Nhp mn Cng ngh sinh hc 59

Chng 3

Cng ngh ln men vi sinh vt

I. M u
Cc c th vi sinh vt c kh nng sinh trng trn nhiu loi c cht
(mi trng dinh dng) khc nhau v c th sn xut nhiu sn phm
thng mi. Gn y, vic p dng cc k thut di truyn in vitro m
rng phm vi cc sn phm c sn xut bi vi sinh vt v cung cp cc
phng php mi tng sn lng ca nhng sn phm . Khai thc
thng mi s a dng ha sinh (biochemical diversity) ca cc vi sinh vt
thc y pht trin cng nghip ln men, v cc k thut di truyn
thit lp mt nn cng nghip u th to c hi pht trin cc qu trnh mi
v ci thin nhng qu trnh ang c.
Thut ng ln men (fermentation) trong cng ngh vi sinh c ngun
gc t ng t Latin fervere ngha l un si, m t s hot ng ca nm
men trn dch chit ca tri cy hoc cc ht ng cc c to mch nha
(malt) trong sn xut ung c ethanol. Tuy nhin, s ln men c cc
nh vi sinh vt hc v ha sinh hc gii thch theo cc cch khc. Theo cc
nh vi sinh vt hc thut ng ln men c ngha l qu trnh sn xut mt sn
phm bng nui cy sinh khi vi sinh vt. Tuy nhin, cc nh ha sinh hc
li cho rng l qu trnh sn sinh ra nng lng trong cc hp cht
hu c hot ng vi vai tr va l cht cho ln cht nhn in t, l qu
trnh ym kh m nng lng c sn xut khng cn s tham gia ca
oxygen hoc cc cht nhn in t v c khc.
Trong chng ny thut ng ln men c s dng theo ngha rng
ca n, gc vi sinh vt hc.

II. Sinh trng ca vi sinh vt
Sinh trng ca vi sinh vt c th to ra s trao i cht, nhng sn
xut mt cht trao i nh mong mun th c th ca chng phi c sinh
trng di nhng iu kin nui cy c bit vi mt tc sinh trng
c trng.
Nhp mn Cng ngh sinh hc 60
Nu vi sinh vt ch c a mt ln vo mi trng sinh trng, th
nui cy ban u (innoculated culture) s tri qua mt s giai on v h
thng ny c gi l nui cy m (batch culture). u tin, s sinh trng
khng xut hin v qu trnh ny c xem nh l pha lag, c th coi y l
thi k thch nghi. Tip theo l khong thi gian m tc sinh trng
ca t bo tng dn, cc t bo sinh trng vi mt tc cc i v khng
i, thi k ny c xem l pha log hoc pha sinh trng theo hm m v
c m t bng phng trnh:
x
dt
dx
=
(1)

Trong : x l nng t bo (mg/mL), t l thi gian nui cy (gi),
v l tc sinh trng c trng (gi). T phng trnh tch phn (1) ta
c:

t
t
e x x

0
=
(2)

Trong : x
0
l nng t bo thi im bt u nui cy v x
t
l
nng t bo sau mt khong thi gian t (gi).
Nh vy, ng cong logarithm t nhin ca nng t bo theo thi
gian t c dc bng tc sinh trng c trng. Tc sinh trng c
trng trong sut pha log t cc i cc iu kin nui cy thng thng
v c m t nh l tc sinh trng cc i c trng (
max
). Phng
trnh (1) v (2) b qua trng hp s sinh trng s lm tiu hao cc cht
dinh dng v tng tch ly c t ca sn phm. Tuy nhin, trong thc t
khi cht dinh dng b hao ht v cc sn phm c c tch ly, th tc
sinh trng ca t bo s khng t cc i v cui cng lm ngng qu
trnh sinh trng, lc ny nui cy i vo pha tnh v sau mt thi gian s i
vo pha cht, dn n gim s lng t bo sng st (Hnh 3.1).
Nh trnh by, hin tng ngng sinh trng trong nui cy m l
do hao ht thnh phn dinh dng hoc tch ly sn phm c. Tuy nhin,
c th khc phc iu ny bng cch b sung mt lng ti thiu mi
trng sch (mi) vo bnh nui. Khi mi trng mi c b sung lin tc
mt tc thch hp (h nui cy lin tc-continuous culture), th sinh
trng ca t bo trong h ny c iu chnh bng s sinh trng gii hn
v thnh phn ca mi trng, v vy h thng ny c xem nh l mt
Nhp mn Cng ngh sinh hc 61
chemostat (th n nh ha tnh). H thng nui cy lin tc cho php t
ti trng thi n nh (steady-state) v vic hao ht sinh khi t bo qua
dng chy ra (output) s c b p bi s sinh trng t bo trong bnh
nui.



Hnh 3.1. ng cong sinh trng c trng ca cc c th n bo trong
nui cy m

Dng chy mi trng qua h thng iu chnh vo bnh nui c
m t bi thut ng tc pha long (dilution rate), k hiu l D, bng tc
b sung mi trng trn th tch lm vic ca bnh nui. S cn bng
gia sinh trng ca t bo (growth) v s hao ht ca chng t h thng
ny c th c m t nh sau:
= dt dx/ growth output
hoc:
Dx x dt dx = /
Di cc iu kin trng thi n nh:
0 / = dt dx
v v th, Dx x = v D =


N

n
g

s
i
n
h

k
h

i

P
h
a

s
i
n
h

t
r

n
g

n
h
a
n
h

P
h
a

s
i
n
h

t
r

n
g

c
h

m

Pha
lag
Pha
log
Pha
tnh
Pha
cht
Thi gian
Nhp mn Cng ngh sinh hc 62
K t y, tc sinh trng ca vi sinh vt c iu chnh bng tc
pha long, v y l mt bin thc nghim. iu ny lu rng di cc
iu kin nui cy m, mt c th s sinh trng tc sinh trng cc
i c trng ca n. V th, nui cy lin tc ch c th hot ng cc tc
pha long pha di tc sinh trng cc i c trng. Nh vy, trong
cc gii hn nht nh, tc pha long c th c dng iu chnh tc
sinh trng ca nui cy chemostat.
C ch v hiu qu iu chnh tc pha long l mi quan h gia
(tc sinh trng c trng) v s (nng c cht gii hn trong
chemostat) c chng minh bi Monod vo nm 1942:

s K
s
s
+
=
max

(3)

Trong : K
s
l hng s s dng hoc bo ha, bng gi tr ca nng
c cht khi bng 1/2 ca
max
. trng thi n nh, =D, v th:
s K
s
D
s
+
=
max

Trong : s l nng c cht trng thi n nh trong chemostat,
v:
D
D K
s
s

=
max

(4)

Phng trnh (4) cho thy nng c cht c xc nh bng tc
pha long. Trong thc t, iu ny xy ra do sinh trng ca t bo lm
tiu hao c cht ti mt nng cn thit tc sinh trng bng tc
pha long. Nu c cht b hao ht di mc cn thit th tc sinh trng
ph thuc tc pha long v mt lot cc kh nng c th xy ra nh sau:
- Tc sinh trng ca t bo km hn tc pha long v chng s
b ra tri khi bnh nui mt tc ln hn tc m chng ang c
sn xut, kt qu l lm gim nng sinh khi t bo.
- Nng c cht trong bnh nui s tng ln do cc t bo c li
t hn trong bnh nui tiu th n.
- Nng c cht c tng ln trong bnh nui s cho kt qu cc t
bo sinh trng mt tc ln hn tc pha long v nng sinh khi
s tng.
Nhp mn Cng ngh sinh hc 63
- Trng thi n nh s c thit lp tr li.
Nh vy, chemostat l h thng nui cy t cn bng c gii hn
cht dinh dng, c th duy tr trng thi n nh trong mt phm vi rng
ca cc tc sinh trng cc i c trng.
Nui cy m c cung cp dinh dng (fed-batch culture) c xem l
h thng trung gian gia qu trnh nui cy m (batch) v nui cy lin tc
(continuous). Thut ng nui cy m c cung cp dinh dng c dng
m t cc nui cy m c cung cp dinh dng lin tc (hoc ni tip
nhau) bng mi trng mi m khng loi b dch nui cy c. Nh vy,
th tch ca loi nui cy ny tng ln theo thi gian. Pirt (1975) m t
ng hc ca h thng ny nh sau: Nu sinh trng ca mt c th b gii
hn bi nng ca c cht trong mi trng th sinh khi pha tnh, x
max
,
s c m t bi phng trnh:
R
YS x =
max

Trong : Y l yu t hiu sut, bng khi lng t bo c sn xut
trn mt gram c cht c s dng, v S
R
l nng ban u ca c cht
gii hn s sinh trng. Nu mi trng mi c b sung vo bnh nui
tc pha long km hn
max
th sau hu nh tt c c cht s c s
dng khi n i vo h thng:
Y
X
FS
R
=
Trong : F l tc dng chy v X l sinh khi tng s trong bnh
nui, v d: nng t bo c nhn ln bi th tch nui cy.
Cho d khi sinh khi tng s (X) trong bnh nui tng ln theo thi
gian th nng t bo (x) hu nh vn khng i; v vy 0 / = dt dx v
D = . Mt h thng nh th c xem l trng thi gn nh n nh
(quasi-steady-state). Khi thi gian v th tch nui cy tng ln, th tc
pha long li gim. Nh vy, gi tr ca D c a ra nh sau:
t
F V
F
D
+
=
0

Trong : F l tc dng chy, V
0
l th tch nui cy ban u, v t
l thi gian. ng hc Monod d bo rng khi D h xung th nng c
cht cn tha cng s gim v kt qu l lm tng sinh khi. Tuy nhin, trn
phm vi cc tc sinh trng hot ng th s tng sinh khi s khng c
Nhp mn Cng ngh sinh hc 64
ngha. S khc nhau gia trng thi n nh ca chemostat v trng thi
gn nh n nh ca fed-batch ch trong chemostat th D (k t y l )
l hng s cn fed-batch th D (k t y l ) li gim theo thi gian. Tc
pha long trong fed-batch c th c gi khng i bng cch tng
(theo hm m) tc dng chy nh s dng mt h thng iu chnh thng
qua computer.

III. Sinh khi vi sinh vt v cng ngh ln men
S ln men vi sinh vt c th c phn loi theo cc nhm chnh sau:
- Sn xut cc t bo vi sinh vt (sinh khi) nh l sn phm.
- Sn xut cc cht trao i ca vi sinh vt.
- Sn xut cc enzyme vi sinh vt.
- Sn xut cc sn phm ti t hp.

1. Sinh khi vi sinh vt
Cng ngh thu sinh khi vi sinh vt l cc qu trnh nui cy cc
chng thun khit hoc hn hp vi chng thu c khi lng t bo
sau khi sinh trng vi cc mc ch:
- Sinh khi giu protein dng lm thc phm cho ngi v thc n
cho gia sc l nhng t bo vi sinh vt (k c sinh khi to) sy kh v
cht, giu protein, cc vitamin nhm B v cht khong. Ngun sinh khi
ny c gi l protein n bo.
- Sinh khi nm men l nhng t bo sng dng trong cng nghip
bnh m-men bnh m, sinh khi vi khun lactic sng c hot tnh enzyme
tiu ha sn xut cc thuc h tr tiu ha nh biolactovin
- Sinh khi c nh m lm phn bn vi sinh, cc loi phn bn vi
sinh vi vi khun sng t do trong t v sng cng sinh vi cy h u.
- Sinh khi vi khun sinh c t i vi cc loi su thn mm ph
hoi rau mu, sn xut thuc tr su vi sinh.
- Sinh khi vi sinh vt c h enzyme phn gii cc cht hu c k c
thuc tr su v hydrocarbon sn xut cc ch phm vi sinh x l nc
thi v nhim trong bo v mi trng.

Nhp mn Cng ngh sinh hc 65
2. Qu trnh ln men
Hnh 3.2 minh ha cc phn ca mt qu trnh ln men tng qut.
Phn trung tm ca h thng l h ln men, trong c th c sinh
trng di cc iu kin ti u to thnh sn phm. Trc khi s ln
men bt u, mi trng phi c pha ch v kh trng, h ln men v
trng, v nui cy khi u phi c mt s lng vi sinh vt va trong
mt trng thi sinh l ph hp cy truyn vo h ln men sn xut. Kt
thc qu trnh ln men cc sn phm phi c tinh sch v x l thm.















Hnh 3.2. S chung ca mt qu trnh ln men

Cc c th vi sinh vt c th sinh trng trong kiu nui cy m (Hnh
3.3), nui cy m c cung cp dinh dng v nui cy lin tc. u im ca
nui cy lin tc i vi sn xut sinh khi l qu r rt (c th xem
nhng tnh ton sau) nhng i vi cc sn phm vi sinh khc th nhc
im ca n li ln hn u im k thut l c kh nng iu chnh ci
thin qu trnh ln men.

Nui cy
trong bnh
tam gic
c lc
Pht trin nguyn liu vi sinh vt
Nui cy
mu gc
H ln men kt ht
Kh trng mi trng
Pha ch mi trng
Nguyn liu chun b mi trng
H ln men sn xut
X l
cht thi
Dch
nui cy
Phn tch
t bo
Sinh khi
Th ni
v bo
Tch chit
sn phm
Tinh sch
sn phm
ng gi
sn phm
Nhp mn Cng ngh sinh hc 66












Hnh 3.3. Cu hnh c bn ca mt h ln men m

Hiu sut ca nui cy m c th c m t bi phng trnh:

ii i
0 max
batch
t t
x x
R
+

=
(5)

Trong : R
batch
l sn lng nui cy trong gii hn nng sinh
khi/gi, x
max
l nng t bo cc i t c pha tnh, x
0
l nng t
bo ban u lc gy nhim, t
i
l thi gian c th sinh trng
max
v t
ii
l
thi gian m c th khng sinh trng
max
bao gm pha lag, pha gim tc
, v cc thi k ca tng m, kh trng v thu hoch.
Hiu sut ca nui cy lin tc c th c biu din nh sau:
R
cont
=
|
.
|

\
|

T
t
x D
iii
1
(6)

Trong : R
cont
l sn lng ca nui cy trong gii hn nng t
bo/gi, t
iii
l thi gian trc khi thit lp trng thi n nh bao gm thi
gian chun b bnh nui, kh trng v hot ng trong nui cy m trc khi
hot ng lin tc. T l thi gian m cc iu kin trng thi n nh chim
u th, v x l nng t bo trng thi n nh.
4 x vch ngn
B phn phun kh
Sensor nhit
Cnh khuy
Trc khuy
Motor
in cc pH
in cc O
2
Nhp mn Cng ngh sinh hc 67
Sn lng cc i ca sinh khi trn mt n v thi gian (v d hiu
sut) trong mt chemostat c th t ti bng cch hot ng tc pha
long cao nht ca x D , gi tr ny c xem nh l D
max
. Hiu sut ln
men m, nh m t trong phng trnh (5), l mt gi tr trung bnh cho
thi gian tng s ca s ln men. Do dx/dt = x, nn hiu sut ca nui cy
tng ln theo thi gian, v nh vy, phn ln sinh khi trong qu trnh nui
cy m c sn xut gn phn kt thc ca pha log. Trong chemostat
trng thi n nh, hot ng (hoc gn) D
max
cho hiu sut duy tr khng
i, v t cc i cho s ln men ton phn. Cng nh vy, mt qu trnh
lin tc c th c hot ng mt thi gian rt lu sao cho thi k khng
sn xut, t
iii
trong phng trnh (6), c th khng c ngha. Tuy nhin, yu
t thi gian khng sn xut cho nui cy m l rt c ngha, c bit khi
h ln men c thit lp li nhiu ln trong sut thi gian vn hnh, v v
th t
ii
s ti din nhiu ln.
Bn cht ca qu trnh lin tc trng thi n nh cng c thun li
do n d dng iu chnh hn h ln men m. Trong sut thi gian ln men
m, sn lng nhit, s sn xut kim hoc acid, v s tiu th oxygen s
bin thin t cc tc rt thp lc bt u ti cc tc rt cao trong
sut pha log mun. V vy, iu chnh mi trng ca mt h thng nh th
kh hn nhiu so vi qu trnh lin tc m trng thi n nh cc tc
sn xut v tiu th l hng s.
Nhc im thng xuyn ca h thng nui cy lin tc l s mn
cm ca chng vi s nhim bn bi cc c th bn ngoi. Ngn cn s
nhim bn l vn hng u khi thit k h ln men, xy dng v vn
hnh, v phi c khc phc bi mt cng ngh tt.
Sn xut cc sn phm ph c kt hp vi s sinh trng (v d
nh ethanol) s hiu qu hn trong nui cy lin tc. Nhng vic ng dng
nui cy lin tc sn xut cc sn phm sinh tng hp ca vi sinh vt
(ngc vi s d ha) gp nhiu hn ch. Mc d, v l thuyt c kh
nng ti u mt h thng lin tc c th tng hiu sut ca s trao i
cht, tuy nhin kh nng n nh trong mt thi gian di ca cc h thng
nh th l rt kh khn do s thoi ha ca chng vi sinh vt. Kho st v
ng hc ca nui cy lin tc cho thy rng h thng ny l s chn lc
cao v thch hp cho vic nhn ging nhng c th thch nghi tt nht trong
Nhp mn Cng ngh sinh hc 68
nui cy. S thch nghi tt nht trong phm vi ny c xem l i lc ca c
th i vi c cht c gii hn tc pha long ang hot ng.
Mc d cng nghip ln men min cng chp nhn nui cy lin
tc sn xut cc cht trao i ca vi sinh vt, nhng nhng tin b rt
ng k li thu c trong s pht trin cc h thng nui cy m c cung
cp dinh dng. Nui cy m c cung cp dinh dng c th c s dng
t ti mt mc rt ng k ca s iu chnh qu trnh v m rng
thi gian sn xut ca qu trnh nui cy m truyn thng m khng c cc
nhc im c hu ca nui cy lin tc c m t trn. u im
chnh ca cung cp thnh phn mi trng vo nui cy l cht dinh dng
c th c duy tr nng rt thp trong sut qu trnh ln men. Nng
cht dinh dng thp c th thun li trong mt s mt sau:
- Duy tr cc iu kin nui cy trong phm vi kh nng thng kh ca
h ln men.
- Loi b cc nh hng khc nghit ca cc thnh phn mi trng,
v d nh s dng nhanh cc ngun nitrogen, carbon v phosphate.
- Trnh cc hiu qu c ca thnh phn mi trng.
- Cung cp mt mc gii hn cht dinh dng cn thit cho cc
chng d dng.

IV. Cc sn phm ln men vi sinh vt
1. Ln men ru
Ru c con ngi sn xut v s dng rt lu, vo khong
6.000 nm trc cng nguyn. Do nhu cu v li ch ca sn phm ny nn
n nay vic nghin cu v m rng sn xut chng ngy cng c quan
tm. C rt nhiu loi ru v mi loi u c thnh phn v quy trnh sn
xut khc nhau, c th tm chia thnh ba loi ch yu sau: Ru trng
(ethanol), ru vang (wine) v ru mi (liquor).

1.1. Ru trng
Ru trng c sn xut bng hai phng php chnh: phng php
ln men vi sinh vt v phng php ha hc. Tuy nhin, phng php ln
men vi sinh vt l phng php ch yu. y l qu trnh ln men ru ca
nm men v mt s vi sinh vt khc, trong nm men l i tng chnh
Nhp mn Cng ngh sinh hc 69
c s dng sn xut ru quy m cng nghip (Hnh 3.4). Ln men
ru l mt qu trnh phc tp chuyn ng thnh ru, c s tham gia
ca nm men trong iu kin ym kh. Phng trnh tng qut ca ln men
ru nh sau:
C
6
H
12
O
6
2C
2
H
5
OH + 2CO
2
+ 27 kcal
Quy trnh sn xut ru trng bng phng php ln men ru bi
nm men c thc hin qua cc bc sau: Ch bin nguyn liu thnh dch
ng, ln men bin ng thnh ru, chng ct v tinh ch ethanol.
Trong , ln men bin ng thnh ru l giai on quan trng nht trong
sn xut ru, quyt nh cht lng sn phm to thnh. Sau khi dch
ng ha c x l, ngi ta b sung thm mt s thnh phn cung
cp thm vitamin v amino acid nh mui ammonium, mui phosphate,
dch thy phn nm men. Mi trng c thnh phn nh trn c th s dng
ln men. Ging c s dng ch yu trong ln men ru l cc chng
nm men Saccharomyces cerevisiae c tc pht trin mnh v hot lc
ln men cao, ln men c nhiu loi ng khc nhau v c tc ln
men nhanh, c kh nng chu c ethanol cao t 10-12%.


Hnh 3.4. Nh my sn xut ethanol quy m nh

Nhp mn Cng ngh sinh hc 70
Mi trng ln men sau khi c kh trng cn c ng t 90-
120 g/L v pH trong khong 4,5-4,8. Thi gian ln men t 65-72 gi, trong
10 gi u c sc kh nm men sinh si ny n, sau cho ln men
tnh (ym kh). Qu trnh ln men ru qua cc bc sau: ng v cc cht
dinh dng ca mi trng ln men c hp th vo trong t bo nm men
qua mng t bo v tham gia vo qu trnh trao i cht, ru ethanol v
CO
2
to thnh lin thot ra khi t bo, ru ethanol tan tt trong nc do
vy n khuch tn rt nhanh vo mi trng chung quanh. Kt thc ln men
ru, sau khi loi b t bo nm men, mun c ru tinh khit cn
chng ct dch ln men loi b tp cht. K thut chng ct ru nh
hng rt ln n cht lng ru thu c.

1.2. Ru vang
,
- .
c (Hnh 3.5).



Hnh 3.5. Mt dy chuyn sn xut ru vang

Nhp mn Cng ngh sinh hc 71
2
ngn cn
cc phn ng
.
Sac.
ellipsoideus, Sac. cerevisiae, Sac. oviformis
bao gm ba g
, b
.
, gn
. Qu trnh
gn lc v ln men ph c th lp li nhiu ln c dung dch trong sut.

. ,
, do cn h th ru ni mt mt thi gian lu
ru c chn v c cht lng hon ho.

2. Sn xut enzyme
ng dng thng mi chnh ca cc enzyme vi sinh vt l trong cng
nghip thc phm v sn xut bia mc d enzyme c tha nhn trong
cc ng dng phn tch v chn on bnh, cng nh trong sn xut bt
git. Hu ht cc loi enzyme c tng hp trong pha log ca nui cy m
v c th, v th, c xem nh cc cht trao i s cp. Tuy nhin, trong
mt s trng hp amylase (Bacillus stearothermophillus) c sn xut
bi nui cy idiophase v th c th xem l tng ng vi cc cht trao
i th cp. Cc enzyme c th c sn xut t ng-thc vt cng nh
cc ngun vi sinh vt, nhng sn xut bng ln men vi sinh vt l phng
php kinh t v thch hp nht. Hn na, hin nay nh cng ngh DNA ti
t hp ngi ta c th chuyn gen vo cc t bo vi sinh vt sn xut cc
enzyme ca ng-thc vt (Hnh 3.6).
Nhp mn Cng ngh sinh hc 72
Cc tin b ca cng ngh DNA ti t hp m rng phm vi cc
sn phm ln men tim tng ca vi sinh vt. C kh nng a cc gen t cc
c th bc cao vo cc t bo vi sinh vt nh l cc t bo nhn tng hp
cc protein (bao gm enzyme) ngoi lai. Cc t bo vt ch dng trong
nhng trng hp ny l E. coli, Sac. cerevisiae v mt s loi nm men
khc.

2.1. Cc loi enzyme vi sinh vt
Trong qu trnh sinh trng, cc enzyme c hnh thnh trong t bo
v mt s c tit ra mi trng xung quanh. Trong sn xut ch yu l
sn phm ca enzyme ngoi bo, cn nu mun tch enzyme ni bo th
phi ph v t bo. Cc vi sinh vt c dng trong sn xut enzyme gm
c vi khun, nm mc, nm men v x khun.
Cc ch phm enzyme c sn xut t vi sinh vt c ng dng
trong nhiu ngnh cng nghip khc nhau, ch yu l cc enzyme thy
phn: amylase, protease, pectinase, cellulase



Hnh 3.6. Sn xut enzyme quy m cng nghip

2.1.1. Amylase nm mc
Nhiu chng nm mc c kh nng sn xut enzyme amylase.
Amylase nm mc c cc loi sau:
- o-amylase c tc dng thy phn tinh bt thnh maltose, glucose v
cc dextrin c phn t lng khc nhau.
Nhp mn Cng ngh sinh hc 73
- Glucoamylase c tc dng thy phn tinh bt, glycogen v
polysaccharide. Enzyme ny c dng trong sn xut ru, chuyn nhng
dextrin c phn t lng cao khng ln men thnh nhng hp cht ln men
c v do nng cao c hiu sut nu ru t cc nguyn liu l tinh
bt.
- o-glucosidase thy phn maltose thnh glucose.
- Dextrinase thy phn isomaltose, panose v dextrin thnh nhng loi
ng c th ln men c.

2.1.2. Amylase vi khun
Mt s vi khun c kh nng sinh ra nhiu enzyme o-amylase.
Amylase vi khun ch c kh nng phn hy tinh bt mnh v to thnh
nhng o-dextrin phn t lng cao bt mu vi iodine. Enzyme o-amylase
vi khun c dng trong sn xut ng mt ng v chocolate, trong sn
xut bia, ch bin dextrin vi dch ng sn xut thc n cho ngi gi
v tr em, trong sn xut nc qu v trong y hc.
Dextrinase nm mc v amylase vi khun cn c s dng rng ri
trong cng nghip dt v giy.

2.1.3. Protease
Protease l nhm enzyme thy phn cc lin kt peptide trong phn t
protein hoc cc polypeptide.
- Protease thy phn protein thnh cc peptide c phn t lng nh
(peptone v polypeptide). Tip theo l s phn hy cc peptide trn thnh
cc amino acid t do di tc dng ca peptidase.
- Protease c dng nng cao gi tr dinh dng ca tht c, thy
phn protein ca sa ch bin nhng mn n king c bit, c dng
trong thuc da, sn xut bt git, phim nh, t si, len d v trong y hc.
Protease vi sinh vt c th s dng cng vi amylase trong ch bin thc n
gia sc.

2.1.4. Pectinase
L nhm enzyme thy phn pectin to thnh galacturonic acid,
glucose, galactose, arabinose, methanol Pectinase c nhiu loi, nhng c
Nhp mn Cng ngh sinh hc 74
hai loi c nghin cu nhiu hn c l pectinesterase v
polygalacturonase.
- Pectinesterase c tc dng thy phn cc lin kt ester trong phn t
pectin, tch nhm metocyl to thnh methanol v polygalacturonic acid.
- Polygalacturonase thy phn pectinic acid v cc polygalacturonic
khc, tch cc gc D-galacturonic acid t do.

2.1.5. Cytolase
Vi sinh vt (c bit l nm mc) sn sinh ra h enzyme c hot tnh
cao c th phn hy hemicellulose, pentozan, lignin Cc enzyme ny
c gi chung l cytolase (bao gm cellulase, hemicelllulase, pentosinase).
Cellulase tc dng phn hy cellulose thnh cellobiose, ri sau tip
tc thy phn ti glucose. Vic phn lp cc chng vi sinh vt sn xut
cellulase c hot tnh cao v tch enzyme ny ra di dng tinh khit vn
cn gp nhiu kh khn. V vy, hin nay cha sn xut c enzyme ny
quy m cng nghip, song vic s dng n trong cc ngnh kinh t v cng
nghip c nhiu tim nng. V d cytolase c th dng trong cng nghip
bia phn hy cc v ht khng phi v mch, trong sn xut nc qu,
trong ch bin bnh m, trong cc qu trnh gia cng thc phm nng cao
gi tr dinh dng, cng nh trong sn xut thc n gia sc.

2.1.6. Invertase
Invertase ca nm mc v nm men u thy phn saccharose, nhng
c ch tc dng ca chng hon ton khc nhau. Invertase ca nm mc l
glucosidase, tc dng ln u glucose ca saccharose. Cn invertase ca
nm men l fructosidase, tc dng ln u fructose ca saccharose.
Invertase l enzyme ni bo v ch thot ra mi trng khi t bo b
phn hy. Enzyme ny c dng rng ri trong sn xut bnh ko, ru
mi, kem, mt ong nhn to. N lm tng v ngt khi thy phn ng
saccharose thnh fructose v glucose, lm tng ha tan ca saccharose
trong sn phm.

2.1.7. Enzyme oxy ha glucosooxydase-catalase
Glucosooxydase l enzyme oxy ha kh, ch tc dng ln |-D-glucose
khi c mt oxygen, n oxy ha glucose thnh gluconic acid v H
2
O
2
. Di
Nhp mn Cng ngh sinh hc 75
tc dng ca catalase (mt enzyme hay i cng vi glucosooxydase) H
2
O
2

s b kh thnh H
2
O v O
2
.
Glucosooxydase-catalase c th loi b oxygen khng kh khi mi
trng. V vy, chng c dng bo v nhng nguyn liu, vt liu
khc nhau trnh oxy ha bi khng kh. S dng nhng enzyme ny cho
php ko di thi gian bo qun thc phm (cc dch c c, cht bo, bia,
ru vang, nc ung, sa). ng thi chng cng c s dng rng ri
trong y hc t nm 1950 cha bnh.

2.2. Sinh tng hp enzyme cm ng
yme
v .
Asper. oryzae
phng p
-
-amylase.
Asper. awamori trn
-
.
-
a .
:
Nhp mn Cng ngh sinh hc 76
.
.
Mun tng hp c enzyme cm ng cn phi c bn iu kin:
- C gen tng ng trong th nhim sc ca t bo.
- C y cc nguyn liu xy dng cc phn t enzyme (cc
amino acid v cc hp cht coenzyme nu enzyme gm hai cu t).
- Nng lng cn thit dng cho vic tng hp enzyme.
- Cht cm ng, nu khng c cht cm ng th d c ba iu kin
trn cng khng th tng hp c enzyme.
Nh vy, c th coi vic c cht cm ng l iu kin rt cn thit
thu c nhng enzyme mong mun. Trong cng nghip sn xut enzyme
cn phi la chn nhng cht cm ng thch hp v xc nh nng ti u
ca n trong mi trng c hiu sut sinh tng hp cao nht.

2.3. Nhng phng php nui cy vi sinh vt sn xut enzyme
Cng ngh sn xut enzyme hin nay trn th gii ng dng hai
phng php: nui cy b mt v nui cy chm.
Trong nui cy b mt, vi sinh vt mc trn b mt mi trng rn
(Hnh 3.7) hoc lng. Cc mi trng rn trc khi nui cy vi sinh vt cn
c lm m. Vi sinh vt pht trin s ly nhng cht dinh dng trong mi
trng v s dng oxygen phn t ca khng kh h hp. m bo
cho vi sinh vt mc u trn b mt mi trng v s dng c nhiu cht
dinh dng sinh ra enzyme, nhng lp mi trng rn cn phi mng (ch
dy khong 2-5 cm). iu ny dn n mt nhc im c bn ca phng
php ny cn phi c mt bng sn xut ln v chi ph lao ng chn tay
nhiu.
Trong nui cy chm, vi sinh vt hiu kh ch s dng c oxygen
ha tan trong mi trng, v vy trong qu trnh nui cy phi sc kh v
khuy lin tc. Phng php nui cy chm hin i hn, d c kh ha v
t ng ha, vic t chc quy m ln tng i d dng v n gin. Vi
phng php ny c th dng cc chng vi sinh vt t bin c kh nng
sinh tng hp enzyme cao v la chn cc thnh phn mi trng thch hp,
cc iu kin nui cy ti u.
Nhp mn Cng ngh sinh hc 77
Phng php nui cy b mt trn mi trng rn cng c mt s u
im so vi phng php nui cy chm, l: nng enzyme to thnh
mi trng rn cao hn nhiu ln, khng cn cc thit b phc tp, ch yu
nui trn khay v bung nui gi nhit v m thch hp, qu trnh
sn xut tiu tn t nng lng. Trong phng php nui cy b mt vi sinh
vt c nui cy trong iu kin khng v trng tuyt i. Nu c vi sinh
vt tp nhim th ch cn loi b phn . Cn trong nui cy chm cn phi
gi v trng tuyt i trong tt c qu trnh, nu b nhim th h hng ton
b v c th phi b i hon ton. Khi nui cy chm khng nhng ch cn
v trng qu trnh nhn ging, ln men, m cn phi m bo v trng i
vi khng kh thi vo mi trng.


Hnh 3.7. Ln men trn mi trng rn. A: ln men k kh trong ni bng t
nung, B: ln men hiu kh.

2.3.1. Phng php nui cy b mt
Nui cy nm mc v mt s vi khun theo phng php b mt
sn xut enzyme thng dng mi trng rn, mt s trng hp c th
dng mi trng lng.
Mi trng rn thng l cc nguyn liu t nhin nh cm, i khi
dng go tm, ng, b bia, b c ci ng, khoai ty, li ng hoc hn
hp nhng nguyn liu ny. Mi trng lng thng l r ng, dch thy
phn t thc mm, nc b ru c pha thm mui khong.
m bo cc cht dinh dng trong mi trng ngi ta c th
b sung cc ngun N, P, K hoc cc cht sinh trng (nc khoai ty, cao
A B
Nhp mn Cng ngh sinh hc 78
ng). m 58-60% tng i thch hp vi nhiu chng nm mc nui
cy b mt trn khay h. Tuy nhin, m 60% vi khun d pht trin, d
gy tp nhim, kh thng kh. Trng hp m t 45-50%, khi nui cy
mi trng s kh nhanh, sinh bo t yu v lm gim hot tnh ca enzyme
to thnh. Trong thi gian nui cy, nn gi m ca mi trng 50-
60%, mun vy m khng kh phng nui cy phi khong 90-100%.
Tuy rng, nui cy b mt khng cn iu kin v trng tuyt i
nhng mi trng (c bit trong qu trnh nhn ging) cng cn c v
trng cho ging pht trin bnh thng nht l giai on u. Trong sn
xut cn phi v trng mi trng rn 1-1,5 atm bng hi nng trong 45-
60 pht. Nu mi trng trc khi v trng c trn vi chlohydric acid
hoc sulfuric acid n pH thch hp, hay thm mt t formalin hoc mt s
cht st trng khc th ch cn v trng di 0,2-0,3 atm. Thm acid v
gi mi trng pH nht nh s gip cho mt vi enzyme to thnh c
nhiu hn.
Mi trng c dn mng ra cc khay v trng dy khong 2-
2,5 cm, ngui ti 30
o
C th tin hnh cy ging. Ging c nhn cng
theo phng php b mt hoc bng bo t thu c theo phng php tch
bo t khi mi trng nhn ging v cha vo cc bnh nt kn hoc trong
cc ti polyethylene. Trong nui cy nhn ging thng mc pht trin
n gi sinh ra nhiu bo t. T l nhn ging khong 0,2-2%. Mi gram
bo t mc c th cy vo 10 kg mi trng. Cc khay c mi trng
cy mc c t vo phng nui c sn cc gi. Phng nui c th iu
chnh c nhit , m v c thng gi. Nhit thch hp vi a s
mc l 30-32
o
C, nu nhit xung di 24
o
C nm pht trin chm, sinh
bo t yu, thi gian nui cy di dn n gim kh nng sinh tng hp
enzyme. Thi gian nui cy nm mc khong 36-60 gi.
Qu trnh nui cy nm mc trn b mt mi trng bao gm ba thi
k:
- Khong 10-14 gi u. Bo t bt u ny mm, thi k ny cha
hnh thnh enzyme khng i hi phi thng kh nhiu, ch cn lm thong
khong 2-3 th tch khng kh/th tch phng/gi. Ging rt nhy cm vi
nhit nhng gi ny, nhit bung nui cn gi 29-31
o
C .
- Thi k gia khong 14-18 gi. Mc pht trin nhanh, h hp
mnh. Si nm c th quan st thy bng mt thng, lc u lp lng c
Nhp mn Cng ngh sinh hc 79
mu trng xm v ngy cng r, lm mi trng kt bnh li. C th phi lt
mi trng, b nh ra si nm mc tt hn. Cc cht dinh dng trong
mi trng tiu hao nhanh phc v cho qu trnh trao i cht trong t
bo v ging h hp mnh ta ra mi trng chung quanh 80-90 kcal/gi,
lm nhit mi trng c th tng ln n 37-40
o
C hoc hn. Thi k ny
cn phi thng kh mnh, ti 60 th tch kh/th tch phng/gi cung cp
O
2
cho mc v ui CO
2
ra khi mi trng, ng thi lm gim nhit
bung nui. Nhit bung nui giai on ny cn gi 28-29
o
C v
m trong phng khong 100%.
- Thi k cui khong 10-20 gi. Cc qu trnh trao i cht vn tip
tc nhng yu dn, nhit mi trng gim xung v vic to thnh
enzyme ca t bo vn tip tc. Nhit lng ta ra khong 15-30
kcal/kg/gi. Thng kh khng qu 20-25 th tch khng kh/th tch
phng/gi, gi nhit bung nui 30
o
C.
Ty thuc vo c tnh sinh l ca tng loi mc, thi gian nui cy
c th kt thc ti im m lng enzyme to thnh ti a.

2.3.2. Phng php nui cy chm
Nui cy vi sinh vt sinh enzyme theo phng php chm c thc
hin trong cc bnh ln men c cnh khuy v sc kh lin tc (Hnh 3.8 v
3.9). Qu trnh tng t nh trong sn xut amino acid, khng sinh
Khng th c mi trng nui cy chung cho tt c cc chng vi sinh
vt, v vy cn phi chn thnh phn mi trng, t l cc cht dinh dng
sao cho thch hp vi tng chng, c bit phi ch ti cht cm ng cn
thit cho vi sinh vt sn sinh ra enzyme mc ti a. Trong nhiu mi
trng nui cy chm,
Aspergillus,
.

Nhp mn Cng ngh sinh hc 80











Hnh 3.8. Ln men bng phng php nui cy chm trong mi trng lng
quy m phng th nghim (5 L)

Thnh phn khong trong mi trng cng rt c ngha. Trong mi
trng nui cy mt s chng Asper. oryzae ngoi tinh bt v nitrate cn
cn thm MnSO
4
. Nu thiu MnSO
4
mc vn pht trin bnh thng, nhng
amylase khng c to thnh (trong phn t amylase c cha nhng amino
acid mang S v Mn). Mi trng c v trng trong thit b ring hoc
trong bnh ln men 121-125
o
C/45-60 pht. Mi trng trc khi v trng
cn c dch ha s b trnh tnh trng tinh bt h ha lm mi trng
c st hoc c dnh cao.
Sau khi lm ngui mi trng n nhit thch hp s tin hnh tip
ging. Ging c cy t ng nghim qua cc bnh tam gic, t trn my
lc, ri nui bnh nhn ging c th tch bng 5-10% th tch bnh ln men
t 24-36 gi. Nh vy, n
. Cy ging mc bo
t theo phng php chm s ko di thi gian ny mm v cng ko di
ton b qu trnh nui cy. Mi trng nhn ging c th dng cc hp cht
nitrogen d tiu i vi vi sinh vt m trong qu trnh nui cy vn nng cao
c hot tnh sinh tng hp. T l tip ging nm trong khong 2-5%,
nhng mt s chng t l ny thp hn nhiu (0,5-0,6%).
Nhp mn Cng ngh sinh hc 81
Sinh tng hp enzyme theo phng php nui cy chm thng
khong t 2-4 ngy. a s cc enzyme thy phn do nm mc, x khun to
thnh c tit ra mi trng xung quanh, phn cn li trong h si sau ba
ngy nui cy khong 10-15%. pH mi trng c mt ngha rt ln,
pH thch hp cho sinh tng hp o-amylase l 7-8, glucoamylase l 4,5-5.
Khi dng cc mui ammonium lm ngun nitrogen, qu trnh pht trin vi
sinh vt s acid ha mi trng cn khi dng nitrate lm ngun nitrogen
mi trng s b kim ha.



Hnh 3.9. Ln men bng phng php nui cy chm trong mi trng lng
quy m pilot (200 L)

S sc kh khng nhng nh hng n sinh trng ca vi sinh vt m
cn nh hng n s to thnh enzyme. Tc s dng oxygen cao nht
ca nm mc sau khong 24 gi nui cy ri gim dn. Tng nng tt c
cc cht dinh dng v oxygen ha tan trong mi trng c th nng cao
c kh nng sinh tng hp o-amylase.

Nhp mn Cng ngh sinh hc 82
2.4. Tch v tinh sch ch phm enzyme
2.4.1. Ch phm enzyme t mi trng nui cy b mt
chit rt enzyme t mi trng rn ngi ta dng nc, cc dung
dch mui trung tnh, cc dung mi hu c (ethanol, acetone). Nhiu nghin
cu cho thy dng nc c kt qu tt v d p dng trong sn xut. C th
chit c lng enzyme trn 90-95% v trong nc chit khng cha cc
tp cht khng tan. Nc thng dng khuch tn ha tan nhit 25-
28
o
C. trnh tp nhim nn thm vo nc mt t formalin hoc cht st
trng khc. Dch chit thu c c mu nu sm, kh trong, cha 10-15%
cht kh ha tan v c lm lnh kp thi xung 10-12
o
C.
Dch chit c c c chn khng ti 50-55% cht kh ha tan. Dch
m c ny c th bo qun lu di m khng mt hot tnh v rt d ha
tan. Dch chit c th khng cn phi c c m a ngay vo my sy phun
v s thu c sn phm dng bt.
Phng php tch chit v lm sch enzyme c s dng rng ri
nht hin nay l phng php kt ta enzyme bng dung mi hu c
(ethanol, isopropanol v acetone). Cc dung mi hu c ny lm gim hng
s in mi ca mi trng. Nh ta bit, lc ht tnh in t l nghch
vi hng s in mi. V vy, cc enzyme-protein cng nh cc cht c
phn t lng thp trong h dung dch nc-dung mi hu c s kt ta v
lng xung. ha tan ca enzyme vo dung dch ethanol-nc ph thuc
vo nng ethanol, nhit , pH, lc ht ion ca dung dch v tnh cht
protein ca enzyme. Thng thng, ngi ta thm 3-4 th tch ethanol vo
mt th tch nc chit enzyme. trnh mt hot tnh ca enzyme tt c
phi c lm lnh xung 3-5
o
C. Khi trn phi khuy mnh, khi cc
enzyme kt ta v lng xung dui cn tch ly tm ngay. Enzyme c ra
2-3 ln bng ethanol cao , ri a vo bnh ht m hoc my ng kh
chn khng, sn phm thu c s c dng bt. Dng isopropanol kt ta
enzyme ch cn 1,5-2 th tch dung mi/1 th tch dch chit. Cc enzyme
tch ra s dng sa c qunh rt kh sy. Dng acetone vi t l nh khi
dng isopropanol, nhng k thut phng trnh chy n trong sn xut l rt
kh khn.
Phng php th hai tch enzyme l dng mui trung tnh kt
ta. Ch phm enzyme thu c c hot lc cao hn so vi vic tch bng
dung mi. Mui trung tnh thng dng l (NH
4
)
2
SO
4
vi t l 50-66% (c
Nhp mn Cng ngh sinh hc 83
khi cao hn) so vi dch chit enzyme. (NH
4
)
2
SO
4
pha thnh dung dch bo
ha ri cho vo dch ln men. Dch ln men c th s b c c trong thit
b chn khng v nh vy s cn dng mt lng (NH
4
)
2
SO
4
t hn. Ch
phm enzyme thu c c ln (NH
4
)
2
SO
4
, v vy mun s dng rng ri cn
phi loi mui bng cch thm tch qua mng bn thm.

2.4.2. Ch phm enzyme t dch nui cy chm
Dch nui cy chm sau khi lc sinh khi vi sinh vt v cc tp cht
rn khng tan cn khong 1-3% cht kh ha tan, trong c cc enzyme.
V nguyn tc tch enzyme t dch lc nui cy chm cng tng t nh
tch t dch chit trong mi trng rn nui cy b mt. Dch lc cn phi
c gim th tch t 4-10 ln trong iu kin chn khng 25-30
o
C, ri
tin hnh tch enzyme.
Ngoi phng php c chn khng, c th tin hnh theo phng
php hp ph qua nha trao i ion hoc cc cht c hot tnh b mt, sau
li tin hnh phn hp ph. Tin hnh nhiu ln v dch thu c cha o-
amylase ca nm mc v vi khun c th c hp ph li bng tinh bt
khoai ty hoc ng c x l s b bng nhit. Trc khi cho hp ph
cn cho thm 20% (NH
4
)
2
SO
4
nng cao kh nng hp ph ca tinh bt.
Tr amylase c hp ph cn cc enzyme khc s li trong dch. Tinh
bt c amylase c sy kh (khng cn phn hp ph) v em s dng
trong k thut sn xut cc sn phm cha tinh bt.
Hin nay, cn mt s phng php tng i phc tp khc kt
tinh v tch enzyme nh lc gel (gel filtration), in di (electrophoresis),
siu ly tm (ultracentrifuge)

3. Sn xut khng sinh
3.1. Penicillin
Penicillin l khng sinh c tm ra u tin v c sn xut sm
nht dng cha mt s bnh nhim khun vo nhng nm u ca Th
chin th 2.
Nhng vi sinh vt sinh penicillin thuc cc ging nm mc Penicillum
v Aspergillus. Nhng cc chng ca Penicillum notatum v Pen.
chrysogenum c hot lc cao v c dng trong cng nghip khng sinh.
Tuy nhin, nhng chng Penicillum c hot lc cao li thng km n nh.
Do , mt vn kh khn c t ra l to c kh nng sinh khng
Nhp mn Cng ngh sinh hc 84
sinh cao nht, gi c n nh trong qu trnh nghin cu v sn xut.
Nhim v ny c mt ngha rt ln trong cng nghip. Ngy nay, nh k
thut di truyn hc ngi ta to c nhng ging n nh, t nht sau
su th h vn khng gim hot tnh khng sinh.
Qu trnh ln men penicillin thuc vo loi ln men hai pha: pha sinh
trng v pha sinh penicillin. Ngun carbon trong ln men penicillin bng
nm Pen. chrysogenum c th l glucose, saccharose, lactose, tinh bt,
dextrin, cc acid hu c (lactic, acetic, formic), cc amino acid... Tuy nhin,
ng lactose cho hiu sut penicillin cao nht v thng c dng trong
cng nghip. Nhng do nm s dng ng lactose chm, v vy trong thc
t lactose c dng phi hp cng vi ng khc (glucose, saccharose...)
trong mi trng dinh dng.
Trong pha ln men th nht ging pht trin mnh, s dng glucose
v lactic acid ca cao ng. Sau , lactose mi c s dng (ch yu trong
pha th hai to thnh penicillin). Khi trong mi trng cn lactose v khng
b sung cc cht dinh dng, h si nm s bt u t phn, nu tip tc ln
men nng penicillin s gim.Trong thc t sn xut cn phi kt thc ln
men trc thi im ny.

Phng php sn xut penicillin
Sn xut penicillin cng nh cc ch phm sinh hc khc, da trn c
s nui cy vi sinh trn mi trng rn hoc lng. Trong qu trnh nui cy,
ging vi sinh vt pht trin s tch t cc sn phm trao i cht trong mi
trng hoc trong sinh khi. Quy trnh cng nghip sn xut penicillin da
trn nm mc Pen. chrysogenum c kh nng sinh penicillin cao, theo hai
phng php:
- Ln men b mt. Phng php ny c p dng trong thi gian u
ca cng nghip khng sinh. Mi trng nui cy b mt c th l cc c
cht rn hoc lng. (1) C cht rn c th l cm hoc cc loi ht, cm
c lm t ri tri ln khay mt lp dy khong 2 cm, ging nm mc
c trn vo mi trng c m 50-60% v trng ngui ti 30
o
C.
Thi gian ln men 6-7 ngy 24-28
o
C trong cc bung c iu chnh
nhit , m v thng gi. Ni chung, phng php ny ging nh ln
men cc enzyme bng nm mc. (2) Mi trng lng dng ngun carbon l
lactose, cao ng v mt s nguyn t khong. Ging c cy vo mi
trng ri ln men 24
o
C khong 6-7 ngy t hiu sut khong
Nhp mn Cng ngh sinh hc 85
193 unit/mL penicillin. Ngy nay, phng php ln men chm thay th
phng php ln men b mt.
- Ln men chm. Thnh phn mi trng gm c cao ng, glucose,
lactose v cc mui khong. Ging dng trong cng nghip thng dng
bo t. Bo t c nui trn cc bnh nhn ging c cnh khuy v sc kh
36-50 m
3
/gi h si nm pht trin, sau chuyn vo cc bnh ln men.
Qu trnh ln men penicillin bng nm mc Pen. chrysogenum 261
o
C
trong khong 120-125 gi. Trong qu trnh ln men pha th nht nm pht
trin h si mnh, sinh khi tng nhanh, cc ngun carbon d ng ha
(glucose, saccharose) cng cc ngun nitrogen c tiu hao nhanh, cng
h hp tng dn n cc i, pH tng v penicillin c to thnh t.
Sang pha th hai h si nm pht trin chm, lactose c tiu hao dn, pH
tng n khong 7-7,5 v penicillin c to thnh ch yu trong pha ny.
Nu ngun carbon trong mi trng cn v sinh khi nm mc bt u t
phn th pH c th tng ti 8 hoc hn, lng penicillin c to thnh
trong mi trng s gim. V vy, qu trnh ln men cn c kt thc trc
thi im h si nm mc bt u t phn. Nm mc sinh penicillin rt hiu
kh, cho nn qu trnh nui cy (nhn ging v ln men) cn phi sc kh v
khuy mi trng m bo ha tan oxygen cn bng vi nhu cu sinh
l ca chng. Nu khng oxygen hiu sut penicillin c th gim ti hai
ln.

3.2. Streptomycin
Streptomycin l mt khng sinh dng ph bin trong y hc, th y v
bo v thc vt. Schatz v cs (1944) pht hin ra streptomycin t dch
nui cy mt chng x khun Streptomyces griseus (cn gi l Actinomyces
streptomycin).
Ging x khun sinh streptomycin khi nui cy chm pht trin thnh
hai pha:
- Pha th nht (pha sinh trng mnh). Cc bo t ny chi v mc
thnh si sau 6-8 gi, mi bo t mc mt chi, khun ty thng thng v
phn nhnh rt yu, t bo cht a kim.
- Pha th hai (khun ty khng pht trin). Cui ngy th ba si x
khun b chia nh v bt u t phn.
Nhng ging sinh streptomycin rt khng n nh. Do , trong tng
lai cn c s can thip ca k thut di truyn to ra nhng ging c hot
lc cao v n nh a vo sn xut. Gi bo t dng ng kh trong
Nhp mn Cng ngh sinh hc 86
khong nm nm c th cn 96-99% hot lc, trong ct thch anh ti ba
nm, trn mi trng thch nc u 5
o
C ti mt nm. Cc ngun carbon
m ging Streptomyces c th ng ha c v sinh khng sinh l glucose,
tinh bt, dextrin, maltose, fructose, galactose, manose. Trong thc t,
glucose v tinh bt c dng lm ngun nguyn liu trong sn xut
streptomycin.

Phng php sn xut streptomycin
Ln men streptomycin c thc hin theo phng php nui cy
chm. Qu trnh ln men ny cng ging nh ln men cc loi khng sinh
khc, bao gm cc giai on: Nhn ging v ln men chnh.
- Nhn ging. Ging x khun c bo qun dng bo t. Cy bo
t vo mi trng nhn ging trong bnh tam gic lc 180-220 vng/pht
26-28
o
C/30-70 gi, sau cho tip vo cc bnh nhn ging (c sc kh v
khuy), nui tip cho pht trin sinh khi 20-40 gi. Nhim v chnh trong
giai on nhn ging l to ra mt khi lng ln khun ty x khun a
kim c kh nng pht trin mnh trong giai on ln men chnh v to
thnh mt lng ln khng sinh.
- Ln men. Ln men streptomycin l qu trnh ln men hai pha in
hnh. Nhit ln men khong 26-28
o
C, thi gian ln men 96 gi. Trong
thi gian ln men cn phi thng kh v khuy trn mi trng. Lng
khng kh thi qua mi trng trung bnh l 1 th tch kh/1 th tch mi
trng. Khuy trn mi trng lin tc trong sut c qu trnh ln men (k
c khi nhn ging) nu ngng khuy ch trong mt thi gian ngn s lm
gim hiu sut streptomycin. pH trong nhng gi u c gim cht t sau
tng dn.

3.3. Tetracycline
Tetracycline l mt dy cc cht khng sinh c cng mt nhn chung
tetracycline (v d: tetracycline, chlotetracycline, oxytetracycline,
dimethyltetracycline) v mt s nhm chung c trong phn t (v d
nhm dimethylamino -N(CH
3
)
2
, nhm amide CONH
2
). Tetracycline c
dng rng ri trong y hc v th y. Tetracycline c th c sn xut bng
ln men x khun Streptomyces aureofaciens. Tetracycline c tm thy
vo nm 1953 bng cch kh halogen trong phn t chlotetracycline. Lc
u phng php ny c dng trong cng nghip nhng gi thnh sn
Nhp mn Cng ngh sinh hc 87
phm rt t, sau ngi ta tm thy cht khng sinh ny c trong dch
nui cy x khun sinh chlotetracycline l Strep. aureofaciens.
Ging x khun c kh nng tng hp tetracycline v chlotetracycline
l Strep. aureofaciens, cn ging sinh oxytetracycline l Strep. rimosus.
Ngun carbon dng trong nui cy Strep. aureofaciens l glucose (tch t
nhiu tetracycline), cn Strep. rimosus cho nhiu oxytetracycline trn mi
trng maltose.
Trong qu trnh ln men, pha th nht cc cht dinh dng tiu hao
nhanh. Trong khong 24-48 gi nui cy khi khun ty c 70-80%
mc ti a v 60-80% cc cht dinh dng c s dng. Bc sang pha
ln men th hai cc ging x khun ny u pht trin chm li, tc s
dng cc cht dinh dng gim i rt nhiu, pht trin khun ty chm li
dn, t ti mc cc i v n nh ri bc vo giai on t phn.
Khng sinh tch t ti a 110-120 gi.

Phng php sn xut tetracycline
Ln men tetracycline (tetracycline, chlotetracycline, oxytetracycline,
dimethyltetracycline) theo phng php nui cy chm. Qu trnh ln men
y ging nh ln men cc ch phm khc, bao gm cc giai on: nhn
ging v ln men.
- X khun Strep. aureofaciens dng trong ln men tetracycline v
chlotetracycline hoc cc halogentetracycline khc. Cy bo t vo mi
trng nhn ging trong bnh tam gic pH 6,8-7,0 lc 220-250 vng/pht
khong 24-40 gi. Sau , c tip tc nhn ging trong ni nh ri
chuyn vo mi trng ln men. Ln men tetracycline v chlotetracycline l
ln men hai pha in hnh.
- Ging Strep. rimosus c nhn ging bnh tam gic lc 220-250
vng/pht 27-28
o
C/48-72 gi, sau nhn tip tc trong ni c sc kh v
khuy ri chuyn sang mi trng ln men c iu kin tng t nhng ko
di t 5-7 ngy.

4. Sn xut acid hu c
4.1. Acetic acid
Acetic acid (CH
3
COOH) c th thu c bng phng php ln men
vi khun acetic. Acid ny (cn gi l dm n) c dng trong ch bin
Nhp mn Cng ngh sinh hc 88
thc phm, p chua rau qu. Qu trnh ln men nh vi khun acetic oxy
ha ru ethanol thnh acetic acid (Hnh 3.10).
C trn 20 loi vi khun c kh nng ln men acetic, chng c gi
mt tn chung l vi khun acetic. Trong mi trng ru ethanol (5-
13%) th sn phm ch yu l acetic acid, nu nng ru thp hn cc vi
khun acetic s oxy ha trit ru thnh CO
2
v H
2
O.
Vi khun acetic l bn a m v rt hiu kh, c tc sinh trng rt
nhanh t mt t bo sau 12 gi c th pht trin thnh 12 triu t bo. Trong
qu trnh sinh trng v pht trin chng to thnh acetic acid v nng
acid thp li kch thch s sinh trng ca chng. V vy, trong sn xut
dm c th dng ru khng cn v trng c b sung mt t acetic
acid ha mi trng, nhit ln men khong 25-32
o
C v sc kh mnh.










Hnh 3.10. Qu trnh ln men acetic acid

Cc loi vi khun acetic c gi tr nh: Acetobacter aceti, Ace.
pasteurianum, Ace. orleaneuse, Ace. xylium, Ace. schiitzenbachii, Ace.
curvum, Ace. suboxydans.
Ngun c cht ch yu trong ln men acetic l ethanol c b sung
thm mt t ng, ngun nitrogen v c hoc hu c, v mt s cht
khong khc. C ba phng php ln men acetic: (1) Phng php chm
cn gi l phng php Orlean hoc phng php Php, dng nc hoa qu
lm nguyn liu vi vi khun Ace. orleaneuse. (2) Phng php nhanh cn
gi l phng php c, phng php ny c p dng ch yu trong
Alcohol-dehydrogenase
CH
3
CH
2
OH CH
3
CHO + 2H
(Ethanol) (Acetaldehyde)
OH

CH
3
CH CH
3
COOH + 2H
(Acetic acid)
OH
(Acetaldehyde)
Aldehyde-dehydrogenase
Nhp mn Cng ngh sinh hc 89
cng nghip sn xut dm n trn th gii, vi khun c s dng l Ace.
schiitzenbachii hoc Ace. curvum. (3) Phng php ln men chm, y l
kiu ln men bn lin tc s dng cc chng vi khun ca loi Ace.
suboxydans.

4.2. Citric acid
Citric acid hay limonic acid (C
6
H
8
O
7
) c nhiu trong thin nhin, c
bit trong cc loi cy n qu c mi (h cam chanh-Rutaceae) c dng
ch yu trong ch bin thc phm v dc phm. Citric acid cng c th
c sn xut quy m cng nghip bng phng php ln men, nm mc
s chuyn ha ng thnh citric acid.
C ch sinh tng hp citric acid vi sinh vt c th biu din bng
phng trnh tng qut nh sau:
2C
6
H
12
O
6
+ 3O
2
2C
6
H
8
O
7
+ 4H
2
O
Cc nm mc sinh citric acid hiu kh, nhit thch hp cho pht
trin v ln men l 30-32
o
C. Ngun carbon tt nht i vi Asper. niger l
saccharose, cn i vi Citromyces l maltose. Nng ng trong mi
trng 10-20% l thch hp hn c. Cc ngun nitrogen v c dng trong
ln men citric acid tt nht l nitrate cn nitrogen hu c l nc chit u
nnh. Trong mi trng ln men cn ch cc nguyn t khong P, Mg, K,
Fe v Zn.
C hai phng php c dng sn xut citric acid l ln men b
mt (trn mi trng lng hoc rn) v ln men chm.
- Phng php ln men b mt trn mi trng lng. Phng php
ny c dng rng ri trong cng nghip sn xut citric acid. Lp vng
nm pht trin trn cc khay ln men cha mi trng dinh dng l dch
ng s chuyn ha ng thnh citric acid.
- Phng php ln men chm. Qu trnh ln men ging nh sn xut
khng sinh, c thc hin trong cc bnh ln men cha mi trng dinh
dng v ging nm mc. Sau khi kt thc ln men dng H
2
SO
4
chuyn
calcium citrate thnh citric acid.

V. Cng ngh ti t hp vi sinh vt
Khng c mt lnh vc no ca cng ngh sinh hc thc nghim li
pht trin nhanh chng nh cng ngh di truyn (genetic engineering) hay
Nhp mn Cng ngh sinh hc 90
cn gi l cng ngh DNA ti t hp (DNA recombinant technology), cng
khng c mt lnh vc no khc c th a ra nhiu loi sn phm mi v
hu ch n nh vy. Nguyn l c bn ca cng ngh ny l thao tc c
nh hng v c ch (a vo hoc loi b) DNA v cc loi nguyn liu
di truyn khc nhm lm thay i c tnh di truyn ca c th sinh vt.
Hu ht cc k thut u bt u bng s la chn mt gen mong
mun, tip theo l phn lp n v ct n bng cc enzyme hn ch. Gen ny
c gn vo mt vector to dng (plasmid) v sau a vo mt vt ch,
n s c dch m thnh mt protein c bit.

1. Cc vi sinh vt ti t hp
Mt trong nhng ng dng u tin ca cng ngh di truyn l to ra
mt chng Pseudomonas syringae. Chng hoang di (wild type) ca vi
khun ny thng thng cha mt gen to bng, gen ny kch thch s to
bng trn cc b mt lnh v m. S bin i gen ny to ra mt th ti t
hp c kh nng ngn nga s to thnh bng gi (frost). c to ra di
tn gi l Frostban, vi khun ny mang li mt thnh cng khim tn
trong vic chng li vic to thnh bng gi trn cc cy du ty v khoai
ty ngoi ng rung. Trong mt th nghim khc, mt chng virus c kh
nng dit su o bp ci c th vo mt tha rung bp ci. N c
thit k c th t ph hy sau mt thi k nht nh. Pseudomonas
fluorescens c ti t hp vi cc gen sn sinh cht dit cn trng sinh
hc Bac. thuringiensis. Cc vi khun ny c th vo t, chng s bm
vo r v gip tiu dit cc cn trng ang tn cng.
Ni chung, mi s phng thch cc th ti t hp vo mi trng u
phi c cc c quan bo v mi trng chun y v c gim st cht
ch. n nay, nhiu nghin cu cho thy cc vi sinh vt ny khng sng
st hoc sinh si trong mi trng v chc chn khng gy ra nhiu him
ha. Cc virus c thit k di truyn c bit c ch trong cc ng dng y
hc, chng hn sn xut vaccine.

2. Cc ng dng trong cng ngh vi sinh
Mt iu lm cng ngh di truyn tr nn c bit hp dn l cc k
thut ca n c th kt hp vi cc k thut ca cng ngh ln men sn
xut ra nhng s lng ln cc cht ging khng sinh hoc steroid. Hu ht
Nhp mn Cng ngh sinh hc 91
cc protein ti t hp ang bn trn th trng hin nay u rt hu ch trong
y hc. Di y l mt s trong cc sn phm ny:
- Insulin, cht thay th hormone dng trong iu tr bnh tiu ng
type I.
- Hormone sinh trng ca ngi, c s dng iu tr nhng tr
em b bnh ln hoc bnh gi trc tui.
- Interferone, mt cht min dch c s dng iu tr mt s loi
ung th, vim gan mn tnh.
- Interleukin-2, mt cht hot ha t bo T v B c dng trong iu
tr ung th.
- Erythropoietin (EPO), mt cht kch thch cc t bo ty sng sinh
hng cu, dng iu tr mt s bnh thiu mu.
- Hot t plasminogen ca m (tPA), mt enzyme tham gia vo qu
trnh lm tan cc cc mu ng (huyt khi).
- Nhn t VIII, mt protein gy ng mu cho nhng ngi a chy
mu.
- Cc vaccine ti t hp cho bnh vim gan, v bnh vim mng no
do Hemophilus influenza B gy ra.
- Octolon, mt cht c ch min dch dng cho cc bnh nhn cy
ghp ni tng.

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1. Trn Th Thanh. 2003. Cng ngh vi sinh. NXB Gio dc, H Ni.
2. Nguyn Vn Uyn v Nguyn Tin Thng. 1999. Nhng kin thc c
bn v cng ngh sinh hc. NXB Gio dc, H Ni.
3. Asenjo JA and Merchuk JC. 1995. Bioreactor System Design. Marcel
Dekker Inc. New York, USA.
4. Bains W. 2003. Biotechnology from A to Z. Oxford University Press Inc.
New York, USA.
5. Klefenz H. 2002. Industrial Pharmaceutical Biotechnology. Wiley-VCH
Verlag GmbH, Weinheim, Germany.
6. Lee JM. 2000. Biochemical Engineering. Prentice Hall Inc. USA.
Nhp mn Cng ngh sinh hc 92
7. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge
University Press, UK.
8. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic Concepts.
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nd
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