhin (thng thu penicillin V hoc
penicillin G) . X l dch ln men tinh ch thu bn thnh phm
penicillin t
nhin.
- 30
Sn xut cc penicillin bn tng hp (t nguyn liu penicillin t
nhin) Pha ch cc loi thuc khng sinh penicillin thng mi
Qui trnh sn xut khng sinh penicillin
2.1
Sn xut penicillin G t nguyn liu t nhin
Phng php sn xut penicillin t nhin l sinh tng hp t nm mc P.
chrysogenum.
- 31
Qu trnh sinh tng hp penicillin nm mc P. chrysogenum c th tm tt
nh sau: t ba tin cht ban u l -aminoadipic, cystein v valin s ngng t
li thnh tripeptit -( - aminoadipyl) - cysteinyl - valin ; tip theo
l qu trnh khp mch to vng -lactam v vng thiazolidin to thnh
izopenicillin-N; ri trao i nhm -aminoadipyl vi phenylacetic (hay
phenooxyacetic) to thnh sn phm penicillin G (hay penicillin V, xem
s tng hp penicillin G trong hnh.
Hnh: S c ch sinh tng hp penicillin t axit L- - aminoadipic,
L-cystein v L-valin. 2.1.1 iu kin ln men:
Nhit : Thng tin hnh nhn ging 300C, ln men 23 250C. pH: pH thch
hp trong khong 6,0 6,5. Trong qu trnh ln men pH mi trng thay i ty
thuc vo tc s dng cc cht cacbon v nit. n nh pH ngi ta cho vo CaCO3
vo mi trng len men.
- 32
Thng kh: P. chrysogenum l chng rt a kh nn trong qu trnh nui cy
cn thi kh ( i vi ln men b mt), lc hoc khuy km tho sc kh ( ln men
chm). Nhu cu cp kh khi c khuy trn lin tc l 1.2 1.5 VVM.
Thi gian: Trong 6-7 ngy.
- 33
2.1.2 Thnh phn mi trng ln men:
2.1.3 Quy trnh ln men: Trong nhng nm 40 ca th k XX vic sn xut
penicillin c thc hin bng phng php ln men b mt c th l c cht rn hoc
lng. C cht rn c th l cc loi ht hoc cm. ln men c cht lng ngi ta nui
trong cc chai l thy tinh c cha mi trng dinh dng. Vng mc sau khi ln
men c th dng cho ln men ln th 2 bng cch nui trong mi trng dinh dng
mi di lp vng. Qu trnh ny tin hnh 240C trong 6-7 ngy. Trong qu trnh
ln men cn thi kh v trng. Hin nay ngi ta s dng ln men chm sn xut ra
cc penicilin. Penicillin G l khng sinh u tin c sn xut bng cng ngh
ln men chm vo nm 1947.Qu trnh ln men to penicillin G c 2 pha: pha
sinh trng sinh- 34
khng sinh. pha ln men th nht nm pht trin h si mnh, sinh khi tng
nhanh, cht dinh dng ng ha nhiu, cng h hp tng dn n cc i, pH tng dn v
penicillin G c to thnh t. pha ln men th hai h si pht trin chm,
lactose c ng ha, pH tng n khong 7 7.5 v penicillin G c to thnh
trong khong ny. Hiu sut sinh tng hp ph thuc nhiu vo lng sinh khi
trong mi trng. 2.1.4 X l dch ln men v tinh ch thu nhn penicillin t
nhin:
Qu trnh tinh ch v tinh chit penicillin t mi trng ln men C 3 phng
php thu nhn v tinh ch penicillin t mi trng nui cy: Trch ly bng dung
mi hu c. Hp ph. Trao i ion. Phng php trch ly bng dung mi hu c c s
dng nhiu nht v c nhng u im: Mui ca penicillin rt tan trong nc.
- 35
Acid penicillin rt tan trong dung mi hu c. Chit penicillin trn
my ly tm siu tc bng dung mi hu c khng trn ln vi nc ( thng dng butyl
acetat). Bc hi chn khng n nng nht nh, thm than hot tnh ( 1%) vo ty
mu. Dch lc sau khi loi than c loi nc bng cch h nhit xung -200C. Kt
tinh penicillin G bng cch b sung trc tip vo dung mi sau khi ty mu
mt lng nh kali acetat ( hay natri acetat ) hoc ly trch sang dung
dch KOH long ( hay NaOH long), tin hnh c chn khng nhit thp, sau b
sung BuOH penicillin t kt tinh. 2.2 Sn xut penicillin bn tng hp t
penicillin G t nhin: V nguyn tc, to ra mt penicillin mi trong ln
men sinh tng hp c th dng phng php thay i mch nhnh bng cch s dng cc
tin cht khc nhau. Tuy nhin bng con ng ln men trc tip hin nay ngi ta
ch mi c kh nng tin hnh vi penicillin G v V. Cn trin khai trong sn
sut cng nghip, ngi ta s dng penicillin G hay V ch to ra 6-APA l
nguyn liu ch yu trong qu trnh bn tng hp ra cc penicillin mi. Theo
cu trc ha hc, cc penicillin t nhin u l nhng hp cht d vng ca cc acid
amin c tn gi l acid 6aminopenicillanic (6-APA). Vic tm ra phn t
6-APA m ra mt hng mi sn xut penicilin bn tng hp bng cch gn vo cc
mch nhnh khc nhau vo phn t 6-APA bng con ng ha hc hay sinh hc. C
hai hng chnh l acyl ha nhm NH 2 v tr s 6 v ester ha nhm COOH v tr s
3. Enzyme penicillinamidase ng vai tr trong vic thy phn penicillin
G hoc V to thnh acid 6- aminopenicillanic (6-APA) v acid phenyl
acetic. 6- APA l tin cht quan trng c s dng sn xut cc khng sinh h
-lactam hot ng cao. 6-APA c th sn xut bng phng php ln men cc chng
penicillinum. ng hc ca qu trnh ln men to 6-APA: ng ha hydratcarbon
nhanh, pH- 36
tng dn v trong giai on to 6- APA t 7.0 -7.4. Thi gian ln men ko
di t 108- 120 gi. Tuy nhin qu trnh ny cho hiu sut thp v 6-APA c to
thnh ng thi vi cc penicillin t nhin , mc khc chi ph cho qu trnh tch
chit cao. V vy trong thc t ngi ta khng s dng phng php ny. Hin nay s
dng enzyme c nh l phng php ph bin sn xut 6-APA trong cng nghip. S
dng enzyme penicillin acylase ct mnh nhnh ca penicillin G . Phng
php ny c nhiu u im, iu kin phn ng d dng nn mang li hiu qu kinh t
cao. Hin nay mt s hng dc phm p dng bin php ny, in hnh l hng dc phm
Snam Progetti (Italia) vi sn lng khong 40 tn/ nm. Phng php enzyme c
nh sn xut 6-APA cho hiu qu tng i cao (9095%) v c p dng rng ri nhiu
nc, mang li hiu qu kinh t cao. Hn 50% 6_APA trn th gii sn xut theo
phng php enzyme c nh. Hin nay mt s nh my sn xut penicillin ch cho
xut xng thnh phm 6-APA.
- 37
3. Cc sn phm khng sinh bn tng hp: Ty theo c ch tc dng cc
penicillin bn tng hp c th chia thnh 3 nhm chnh: Nhm 1: Ph hot tnh
hp nh cc penicillin t nhin, ch yu
tc dng ln vi khun Gram (+) nh lin cu, t cu khng c kh nng tit ra
enzym penicillinase v mt s vi khun Gram (-) nh lu cu. Thng dng ung,
i din cho nhm ny l phenethicillin v azidocollin.
- 38
Nhm 2: Ph hot ng hp nhng c u dim khng li
penicillinase nn dng iu tr cc bnh do vi khun khng li cc
penicillin nhm 1 gy ra. Nhm 3: Gm cc cht c ph hot ng mnh ln nhiu
loi vi
khun Gram (+) v Gram (-) v bn vng trong ng tiu ha. c dng iu tr
cc bnh nhim trng ng h hp, tit niu, sinh dc v nhim khun mu. Tuy nhin
chng c nhc im l nhanh chng b ph hy bi cc vi khun c kh nng sinh ra
penicillinase ( hay
-lactam ) nh amonicillin,
ampicillin. Nhm penicillin ny cng c gi l cc penicillin khng
pseudomonas v c chia theo cu trc thnh 2 nhm: nhm carboxypenicillin
( ticarcillin v carbenicillin) v cc nhm acylureido penicillin (
piperacillin v mezlocillizz) Cc khng sinh penicillin bn tng hp
- 39
III/ KT LUN Penicillin,khng sinh nhm beta-lactam,ngn chn s tng
hp thnh t bo, mt thnh phn quan trng ca thnh t bo vi trng l lp
peptidoglycan.- 40
Cc enzyme thnh t bo xc tc to ra phn ng cn thit cho cc chuyn i,
to thnh cc cu ni peptidoglycan,cn thit cho s tng hp thnh t bo.
Penicillin tc ng ln cc enzyme ny, ngn cn s tng hp mng t bo, lm t bo
b cht. Penicillin V v G tc ng rt hiu qu trn cc vi trng Gram dng.
Penicillin cn l loi thuc c chn dng trong cc trng hp nhim trng gm
vim hu hng do strepto,vim mng no do meningococcus,actinomycosis,
giang mai, v cc lu ,st thp khp Penicillin c thi ra rt nhanh nhng
ngi bnh nhn c chc nng thn bnh thng, v c a vo c th qua con ng tnh
mch nhng bnh nhn b nhim trng nng. tc dng ngc quan trng v ph bin nht
i vi Penicillin l d ng. \
- 41
Ti liu tham kho:1. Phm Th Trn Chu, Phan Tun Ngha Cng ngh sinh hc
Tp 3. Nh xut bn gio dc 2007.2. T Minh Kong K thut sn xut dc phm Tp
2. Nh xut bn y
hc 2004. 3. Mai Xun Lng - Gio trnh enzyme . i Hc Lt 2005. 4. c
Lng Cng ngh enzyme Tp 3. Nh xut bn 5. United States Patent, s
4554250,nov,19,1985 6. David w.spence and martin ramsden.glaxo
SmithKline 7. Attila Szentirmai,applided
microbiology,Vol.12,No.3,p. 185-187 may,1964.
42 -
